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Sample records for agrobacterium-mediated transient gene

  1. AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings

    PubMed Central

    2014-01-01

    Background Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. Results We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. Conclusions AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous

  2. Agrobacterium mediated transient gene silencing (AMTS) in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

    PubMed

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

    Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

  3. Agrobacterium Mediated Transient Gene Silencing (AMTS) in Stevia rebaudiana: Insights into Steviol Glycoside Biosynthesis Pathway

    PubMed Central

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

    Background Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi) based Agrobacterium mediated transient gene silencing (AMTS) approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. Methodology/Principal Findings RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3) content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. Conclusions SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route. PMID:24023961

  4. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression

    PubMed Central

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area–time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  5. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression.

    PubMed

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area-time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  6. Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.

    PubMed

    Nanjareddy, Kalpana; Arthikala, Manoj-Kumar; Blanco, Lourdes; Arellano, Elizabeth S; Lara, Miguel

    2016-06-24

    Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. We present simple and efficient methodologies for protoplast isolation from multiple P

  7. Cloning and functional characterization of the promoter of PsSEOF1 gene from Pisum sativum under different stress conditions using Agrobacterium-mediated transient assay.

    PubMed

    Srivastava, Vineet Kumar; Raikwar, Shailendra; Tuteja, Narendra

    2014-01-01

    PsSEOF1, a SEO (sieve element occlusion) gene family protein (forisome) is calcium powered motor protein and is located close to plasma membrane of sieve element. In sieve element (SE) it senses the calcium ion levels and undergoes ATP-independent conformational shifts. Forisome, meaning gate-bodies (Latin foris: wing of a gate; Greek soma: body). Recent reports show that SEO gene family protein can prevent the loss of nutrient rich photoassimilate upon wound injury. The regulation of SEO protein forisome under abiotic/ biotic stress is still unknown. The analysis of cis-regulatory element present in the upstream region is not well understood. Tissue specific promoters guarantee correct expression when it perceives particular stimuli. Here we report isolation of tissue specific promoter of PsSEOF1 was isolated by gene walking PCR from P. sativum (pea) genomic DNA library constructed by BD genome walker kit. In silico analysis revealed several putative cis element within this promoter sequence like wound response, cold, dehydration. Putative elements which might be required for its vascular tissue specificity has also been identified. The GUS activities of PsSEOF1 promoter-GUS chimeric construct in the agroinfiltrated leaves under different environmental stress abiotic and biotic like wound, cold, salt and phytohormones has shown high level of GUS activity. To identify the activity of PsSEOF1 promoter under different stress condition an Agrobacterium-mediated transient expression of tobacco plants were subjected to histochemical GUS staining. Stress-inducible nature of PsSEOF1 promoter opens possibility for the study of the PsSEOF1 gene regulation under stress condition. The isolated promoter sequence could serve as an important candidate for tissue specific promoter in genetic engineering of plant under stress conditions.

  8. Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium-mediated transient gene expression system using Nicotiana benthamiana plants.

    PubMed

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2017-08-01

    Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM(-1) ) and recombinant protein productivity per unit area-time (g m(-2) /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m(-2) than at a low plant density of 100 plants m(-2) . The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area-time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium-mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762-1770. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Testing the utility of fluorescent proteins in Mimulus lewisii by an Agrobacterium-mediated transient assay.

    PubMed

    Ding, Baoqing; Yuan, Yao-Wu

    2016-04-01

    The Agrobacterium -mediated transient expression assay by leaf infiltration in Mimulus lewisii is robust. Fluorescent proteins EGFP, EYFP and DsRed give bright fluorescence signals in the infiltrated tissue. Mimulus lewisii is an emerging developmental genetic model system. Recently developed genomic and genetic resources and a stable transformation protocol have greatly facilitated the identification and functional characterization of genes controlling the development of ecologically important floral traits using this species. To further expedite gene and protein function analyses in M. lewisii, we adopted and simplified the Agrobacterium-mediated transient gene expression method routinely used in tobacco plants. With the validated transient assay, we examined the performance of fluorescent proteins EGFP, EYFP and DsRed in M. lewisii. All three proteins gave bright fluorescence signals when transiently expressed in agroinfiltrated leaves. Furthermore, we demonstrated the utility of fluorescent proteins in M. lewisii by showing the nuclear localization of Reduced Carotenoid Pigmentation 1 (RCP1), a recently discovered R2R3-MYB transcription factor that regulates carotenoid pigmentation during flower development. Both the transient assay and the fluorescent proteins are valuable additions to the M. lewisii toolbox, making this emerging genetic and developmental model system even more powerful.

  10. A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

    PubMed

    Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

  11. A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

    PubMed Central

    Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale. PMID:24416168

  12. Agrobacterium-mediated virus-induced gene silencing assay in cotton.

    PubMed

    Gao, Xiquan; Britt, Robert C; Shan, Libo; He, Ping

    2011-08-20

    Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation(1). To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions. Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation(2,3). Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies(3,4). As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation. In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development(6), and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves(7), providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration

  13. Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

    PubMed

    Buschmann, Henrik

    2016-01-01

    The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

  14. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  15. Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

    PubMed

    Wu, Su-Li; Yang, Xiao-Bing; Liu, Li-Qun; Jiang, Tao; Wu, Hai; Su, Chao; Qian, Yong-Hua; Jiao, Feng

    2015-01-01

    To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

  16. Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

    PubMed

    Bhagwat, Basdeo; Chi, Ming; Han, Dianwei; Tang, Haifeng; Tang, Guiliang; Xiang, Yu

    2016-01-01

    Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.

  17. ipt Gene transformation in petunia by an Agrobacterium mediated method.

    PubMed

    Bai, L J; Ye, C J; Lu, J Y; Yang, D E; Xue, H; Pan, Y; Cao, P X; Wang, B; Liu, M

    2009-01-01

    To prevent leaf senescence of petunia, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of 35S promoter and introduced into petunia. PCR analysis showed an expected 0.5 Kb fragment of ipt gene in transgenic petunia. RT-PCR analysis indicated the expression of ipt gene in the transgenic lines. Leaves from transgenic plants remained green and healthy in normal culture condition, while the non-transformed plants turned to yellow. Transgenic plants showed a reduction in height and smaller leaf sizes. In transgenic lines, the internodes were shorter, and the roots grew slower than the non-transformed plants.

  18. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    SciTech Connect

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  19. Agrobacterium-mediated transformation of Malus robusta with tomato iron transporter gene.

    PubMed

    Qu, Shen-Chun; Huang, Xiao-De; Zhang, Zhen; Yao, Quan-Hong; Tao, Jian-Min; Qiao, Yu-Shan; Zhang, Jun-Yi

    2005-06-01

    The tomato iron transporter gene (LeIRT2) was introduced to Malus robusta Rehd. via Agrobacterium-mediated transformation to produce iron-deficiency tolerant apple rootstock. A total of 19 putative transformants were obtained, 11 of which were verified by PCR amplification to carry a fragment of the transgene. Among them, nine were confirmed to carry the transgene by Southern blot analysis with one to three copies of the transgene integrated into the plant genome. Two transgenic plants, one carrying one copy and the other three copies of the transgene, were hydroponically cultured to test their tolerance to iron-deficiency, which was found only in the transgenic plant with a single copy, which weighted 21%-4% greater than those of the control plants.

  20. [Agrobacterium-mediated transformation of LJAMP2 gene into 'Red Sun' kiwifruit and its molecular identification].

    PubMed

    Zhou, Yue; Zhao, Xupeng; Wu, Xiuhua; Zhang, Yanling; Zhang, Lin; Luo, Keming; Tang, Shaohu

    2014-06-01

    Bacterial canker caused by Pseudomonas syringae pv. Actinidiae is one of the most important diseases of kiwifruit (Actinidia chinensis) and leads to considerable yield losses. In order to obtain transgenic plants with resistance for 'Red Sun' kiwifruit to canker disease, a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into 'Red Sun' kiwifruit through Agrobacterium-mediated transformation. After two days of co-cultivation with A. tumefaciens strain LBA4404 harboring 35S:LJAMP2, the transformed explants were transferred to the selection medium containing 25 mg/L kanamycin+3.0 mg/L BA+1.0 mg/L NAA. The regeneration efficiency of kanamycin-resistant shoots reached to 85%. All (100%) of kanamycin-resistant shoots rooted on half-strength MS medium supplemented with 0.8 mg/L IBA and a total of 40 regenerated plantlets were obtained. PCR and histochemical GUS activity analysis show that 23 of 40 lines (57.50%) were positive, suggesting that the LJAMP2 gene was integrated into the genome of 'Red Sun' kiwifruit. Taken together, we established an efficient genetic transformation method for 'Red Sun' kiwifruit using A. tumefaciens and the transformation frequency reached 5.11%. This protocol will be useful for the genetic breeding of 'Red Sun' kiwifruit for improvement of disease resistance.

  1. Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches.

    PubMed

    de Boer, Paulo; Bronkhof, Jurian; Dukiќ, Karolina; Kerkman, Richard; Touw, Hesselien; van den Berg, Marco; Offringa, Remko

    2013-12-01

    The industrial production of β-lactam antibiotics by Penicillium chrysogenum has increased tremendously over the last decades, however, further optimization via classical strain and process improvement has reached its limits. The availability of the genome sequence provides new opportunities for directed strain improvement, but this requires the establishment of an efficient gene targeting (GT) system. Recently, mutations affecting the non-homologous end joining (NHEJ) pathway were shown to increase GT efficiencies following PEG-mediated DNA transfer in P. chrysogenum from 1% to 50%. Apart from direct DNA transfer many fungi can efficiently be transformed using the T-DNA transfer system of the soil bacterium Agrobacterium tumefaciens, however, for P. chrysogenum no robust system for Agrobacterium-mediated transformation was available. We obtained efficient AMT of P. chrysogenum spores with the nourseothricin acetyltransferase gene as selection marker, and using this system we investigated if AMT in a NHEJ mutant background could further enhance GT efficiencies. In general, AMT resulted in higher GT efficiencies than direct DNA transfer, although the final frequencies depended on the Agrobacterium strain and plasmid backbone used. Providing overlapping and complementing fragments on two different plasmid backbones via the same Agrobacterium host was shown to be most effective. This so-called split-marker or bi-partite method resulted in highly efficient GT (>97%) almost exclusively without additional ectopic T-DNA insertions. As this method provides for an efficient GT method independent of protoplasts, it can be applied to other fungi for which no protoplasts can be generated or for which protoplast transformation leads to varying results.

  2. Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement

    PubMed Central

    Nyaboga, Evans; Tripathi, Jaindra N.; Manoharan, Rajesh; Tripathi, Leena

    2014-01-01

    Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important

  3. Isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 using Agrobacterium-mediated transient assay.

    PubMed

    Banu, Sufara Akhter; Huda, Kazi Md Kamrul; Tuteja, Narendra

    2014-04-30

    Helicases are molecular motor proteins that perform a variety of cellular functions including transcription, translation, DNA replication and repair, RNA maturation, ribosome synthesis, nuclear export and splicing processes. The p68 is an evolutionarily conserved protein which plays pivotal roles in all aspect RNA metabolism processes. It is well established that helicases provides abiotic stress adaptation in plants but analysis of cis-regulatory elements present in the upstream regions is still infancy. Here we report isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 in response to abiotic stress and hormonal regulation. The promoter of Psp68 was isolated by gene walking PCR from pea genomic DNA library constructed in BD genome walker kit. In silico analysis revealed that promoter of Psp68 contained a TATA, a CAAT motif and also harbors some important stress and hormone associated cis regulatory elements, including E-box, AGAAA, GATA-box, ACGT, GAAAA and GTCTC. Functional analyses were performed by Agrobacterium-mediated transient assay in tobacco leaves. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the Psp68 promoter::GUS, subjected to abiotic stress and exogenous hormonal treatments. Stress-inducible nature of Psp68 promoter opens possibility for the study of the gene regulation under stress condition. Therefore, may be useful in the field of agriculture and biotechnology.

  4. Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995

    SciTech Connect

    Marton, L.

    1996-02-01

    Genetic manipulation of plants often involves the introduction of homologous or partly homologous genes. Ectropic introduction of homologous sequences into plant genomes may trigger epigenetic changes, making expression of the genes unpredictable. The main project objective was to examine the feasibility of using Agrobacterium-mediated gene transfer for homologous gene targeting in plants.

  5. Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing.

    PubMed

    Gao, Xiquan; Shan, Libo

    2013-01-01

    Cotton (Gossypium spp.) is one of the most agronomically important crops worldwide for its unique textile fiber production and serving as food and feed stock. Molecular breeding and genetic engineering of useful genes into cotton have emerged as advanced approaches to improve cotton yield, fiber quality, and resistance to various stresses. However, the understanding of gene functions and regulations in cotton is largely hindered by the limited molecular and biochemical tools. Here, we describe the method of an Agrobacterium infiltration-based virus-induced gene silencing (VIGS) assay to transiently silence endogenous genes in cotton at 2-week-old seedling stage. The genes of interest could be readily silenced with a consistently high efficiency. To monitor gene silencing efficiency, we have cloned cotton GrCla1 from G. raimondii, a homolog gene of Arabidopsis Cloroplastos alterados 1 (AtCla1) involved in chloroplast development, and inserted into a tobacco rattle virus (TRV) binary vector pYL156. Silencing of GrCla1 results in albino phenotype on the newly emerging leaves, serving as a visual marker for silencing efficiency. To further explore the possibility of using VIGS assay to reveal the essential genes mediating disease resistance to Verticillium dahliae, a fungal pathogen causing severe Verticillium wilt in cotton, we developed a seedling infection assay to inoculate cotton seedlings when the genes of interest are silenced by VIGS. The method we describe here could be further explored for functional genomic analysis of cotton genes involved in development and various biotic and abiotic stresses.

  6. SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation.

    PubMed

    Liu, Yiming; Miao, Jiamin; Traore, Sy; Kong, Danyu; Liu, Yi; Zhang, Xunzhong; Nimchuk, Zachary L; Liu, Zongrang; Zhao, Bingyu

    2016-01-01

    Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor.

  7. SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation

    PubMed Central

    Liu, Yiming; Miao, Jiamin; Traore, Sy; Kong, Danyu; Liu, Yi; Zhang, Xunzhong; Nimchuk, Zachary L.; Liu, Zongrang; Zhao, Bingyu

    2016-01-01

    Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor. PMID:27833912

  8. Production of purple-colored creeping bentgrass using maize transcription factor genes Pl and Lc through Agrobacterium-mediated transformation.

    PubMed

    Han, Yun-Jeong; Kim, Yong-Min; Lee, Jee-Yeon; Kim, Soo Jung; Cho, Kyu-Chang; Chandrasekhar, Thummala; Song, Pill-Soon; Woo, Young-Min; Kim, Jeong-Il

    2009-03-01

    Purple-colored transgenic creeping bentgrass (Agrostis stolonifera L.) plants were developed for ornamental purpose by means of Agrobacterium-mediated transformation. Embryogenic creeping bentgrass calli were transformed with the pCAMBIA 3301 vector harboring maize (Zea mays) flavonoid/anthocyanin biosynthetic pathway transcription factor genes, Lc (Leaf color) and Pl (Purple leaf), individually and in combination, and three types of putative transgenic plants (Lc, Pl, and Lc + Pl) were generated. Genomic integration and expression of the transgenes were confirmed by Southern and northern blot analyses, respectively. The transgenic creeping bentgrass plants expressing both Lc and Pl genes were entirely purple, whereas those expressing Pl alone had purple stems and those expressing Lc alone lacked purple pigmentation in adult plants. The anthocyanin content was estimated in all the three types of transgenic plant and correlated well with the degree of purple coloration observed. These results suggest that both Lc and Pl genes are necessary and sufficient to confer purple coloration to creeping bentgrass.

  9. Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene.

    PubMed

    Li, D D; Shi, W; Deng, X X

    2003-12-01

    Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens. Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS). The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium. Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C. Almost no resistant callus was obtained if the cocultivation medium lacked AS. The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting. Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods.

  10. The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum).

    PubMed

    Lamblin, Frédéric; Aimé, Aurélie; Hano, Christophe; Roussy, Isabelle; Domon, Jean-Marc; Van Droogenbroeck, Bart; Lainé, Eric

    2007-06-01

    In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants, we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L(-1) sucrose and 10 g L(-1) mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached 6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully used for the recovery of flax transgenic plants under safe conditions for human health and the environment.

  11. Functional Characterization of a Bidirectional Plant Promoter from Cotton Leaf Curl Burewala Virus Using an Agrobacterium-Mediated Transient Assay

    PubMed Central

    Ashraf, Muhammad Aleem; Shahid, Ahmad Ali; Rao, Abdul Qayyum; Bajwa, Kamran Shehzad; Husnain, Tayyab

    2014-01-01

    The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional genepromoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant. PMID:24424501

  12. Scale-up of Agrobacterium-mediated transient protein expression in bioreactor-grown Nicotiana glutinosa plant cell suspension culture.

    PubMed

    O'Neill, Kristin M; Larsen, Jeffrey S; Curtis, Wayne R

    2008-01-01

    The reporter gene beta-glucuronidase was transiently expressed in a 51-L bioreactor-grown plant cell suspension culture of Nicotiana glutinosa at a yield of approximately 1.1 mg through co-culture with an auxotrophic strain of Agrobacterium tumefaciens. The three order of magnitude scale-up involved the investigation of factors contributing to transient expression including the timing of Agrobacterium inoculation relative to the plant cell growth phase, plant tissue culture hormonal triggers and plant cell cycle synchronization. The co-culture process was simplified to facilitate implementation in a pilot-scale bioreactor. At the shake flask scale it was determined that elevated concentrations of oxygen in the headspace were detrimental to transient expression levels and the addition of acetosyringone to the co-culture had a negligible effect. The bacterial preparation process was also streamlined, permitting the direct transfer of the Agrobacterium culture from a bench-scale fermentor to the pilot-scale plant cell culture bioreactor. Increasing expression levels and overcoming batch-to-batch variability despite extensive procedure systemization remain the major technical hurdles.

  13. Agrobacterium-Mediated Co-transformation of Multiple Genes in Metarhizium robertsii

    PubMed Central

    Bidochka, Michael J.

    2017-01-01

    Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii. PMID:28781540

  14. Agrobacterium-mediated transformation of rough lemon (Citrus jambhiri Lush) with yeast HAL2 gene.

    PubMed

    Ali, Shawkat; Mannan, Abdul; El Oirdi, Mohamed; Waheed, Abdul; Mirza, Bushra

    2012-06-12

    Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants. Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100 mg L(-1) along with 250 mg L(-1) each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200 μM acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3 mg L(-1) of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis. Rough lemon has been successfully transformed via Agrobacterium tumefaciens with β-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated.

  15. Agrobacterium-mediated transformation of rough lemon (Citrus jambhiri Lush) with yeast HAL2 gene

    PubMed Central

    2012-01-01

    Background Rough lemon (Citrus jambhiri Lush.) is the most commonly used Citrus rootstock in south Asia. It is extremely sensitive to salt stress that decreases the growth and yield of Citrus crops in many areas worldwide. Over expression of the yeast halotolerant gene (HAL2) results in increasing the level of salt tolerance in transgenic plants. Results Transformation of rough lemon was carried out by using Agrobacterium tumefaciens strains LBA4404 harboring plasmid pJRM17. Transgenic shoots were selected on kanamycin 100 mg L-1 along with 250 mg L-1 each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200 μM acetoseryngone (AS) proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3 mg L-1 of 6-benzylaminopurine (BA) showed maximum regeneration efficiency of the transformed explants. The final selection of the transformed plants was made on the basis of PCR and Southern blot analysis. Conclusion Rough lemon has been successfully transformed via Agrobacterium tumefaciens with β-glucuronidase (GUS) and HAL2. Various factors affecting gene transformation and regeneration efficiency were also investigated. PMID:22691292

  16. SacB-SacR gene cassette as the negative selection marker to suppress Agrobacterium overgrowth in Agrobacterium-mediated plant transformation

    USDA-ARS?s Scientific Manuscript database

    Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce ...

  17. Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker.

    PubMed

    Chakraborty, M; Sairam Reddy, P; Laxmi Narasu, M; Krishna, Gaurav; Rana, Debashis

    2016-01-01

    Transformation of commercially important indica cultivars remains challenging for the scientific community even though Agrobacterium-mediated transformation protocols for a few indica rice lines have been well established. We report successful transformation of a commercially important restorer line JK1044R of indica rice hybrid JKRH 401. While following existing protocol, we optimized several parameters for callusing, regeneration and genetic transformation of JK1044R. Calli generated from the rice scutellum tissue were used for transformation by Agrobacterium harboring pCAMBIA2201. A novel two tire selection scheme comprising of Geneticin (G418) and Paramomycin were deployed for selection of transgenic calli as well as regenerated plantlets that expressed neomycin phosphotransferase-II gene encoded by the vector. One specific combination of G418 (30 mg l(-1)) and Paramomycin (70 mg l(-1)) was very effective for calli selection. Transformed and selected calli were detected by monitoring the expression of the reporter gene uidA (GUS). Regenerated plantlets were confirmed through PCR analysis of nptII and gus genes specific primers as well as dot blot using gus gene specific as probe.

  18. Barley Transformation Using Agrobacterium-Mediated Techniques

    NASA Astrophysics Data System (ADS)

    Harwood, Wendy A.; Bartlett, Joanne G.; Alves, Silvia C.; Perry, Matthew; Smedley, Mark A.; Leyland, Nicola; Snape, John W.

    Methods for the transformation of barley using Agrobacterium-mediated techniques have been available for the past 10 years. Agrobacterium offers a number of advantages over biolistic-mediated techniques in terms of efficiency and the quality of the transformed plants produced. This chapter describes a simple system for the transformation of barley based on the infection of immature embryos with Agrobacterium tumefaciens followed by the selection of transgenic tissue on media containing the antibiotic hygromycin. The method can lead to the production of large numbers of fertile, independent transgenic lines. It is therefore ideal for studies of gene function in a cereal crop system.

  19. Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L.

    PubMed

    Sankari, Mohan; Hemachandran, Hridya; Anantharaman, Amirtha; Babu, Subramanian; Madrid, Renata Rivera; C, George Priya Doss; Fulzele, Devanand P; Siva, Ramamoorthy

    2016-07-01

    Carotenoids are metabolized to apocarotenoids through the pathway catalysed by carotenoid cleavage oxygenases (CCOs). The apocarotenoids are economically important as it is known to have therapeutic as well as industrial applications. For instance, bixin from Bixa orellana and crocin from Crocus sativus are commercially used as a food colourant and cosmetics since prehistoric time. In our present study, CCD4a gene has been identified and isolated from leaves of B. orellana for the first time and named as BoCCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, BoCCD4a contains two exons and one intron, which was compared with the selected AtCCD4, RdCCD4, GmCCD4 and CmCCD4a gene. Further, the BoCCD4a gene was cloned into pCAMBIA 1301, transformed into Agrobacterium tumefaciens EHA105 strain and subsequently transferred into hypocotyledons and callus of B. orellana by agro-infection. Selection of stable transformation was screened on the basis of PCR detection by using GUS and hptII specific primer, which was followed by histochemical characterization. The percent transient GUS expression in hypocotyledons and callus was 84.4 and 80 %, respectively. The expression of BoCCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of BoCCD4a gene of B. orellana was closely related to the CsCCD4 gene of C. sativus, which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination.

  20. Development of Agrobacterium-Mediated Virus-Induced Gene Silencing and Performance Evaluation of Four Marker Genes in Gossypium barbadense

    PubMed Central

    Pang, Jinhuan; Zhu, Yue; Li, Qing; Liu, Jinzhi; Tian, Yingchuan; Liu, Yule; Wu, Jiahe

    2013-01-01

    Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species). These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS) system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV) vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum. PMID:24023833

  1. Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.

    PubMed

    Pang, Jinhuan; Zhu, Yue; Li, Qing; Liu, Jinzhi; Tian, Yingchuan; Liu, Yule; Wu, Jiahe

    2013-01-01

    Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species). These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS) system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV) vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum.

  2. Rapid and efficient Agrobacterium-mediated transformation of sorghum (Sorghum bicolor) employing standard binary vectors and bar gene as a selectable marker.

    PubMed

    Do, Phat T; Lee, Hyeyoung; Mookkan, Muruganantham; Folk, William R; Zhang, Zhanyuan J

    2016-10-01

    A rapid and efficient Agrobacterium -mediated transformation system in sorghum has been developed employing standard binary vectors and bar gene as a selectable marker. Sorghum (Sorghum bicolor) is an important food and biofuel crop worldwide, for which improvements in genetic transformation are needed to study its biology and facilitate agronomic and commercial improvement. Here, we report optimization of regeneration and transformation of public sorghum genotype P898012 using standard binary vectors and bar gene as a selectable marker. The tissue culture regeneration time frame has been reduced to 7-12 weeks with a yield of over 18 plants per callus, and the optimized transformation system employing Agrobacterium tumefaciens strain AGL1 and the bar with a MAS promoter achieved an average frequency over 14 %. Of randomly analyzed independent transgenic events, 40-50 % carry single copy of integrated T-DNA. Some independent transgenic events were derived from the same embryogenic callus lines, but a 3:1 Mendelian segregation ratio was found in all transgenic events with single copy as estimated by Southern blots. The system described here should facilitate studies of sorghum biology and agronomic improvement.

  3. RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

    PubMed Central

    2012-01-01

    Background Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures. Results Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene β-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass. Conclusions For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens. PMID:22559055

  4. Functional analysis of autophagy genes via Agrobacterium-mediated transformation in the vascular Wilt fungus Verticillium dahliae.

    PubMed

    Zhou, Lei; Zhao, Jun; Guo, Wangzhen; Zhang, Tianzhen

    2013-08-20

    Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agrobacterium tumefaciens-mediated transformation (ATMT) system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant transformants per 1 × 10(6) conidia. V. dahliae mutants lacking either VdATG8 or VdATG12 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild-type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in V. dahliae.

  5. The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes.

    PubMed

    Rakosy-Tican, Elena; Aurori, Cristian M; Dijkstra, Camelia; Thieme, Ramona; Aurori, Adriana; Davey, Michael R

    2007-05-01

    Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.

  6. Agrobacterium-mediated transformation of cotton (Gossypium hirsutum) shoot apex with a fungal phytase gene improves phosphorus acquisition.

    PubMed

    Ma, Zhiying; Liu, Jianfeng; Wang, Xingfen

    2013-01-01

    Cotton is an important world economic crop plant. It is considered that cotton is recalcitrant to in vitro proliferation. Somatic embryogenesis and plant regeneration has been successful by using hypocotyl, whereas it is highly genotype dependent. Here, a genotype-independent cotton regeneration protocol from shoot apices is presented. Shoot apices from 3- to 5-day-old seedlings of cotton are infected with an Agrobacterium strain, EHA105, carrying the binary vector pC-KSA contained phytase gene (phyA) and the marker gene neomycin phosphotransferase (NPTII), and directly regenerated as shoots in vitro. Rooted shoots can be obtained within 6-8 weeks. Plants that survived by leaf painting kanamycin (kan) were -further analyzed by DNA and RNA blottings. The transgenic plants with increased the phosphorus (P) acquisition efficiency were obtained following the transformation method.

  7. [Effective agrobacterium-mediated transformation of chicory (Cichorium intybus L.) using vector with gene of tuberculosis antigene ESAT6].

    PubMed

    Matveeva, N A; Vasilenko, M Iu; Shakhovskiĭ, A M; Bannikova, M A; Kvasko, O Iu; Kuchuk, N V

    2011-01-01

    The conditions of high efficient chicory transformation with Mycobacterium tuberculosis antigene ESAT6 have been determined. Transformation frequency was up to 86% when the cotyledons were cultivated within 3 days without cefotaxime and then 1 day without kanamycine. DNA PCR-analysis has shown the presence both of selective nptII and target esxA genes in all analysed plants. At the same time RT-PCR has shown the presence of nptII transcripts for eight analysed lines and esxA transcripts for only five analysed lines.

  8. Optimization of Agrobacterium-Mediated Transformation in Soybean

    PubMed Central

    Li, Shuxuan; Cong, Yahui; Liu, Yaping; Wang, Tingting; Shuai, Qin; Chen, Nana; Gai, Junyi; Li, Yan

    2017-01-01

    High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens-mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium-mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA3) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR, herbicide

  9. Agrobacterium-mediated transformation of three freshwater microalgal strains.

    PubMed

    Sanitha, Mary; Radha, Sudhakar; Fatima, Anwar Aliya; Devi, Selvaraju Gayathri; Ramya, Mohandass

    2014-01-01

    Microalgal transformation has gained interest in recent years. Agrobacterium-mediated transformation remains as the most efficient method for the development of transgenic plants and microalgae due to its wide host range, inexpensive procedure and transfer of large segments of DNA. In the present study, three different microalgal species were isolated from freshwater environment and identified based on the morphological characteristics and ITS-2 region amplification. Agrobacterium-mediated transformation was successful for the isolates Chlorella sp., Ankistrodesmus sp and Scenedesmus bajacalifornicus. Gene integration and expression was confirmed by PCR amplification of hptII and GUS histochemical assay. A. tumifaciens contamination was checked by amplification of npt II gene (kanamycin resistant) which lies outside the T-border. Based on GUS assay, transformation efficiencies were found to be 12.25% for Chlorella sp. 2.96% for Scenedesmus bajacalifornicus and 3.5% for Ankistrodesmus sp.

  10. Agrobacterium-mediated transformation of Fusarium proliferatum.

    PubMed

    Bernardi-Wenzel, J; Quecine, M C; Azevedo, J L; Pamphile, J A

    2016-06-03

    Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacterium-mediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process.

  11. Development of an efficient agrobacterium-mediated gene targeting system for rice and analysis of rice knockouts lacking granule-bound starch synthase (Waxy) and β1,2-xylosyltransferase.

    PubMed

    Ozawa, Kenjirou; Wakasa, Yuhya; Ogo, Yuko; Matsuo, Kouki; Kawahigashi, Hiroyuki; Takaiwa, Fumio

    2012-04-01

    We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (β1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.

  12. A comparison of Agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius.

    PubMed

    Weyda, István; Yang, Lei; Vang, Jesper; Ahring, Birgitte K; Lübeck, Mette; Lübeck, Peter S

    2017-04-01

    In recent years, versatile genetic tools have been developed and applied to a number of filamentous fungi of industrial importance. However, the existing techniques have limitations when it comes to achieve the desired genetic modifications, especially for efficient gene targeting. In this study, we used Aspergillus carbonarius as a host strain due to its potential as a cell factory, and compared three gene targeting techniques by disrupting the ayg1 gene involved in the biosynthesis of conidial pigment in A. carbonarius. The absence of the ayg1 gene leads to phenotypic change in conidia color, which facilitated the analysis on the gene targeting frequency. The examined transformation techniques included Agrobacterium-mediated transformation (AMT) and protoplast-mediated transformation (PMT). Furthermore, the PMT for the disruption of the ayg1 gene was carried out with bipartite gene targeting fragments and the recently adapted CRISPR-Cas9 system. All three techniques were successful in generating Δayg1 mutants, but showed different efficiencies. The most efficient method for gene targeting was AMT, but further it was shown to be dependent on the choice of Agrobacterium strain. However, there are different advantages and disadvantages of all three gene targeting methods which are discussed, in order to facilitate future approaches for fungal strain improvements.

  13. High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

    NASA Technical Reports Server (NTRS)

    Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

    1999-01-01

    Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

  14. High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

    NASA Technical Reports Server (NTRS)

    Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

    1999-01-01

    Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

  15. Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation

    PubMed Central

    Kwon, Tackmin

    2016-01-01

    The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection. PMID:27643450

  16. Infiltration-RNAseq: transcriptome profiling of Agrobacterium-mediated infiltration of transcription factors to discover gene function and expression networks in plants.

    PubMed

    Bond, Donna M; Albert, Nick W; Lee, Robyn H; Gillard, Gareth B; Brown, Chris M; Hellens, Roger P; Macknight, Richard C

    2016-01-01

    Transcription factors (TFs) coordinate precise gene expression patterns that give rise to distinct phenotypic outputs. The identification of genes and transcriptional networks regulated by a TF often requires stable transformation and expression changes in plant cells. However, the production of stable transformants can be slow and laborious with no guarantee of success. Furthermore, transgenic plants overexpressing a TF of interest can present pleiotropic phenotypes and/or result in a high number of indirect gene expression changes. Therefore, fast, efficient, high-throughput methods for assaying TF function are needed. Agroinfiltration is a simple plant biology method that allows transient gene expression. It is a rapid and powerful tool for the functional characterisation of TF genes in planta. High throughput RNA sequencing is now a widely used method for analysing gene expression profiles (transcriptomes). By coupling TF agroinfiltration with RNA sequencing (named here as Infiltration-RNAseq), gene expression networks and gene function can be identified within a few weeks rather than many months. As a proof of concept, we agroinfiltrated Medicago truncatula leaves with M. truncatula LEGUME ANTHOCYANIN PRODUCITION 1 (MtLAP1), a MYB transcription factor involved in the regulation of the anthocyanin pathway, and assessed the resulting transcriptome. Leaves infiltrated with MtLAP1 turned red indicating the production of anthocyanin pigment. Consistent with this, genes encoding enzymes in the anthocyanin biosynthetic pathway, and known transcriptional activators and repressors of the anthocyanin biosynthetic pathway, were upregulated. A novel observation was the induction of a R3-MYB transcriptional repressor that likely provides transcriptional feedback inhibition to prevent the deleterious effects of excess anthocyanins on photosynthesis. Infiltration-RNAseq is a fast and convenient method for profiling TF-mediated gene expression changes. We utilised this method

  17. [Agrobacterium-mediated sunflower transformation (Helianthus annuus L.) in vitro and in Planta using strain of LBA4404 harboring binary vector pBi2E with dsRNA-suppressor proline dehydrogenase gene].

    PubMed

    Tishchenko, E N; Komisarenko, A G; Mikhal'skaia, S I; Sergeeva, L E; Adamenko, N I; Morgun, B V; Kochetov, A V

    2014-01-01

    To estimate the efficiency of proline dehydrogenase gene suppression towards increasing of sunflower (Helianthus annuus L.) tolerance level to water deficit and salinity, we employed strain LBA4404 harboring pBi2E with double-stranded RNA-suppressor, which were prepared on basis arabidopsis ProDH1 gene. The techniques of Agrobacterium-mediated transformation in vitro and in planta during fertilization sunflower have been proposed. There was shown the genotype-depended integration of T-DNA in sunflower genome. PCR-analysis showed that ProDH1 presents in genome of inbred lines transformed in planta, as well as in T1- and T2-generations. In trans-genic regenerants the essential accumulation of free L-proline during early stages of in vitro cultivation under normal conditions was shown. There was established the essential accumulation of free proline in transgenic regenerants during cultivation under lethal stress pressure (0.4 M mannitol and 2.0% sea water salts) and its decline upon the recovery period. These data are declared about effectiveness of suppression of sunflower ProDH and gene participation in processes connected with osmotolerance.

  18. Agrobacterium-mediated sorghum transformation.

    PubMed

    Zhao, Z Y; Cai, T; Tagliani, L; Miller, M; Wang, N; Pang, H; Rudert, M; Schroeder, S; Hondred, D; Seltzer, J; Pierce, D

    2000-12-01

    Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a 'Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T0 plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T1 generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.

  19. Agrobacterium-mediated transformation of maize (Zea mays) immature embryos.

    PubMed

    Lee, Hyeyoung; Zhang, Zhanyuan J

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is one of the most efficient and simple gene delivery systems for genetic improvement and biology studies in maize. This system has become more widely used by both public and private laboratories. However, transformation efficiencies vary greatly from laboratory to laboratory for the same genotype. Here, we illustrate our advanced Agrobacterium-mediated transformation method in Hi-II maize using simple binary vectors. The protocol utilizes immature embryos as starting explants and the bar gene as a selectable marker coupled with bialaphos as a selective agent. The protocol offers efficient transformation results with high reproducibility, provided that some experimental conditions are well controlled. This transformation method, with minor modifications, can be also employed to transform certain maize inbreds.

  20. Selectable marker elimination in the T0 generation by Agrobacterium-mediated co-transformation involving Mungbean yellow mosaic virus TrAP as a non-conditional negative selectable marker and bar for transient positive selection.

    PubMed

    RamanaRao, Mangu Venkata; Veluthambi, Karuppannan

    2010-05-01

    Transient selection involving the bar gene and non-conditional negative selection against stable T-DNA integration through the use of the Mungbean yellow mosaic virus (MYMV) transcriptional activator protein gene (TrAP) were used in a novel co-transformation strategy to generate selectable marker gene (SMG)-eliminated transgenic tobacco plants in the T(0) generation itself. Two compatible binary plasmids, pCam-bar-TrAP-gus harbouring bar as an SMG and the MYMV TrAP gene as a non-conditional negative selectable marker, and pGA472 with the nptII gene as an unselected experimental gene of interest (GOI) were placed in the Agrobacterium tumefaciens strain EHA105 and used for co-transformation. Transient selection with 5 mg l(-1) phosphinothricin (PPT) for 2-4 weeks and subsequent establishment in a PPT-minus medium yielded 114 plants from 200 leaf discs. The unselected nptII gene was detected by Southern blot analysis in 13 plants, revealing a co-transformation efficiency of 11.5%. Five of these plants harboured only the nptII gene (GOI) and not the bar gene (SMG). Thus, SMG elimination was achieved in the T(0) generation itself in 4.4% (5/114) of plants, which were transiently selected for 2-4 weeks on PPT. MYMV TrAP, a non-conditional negative selectable marker, effectively reduced the recovery of plants with stable integration of the SMG (bar).

  1. Agrobacterium-mediated in planta genetic transformation of sugarcane setts.

    PubMed

    Mayavan, Subramanian; Subramanyam, Kondeti; Jaganath, Balusamy; Sathish, Dorairaj; Manickavasagam, Markandan; Ganapathi, Andy

    2015-10-01

    An efficient, reproducible, and genotype-independent in planta transformation has been developed for sugarcane using setts as explant. Traditional Agrobacterium-mediated genetic transformation and in vitro regeneration of sugarcane is a complex and time-consuming process. Development of an efficient Agrobacterium-mediated transformation protocol, which can produce a large number of transgenic plants in short duration is advantageous. Hence, in the present investigation, we developed a tissue culture-independent in planta genetic transformation system for sugarcane using setts collected from 6-month-old sugarcane plants. The sugarcane setts (nodal cuttings) were infected with three Agrobacterium tumefaciens strains harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA(®). Several parameters influencing the in planta transformation such as A. tumefaciens strains, acetosyringone, sonication and exposure to vacuum pressure, have been evaluated. The putatively transformed sugarcane plants were screened by GUS histochemical assay. Sugarcane setts were pricked and sonicated for 6 min and vacuum infiltered for 2 min at 500 mmHg in A. tumefaciens C58C1 suspension containing 100 µM acetosyringone, 0.1 % Silwett L-77 showed the highest transformation efficiency of 29.6 % (with var. Co 62175). The three-stage selection process completely eliminated the chimeric transgenic sugarcane plants. Among the five sugarcane varieties evaluated using the standardized protocol, var. Co 6907 showed the maximum transformation efficiency (32.6 %). The in planta transformation protocol described here is applicable to transfer the economically important genes into different varieties of sugarcane in relatively short time.

  2. Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

    USDA-ARS?s Scientific Manuscript database

    Citrus paradisi Macf. cv. Duncan was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from CTV genome and used to gener...

  3. Agrobacterium-mediated transformation of potato using PLRV-rep and PVY CP genes and assessment of replicase mediated resistance against natural infection of PLRV

    USDA-ARS?s Scientific Manuscript database

    Replicase-and coat protein gene-mediated resistances against potato leafroll virus (PLRV) and potato virus Y (PVY), respectively, demonstrated to be an effective way of protecting potato against two major virus problems (PLRV & PVY) world-wide. Potato cultivar Desiree was transformed using Agrobacte...

  4. Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens

    PubMed Central

    Arshad, Waheed; Haq, Ihsan-ul-; Waheed, Mohammad Tahir; Mysore, Kirankumar S.; Mirza, Bushra

    2014-01-01

    Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens. PMID:24817272

  5. Complementary DNA cloning of the pear 1-aminocyclopropane-1-carboxylic acid oxidase gene and agrobacterium-mediated anti-sense genetic transformation.

    PubMed

    Qi, Jing; Dong, Zhen; Zhang, Yu-Xing

    2015-12-01

    The aim of the present study was to genetically modify plantlets of the Chinese yali pear to reduce their expression of ripening-associated 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and therefore increase the shelf-life of the fruit. Primers were designed with selectivity for the conserved regions of published ACO gene sequences, and yali complementary DNA (cDNA) cloning was performed by reverse transcription quantitative polymerase chain reaction (PCR). The obtained cDNA fragment contained 831 base pairs, encoding 276 amino acid residues, and shared no less than 94% nucleotide sequence identity with other published ACO genes. The cDNA fragment was inversely inserted into a pBI121 expression vector, between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, in order to construct the anti‑sense expression vector of the ACO gene; it was transfected into cultured yali plants using Agrobacterium LBA4404. Four independent transgenic lines of pear plantlets were obtained and validated by PCR analysis. A Southern blot assay revealed that there were three transgenic lines containing a single copy of exogenous gene and one line with double copies. The present study provided germplasm resources for the cultivation of novel storage varieties of pears, therefore providing a reference for further applications of anti‑sense RNA technology in the genetic improvement of pears and other fruit.

  6. An efficient method for Agrobacterium-mediated genetic transformation and plant regeneration in cumin (Cuminum cyminum L.).

    PubMed

    Pandey, Sonika; Mishra, Avinash; Patel, Manish Kumar; Jha, Bhavanath

    2013-09-01

    Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg's B5 medium supplemented with 0.5-μM 6-benzyladenine and 2.0-μM α-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD600, a co-cultivation time of 72 h, 300-μM acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient β-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T0 plantlets showed β-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and β-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin.

  7. Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize.

    PubMed

    Sivamani, Elumalai; Li, Xianggan; Nalapalli, Samson; Barron, Yoshimi; Prairie, Anna; Bradley, David; Doyle, Michele; Que, Qiudeng

    2015-12-01

    Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments.

  8. Agrobacterium-mediated transformation of embryogenic cultures and plant regeneration in Vitis rotundifolia Michx. (muscadine grape).

    PubMed

    Dhekney, S A; Li, Z T; Dutt, M; Gray, D J

    2008-05-01

    A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM) and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l(-1) in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of transgenic cells, compared to 50 or 75 mg l(-1), which permitted the proliferation of more non-transformed cells. Transgenic plants of "Alachua" and "Carlos" were recovered after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization. Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots, inflorescences and the embryo and endosperm of developing berries.

  9. Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.).

    PubMed

    Han, J-S; Kim, C K; Park, S H; Hirschi, K D; Mok, I- G

    2005-03-01

    We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the beta-D-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l L-alpha-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l DL-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l DL-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.

  10. Efficient Agrobacterium-mediated transformation of the liverwort Marchantia polymorpha using regenerating thalli.

    PubMed

    Kubota, Akane; Ishizaki, Kimitsune; Hosaka, Masashi; Kohchi, Takayuki

    2013-01-01

    The thallus, the gametophyte body of the liverwort Marchantia polymorpha, develops clonal progenies called gemmae that are useful in the isolation and propagation of isogenic plants. Developmental timing is critical to Agrobacterium-mediated transformation, and high transformation efficiency has been achieved only with sporelings. Here we report an Agrobacterium-mediated transformation system for M. polymorpha using regenerating thalli. Thallus regeneration was induced by cutting the mature thallus across the apical-basal axis and incubating the basal portion of the thallus for 3 d. Regenerating thalli were infected with Agrobacterium carrying binary vector that contained a selection marker, the hygromycin phosphotransferase gene, and hygromycin-resistant transformants were obtained with an efficiency of over 60%. Southern blot analysis verified random integration of 1 to 4 copies of the T-DNA into the M. polymorpha genome. This Agrobacterium-mediated transformation system for M. polymorpha should provide opportunities to perform genetic transformation without preparing spores and to generate a sufficient number of transformants with isogenic background.

  11. Efficient method for Agrobacterium mediated transformation of Artemisia annua L.

    PubMed

    Alam, Pravej; Mohammad, Anis; Ahmad, M M; Khan, Mather Ali; Nadeem, Mohd; Khan, Riyazuddeen; Akmal, Mohd; Ahlawat, Seema; Abdin, M Z

    2014-01-01

    Artemisinin, a potent antimalarial natural products isolated from aerial parts of Artemisia annua L. Many patents have been reported that the demand for artemisinin is exponentially increasing year after year due to increased incidences of drug resistant malaria throughout the world. Leaf explants were used frequently as target tissue to generate transgenic of Artemisia. annua L. However, obtaining a large number of transgenic lines through out the year is a laborious and delicate process. To circumvent this, we have developed a highly efficient leaf explant based Agrobacterium mediated transformation of A. annua L. plant. The gus gene was used as screenable marker to assess and optimize the performance of T-DNA delivery. The age of explant, kind of bacterial inoculation, suspension duration, infection times and co-culture conditions were optimized. The co-culture was carried out with Agrobacterium tumefaciens strain EHA105 under desiccation condition in the dark at 25-28 0C for 2-4 days. Complete analysis of transgene insertion demonstrated that the optimized method of transformation from leaf explants of A. annua L. was efficient and highly reproducible.

  12. Transgenic sugar beet tolerant to imidazolinone obtained by Agrobacterium-mediated transformation.

    PubMed

    Kishchenko, E M; Komarnitskii, I K; Kuchuk, N V

    2011-01-01

    Sugar beet is highly sensitive to imidazolinone herbicides thus rotational restrictions exist. In order to develop imidazolinone tolerant sugar beets als gene from Arabidopsis thaliana encoding acetolactate synthase with S653N mutation was used for genetic transformation. Transgenic sugar beet plants were obtained by Agrobacterium-mediated transformation of aseptic seedlings using vacuum-infiltration. The efficiency of genetic transformation was 5.8%. RT-PCR analysis of obtained plants revealed accumulation of specific als transcript. The resistance to imidazolinone was proved for developed transgenic sugar beet plants in vitro and in greenhouse conditions after spraying with imazethapyr (Pursuit, BASF).

  13. Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection.

    PubMed

    Hu, T; Metz, S; Chay, C; Zhou, H P; Biest, N; Chen, G; Cheng, M; Feng, X; Radionenko, M; Lu, F; Fry, J

    2003-06-01

    An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study-selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50-80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of theAROA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.

  14. AgarTrap: a simplified Agrobacterium-mediated transformation method for sporelings of the liverwort Marchantia polymorpha L.

    PubMed

    Tsuboyama, Shoko; Kodama, Yutaka

    2014-01-01

    The liverwort Marchantia polymorpha L. is being developed as an emerging model plant, and several transformation techniques were recently reported. Examples are biolistic- and Agrobacterium-mediated transformation methods. Here, we report a simplified method for Agrobacterium-mediated transformation of sporelings, and it is termed Agar-utilized Transformation with Pouring Solutions (AgarTrap). The procedure of the AgarTrap was carried out by simply exchanging appropriate solutions in a Petri dish, and completed within a week, successfully yielding sufficient numbers of independent transformants for molecular analysis (e.g. characterization of gene/protein function) in a single experiment. The AgarTrap method will promote future molecular biological study in M. polymorpha.

  15. An efficient regeneration protocol for Agrobacterium-mediated transformation of melon (Cucumis melo L.).

    PubMed

    Zhang, H J; Gao, P; Wang, X Z; Luan, F S

    2014-01-08

    An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation, using cotyledon node zone-stem connection region of melon, has been developed. The new Agrobacterium-mediated transformation methodology, independent of organ culture, used the entire germinated seed as explants. The transformation system was maximized to maintain the integrity of melon itself, thus avoiding the limitations of traditional tissue culture methods. The transformation was carried out under a non-sterile environment. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed by PCR and Southern blot analyses. The transformation frequency based on the PCR was 13%. Transgenic melon plants were usually detected by PCR in less than 1 month after Agrobacterium inoculation, and seeds could be harvested in 3 months. The growth characteristics and morphology of the transgenic plants were identical to the untransformed wild-type plants. This method would be beneficial for facilitating the characteristics of gene functions and for boosting the manipulation of melon transformation for commercial purposes.

  16. Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology.

    PubMed

    Ishizaki, Kimitsune; Chiyoda, Shota; Yamato, Katsuyuki T; Kohchi, Takayuki

    2008-07-01

    Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the beta-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.

  17. Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient Agrobacterium-mediated transformation of the unicellular green alga, Nannochloropsis sp.

    PubMed

    Cha, Thye-San; Chen, Chin-Fong; Yee, Willy; Aziz, Ahmad; Loh, Saw-Hong

    2011-03-01

    The use of acetosyringone in Agrobacterium-mediated gene transfer into plant hosts has been favored for the past few decades. The influence of other phenolic compounds and their effectiveness in Agrobacterium-mediated plant transformation systems has been neglected. In this study, the efficacy of four phenolic compounds on Agrobacterium-mediated transformation of the unicellular green alga Nannochloropsis sp. (Strain UMT-M3) was assessed by using β-glucuronidase (GUS) assay. We found that cinnamic acid, vanillin and coumarin produced higher percentages of GUS positive cells as compared to acetosyringone. These results also show that the presence of methoxy group in the phenolic compounds may not be necessary for Agrobacterium vir gene induction and receptor binding as suggested by previous studies. These findings provide possible alternative Agrobacterium vir gene inducers that are more potent as compared to the commonly used acetosyringone in achieving high efficiency of Agrobacterium-mediated transformation in microalgae and possibly for other plants. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Agrobacterium-mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis.

    PubMed

    Zhong, Yao Hua; Wang, Xiao Li; Wang, Tian Hong; Jiang, Qiao

    2007-01-01

    Filamentous fungus Trichoderma reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell division. The integration of the hph gene into T. reesei genome was determined by PCR and dot blot analysis. Transgenic T. reesei strains were analyzed using TAIL-PCR for their T-DNA contents. The results showed that T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination, which suggests that Agrobacterium-mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene transfer technology for T. reesei.

  19. A high-efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.).

    PubMed

    Ozawa, Kenjirou

    2012-01-01

    Agrobacterium-mediated transformation of rice has been routinely performed according to the protocol reported by Hiei et al. (Plant J. 6:271-282, 1994). However, several elite japonica and many indica varieties cannot be efficiently transformed by Agrobacterium system. Also a large number of transformants are required to generate T-DNA insertion and FOX libraries as well as gene-targeting studies. To overcome these challenges, we established a high-efficiency transformation system in rice by cocultivating rice calli with Agrobacterium on filter papers moistened with enriched liquid media instead of using solid media (Ozawa, Plant Sci. 176:522-527, 2009; Ozawa and Takaiwa, Plant Sci. 179:333-337, 2010). In this system, the transformation efficiency of the calli is almost 100% in many varieties.

  20. A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L.

    PubMed

    Păcurar, Daniel Ioan; Thordal-Christensen, Hans; Nielsen, Klaus Kristian; Lenk, Ingo

    2008-10-01

    In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA, while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T(0) plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.

  1. Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency

    PubMed Central

    He, Y.; Jones, H. D.; Chen, S.; Chen, X. M.; Wang, D. W.; Li, K. X.; Wang, D. S.; Xia, L. Q.

    2010-01-01

    An efficient Agrobacterium-mediated durum wheat transformation system has been developed for the production of 121 independent transgenic lines. This improved system used Agrobacterium strain AGL1 containing the superbinary pGreen/pSoup vector system and durum wheat cv Stewart as the recipient plant. Acetosyringone at 400 μM was added to both the inoculation and cultivation medium, and picloram at 10 mg l−1 and 2 mg l−1 was used in the cultivation and induction medium, respectively. Compared with 200 μM in the inoculation and cultivation media, the increased acetosyringone concentration led to significantly higher GUS (β-glucuronidase) transient expression and T-DNA delivery efficiency. However, no evident effects of acetosyringone concentration on regeneration frequency were observed. The higher acetosyringone concentration led to an improvement in average final transformation efficiency from 4.7% to 6.3%. Furthermore, the concentration of picloram in the co-cultivation medium had significant effects on callus induction and regeneration. Compared with 2 mg l−1 picloram in the co-cultivation medium, increasing the concentration to 10 mg l−1 picloram resulted in improved final transformation frequency from 2.8% to 6.3%, with the highest frequency of 12.3% reached in one particular experiment, although statistical analysis showed that this difference in final transformation efficiency had a low level of significance. Stable integration of foreign genes, their expression, and inheritance were confirmed by Southern blot analyses, GUS assay, and genetic analysis. Analysis of T1 progeny showed that, of the 31 transgenic lines randomly selected, nearly one-third had a segregation ratio of 3:1, while the remainder had ratios typical of two or three independently segregating loci. PMID:20202997

  2. Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.).

    PubMed

    Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

    2013-08-01

    The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.

  3. Aboveground insect infestation attenuates belowground Agrobacterium-mediated genetic transformation.

    PubMed

    Song, Geun Cheol; Lee, Soohyun; Hong, Jaehwa; Choi, Hye Kyung; Hong, Gun Hyong; Bae, Dong-Won; Mysore, Kirankumar S; Park, Yong-Soon; Ryu, Choong-Min

    2015-07-01

    Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be popularly used for genetic engineering, the influence of aboveground insect infestation on Agrobacterium induced gall formation has not been investigated. Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation affects crown gall disease. Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation indicating an involvement of SA in whitefly-derived plant defence against Agrobacterium. Endogenous SA content was augmented in whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was three times higher in root exudates from whitefly-infested plants. As a consequence, Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhibited when compared to control plants. These results suggest that aboveground whitefly infestation elicits systemic defence responses throughout the plant. Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand interactions between three organisms: whitefly, N. benthamiana and Agrobacterium. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  4. Stable Agrobacterium-mediated transformation of maritime pine based on kanamycin selection.

    PubMed

    Alvarez, José M; Ordás, Ricardo J

    2013-01-01

    An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β -glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL(-1) kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD(600 nm)) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.

  5. Stable Agrobacterium-Mediated Transformation of Maritime Pine Based on Kanamycin Selection

    PubMed Central

    Alvarez, José M.; Ordás, Ricardo J.

    2013-01-01

    An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β-glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL−1 kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD600 nm) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth. PMID:24376383

  6. Deletion of host histone acetyltransferases and deacetylases strongly affects Agrobacterium-mediated transformation of Saccharomyces cerevisiae.

    PubMed

    Soltani, Jalal; van Heusden, Gerard Paul H; Hooykaas, Paul J J

    2009-09-01

    Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells by transferring a part of its Ti-plasmid, the T-strand, to the host cell. Under laboratory conditions, it can also transform cells from many different nonplant organisms, including the yeast Saccharomyces cerevisiae. Collections of S. cerevisiae strains have been developed with systematic deletion of all coding sequences. Here, we used these collections to identify genes involved in the Agrobacterium-mediated transformation (AMT) of S. cerevisiae. We found that deletion of genes (GCN5, NGG1, YAF9 and EAF7) encoding subunits of the SAGA, SLIK, ADA and NuA4 histone acetyltransferase complexes highly increased the efficiency of AMT, while deletion of genes (HDA2, HDA3 and HST4) encoding subunits of histone deacetylase complexes decreased AMT. These effects are specific for AMT as the efficiency of chemical (lithium acetate) transformation was not or only slightly affected by these deletions. Our data are consistent with a positive role of host histone deacetylation in AMT.

  7. AGROBACTERIUM-MEDIATED TRANSFORMATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE, VOLVOCALES)(1).

    PubMed

    Kathiresan, S; Chandrashekar, A; Ravishankar, G A; Sarada, R

    2009-06-01

    The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis Flot. using the binary vectors hosting the genes coding for GUS (β-glucuronidase), GFP (green fluorescent protein), and hpt (hygromycin phosphotransferase) is reported here. Colonies resistant to hygromycin at 10 mg · L(-1) expressed β-glucuronidase. The greenish yellow fluorescence of GFP was observed when the hygromycin-resistant cells were viewed with a fluorescent microscope. PCR was used to successfully amplify fragments of the hpt (407 bp) and GUS (515 bp) genes from transformed cells, while Southern blots indicated the integration of the hygromycin gene into the genome of H. pluvialis. SEM indicated that the cell wall of H. pluvialis was altered on infection with Agrobacterium. The transformation achieved here by Agrobacterium does not need treatment with acetosyringone or the wounding of cells. A robust transformation method for this alga would pave the way for manipulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries.

  8. Efficient soybean regeneration and Agrobacterium-mediated transformation using a whole cotyledonary node as an explant.

    PubMed

    Zhang, Fuli; Chen, Can; Ge, Honglian; Liu, Jinmei; Luo, Yunling; Liu, Kun; Chen, Long; Xu, Kedong; Zhang, Yi; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3))]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a β-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  9. Agrobacterium-mediated genetic transformation of pineapple (Ananas comosus L., Merr.).

    PubMed

    Mhatre, Minal

    2013-01-01

    Pineapple (Ananas comosus L., Merr.) is a commercially important crop, grown in the tropical and subtropical regions. However, the crop is faced with postharvest damage and poor varietal and nutritional improvement. Being a vegetatively propagated crop, conventional breeding programs take longer time for genetic improvement, which may not necessarily successfully develop an improved cultivar. Hence, the genetic modification of pineapple is an alternative handy approach to improve pineapple. We have established an Agrobacterium-mediated transformation system using leaf bases from in vitro-grown pineapple plants. Being a monocot, acetosyringone is added to the culture medium for overnight growth of Agrobacterium and transformation to transfer a gene of interest MSI99 soybean ferritin. Leaf bases isolated from in vitro shoot cultures are treated with Agrobacterium suspension at two dilutions, 10× and 20×, for 30 min. Explants are subsequently blot dried and cultured on gelrite solidified hormone-free Pin1 medium for 2 days (cocultivation). Periodic transfer is first done to the regeneration medium (Pin1) containing cefotaxime for the suppression of Agrobacterium growth. The transformants are selected by culturing on Pin1 medium containing cefotaxime and kanamycin. Multiple shoots, regenerated in leaf bases, are further multiplied and individually rooted in the liquid RM medium amended with antibiotics to recover plants. Putative transformants are analyzed for transgene integration and expression using standard molecular biological methods of PCR, RT-PCR, and genomic Southern.

  10. Agrobacterium-mediated transformation of the endophytic fungus Acremonium implicatum associated with Brachiaria grasses.

    PubMed

    Abello, Javier; Kelemu, Segenet; García, Celsa

    2008-03-01

    Acremonium implicatum is a seed-transmitted endophytic fungus that forms symbiotic associations with the economically significant tropical forage grasses, Brachiaria species. To take advantage of the endophyte's plant protective properties, we developed an efficient Agrobacterium-mediated transformation system for Acremonium implicatum, using green fluorescent protein (GFP) expression and vector pSK1019 (trpC promoter) or pCAMBIA1300 (CaMV35S promoter). We found that transformation efficiency doubled for both mycelial and conidial transformation as the co-cultivation period for Agrobacterium tumefaciens and Acremonium implicatum was increased from 48 to 72h. Significantly, optimal results were obtained for either mycelial or conidial transformation with Agrobacterium tumefaciens strain AGL-1 and vector pSK1019 under the control of the trpC promoter. However, mycelial transformation consistently generated a significantly higher number of transformants than did conidial transformation. The mitotic stability of the transferred DNA was confirmed by growing ten transformants in liquid and agar media for six generations. In all cases, resistance to the selection pressure (hygromycin B) was maintained. Fluorescence emission was retained by the transformants and also expressed in Brachiaria tissues from plants inoculated with GFP-transformed A. implicatum. This technology will help in the transfer and expression of agronomically important genes in host plants.

  11. Transgenic plants from shoot apical meristems of Vitis vinifera L. "Thompson Seedless" via Agrobacterium-mediated transformation.

    PubMed

    Dutt, M; Li, Z T; Dhekney, S A; Gray, D J

    2007-12-01

    Shoot apical meristem explants of Vitis vinifera "Thompson Seedless" were used for Agrobacterium-mediated genetic transformation. It was determined that the meristems had to be subjected to a dark growth phase then wounded to obtain transgenic plants. Morphological and histological studies illustrated the role of wounding to expose apical meristem cells for transformation. A bifunctional egfp/nptII fusion gene was used to select kanamycin resistant plants that expressed green fluorescent protein (GFP). Kanamycin at a concentration of 16 mg L(-1) in selection medium resulted in recovery of non-chimeric transgenic plants that uniformly expressed GFP, whereas 8 mg L(-1) kanamycin allowed non-transgenic and/or chimeric plants to develop. Polymerase chain reaction (PCR) and Southern blot analyses confirmed the presence of transgenes and their stable integration into the genome of regenerated plants. Up to 1% of shoot tips produced stable transgenic cultures within 6 weeks of treatment, resulting in a total of 18 independent lines.

  12. Improvement of Agrobacterium-mediated transformation and rooting of black cherry

    Treesearch

    Ying Wang; Paula M. Pijut

    2014-01-01

    An improved protocol for Agrobacterium-mediated transformation of an elite, mature black cherry genotype was developed. To increase transformation efficiency, vacuum infiltration, sonication, and a combination of the two treatments were applied during the cocultivation of leaf explants with Agrobacterium tumefaciens strain EHA105...

  13. Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.

    PubMed

    Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

    2014-01-01

    The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.

  14. Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica

    PubMed Central

    Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

    2014-01-01

    The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

  15. An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei.

    PubMed

    Kummasook, Aksarakorn; Cooper, Chester R; Vanittanakom, Nongnuch

    2010-12-01

    We have developed an improved Agrobacterium-mediated transformation (AMT) system for the functional genetic analysis of Penicillium marneffei, a thermally dimorphic, human pathogenic fungus. Our AMT protocol included the use of conidia or pre-germinated conidia of P. marneffei as the host recipient for T-DNA from Agrobacterium tumefaciens and co-cultivation at 28°C for 36 hours. Bleomycin-resistant transformants were selected as yeast-like colonies following incubation at 37°C. The efficiency of transformation was approximately 123 ± 3.27 and 239 ± 13.12 transformants per plate when using 5 × 10(4) conidia and pre-germinated conidia as starting materials, respectively. Southern blot analysis demonstrated that 95% of transformants contained single copies of T-DNA. Inverse PCR was employed for identifying flanking sequences at the T-DNA insertion sites. Analysis of these sequences indicated that integration occurred as random recombination events. Among the mutants isolated were previously described stuA and gasC defective strains. These AMT-derived mutants possessed single T-DNA integrations within their particular coding sequences. In addition, other morphological and pigmentation mutants possessing a variety of gene-specific defects were isolated, including two mutants having T-DNA integrations within putative promoter regions. One of the latter integration events was accompanied by the deletion of the entire corresponding gene. Collectively, these results indicated that AMT could be used for large-scale, functional genetic analyses in P. marneffei. Such analyses can potentially facilitate the identification of those genetic elements related to morphogenesis, as well as pathogenesis in this medically important fungus.

  16. High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco

    PubMed Central

    Pathi, Krishna Mohan; Tula, Suresh; Tuteja, Narendra

    2013-01-01

    A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis. PMID:23518589

  17. Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori.

    PubMed

    Michielse, C B; Ram, A F J; Hooykaas, P J J; Hondel, C A M J J van den

    2004-05-01

    The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system. Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer. This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants. The results indicate that the Agrobacterium-mediated transformation of A. awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important.

  18. Development of a phosphomannose isomerase-based Agrobacterium-mediated transformation system for chickpea (Cicer arietinum L.).

    PubMed

    Patil, Gunvant; Deokar, Amit; Jain, P K; Thengane, R J; Srinivasan, R

    2009-11-01

    To develop an alternative genetic transformation system that is not dependent on an antibiotic selection strategy, the phosphomannose isomerase gene (pmi) system was evaluated for producing transgenic plants of chickpea (Cicer arietinum L.). A shoot morphogenesis protocol based on the thidiazuron (TDZ)-induced shoot morphogenesis system was combined with Agrobacterium-mediated transformation of the pmi gene and selection of transgenic plants on mannose. Embryo axis explants of chickpea cv. C-235 were grown on a TDZ-supplemented medium for shoot proliferation. Embryo axis explants from which the first and second flush of shoots were removed were transformed using Agrobacterium carrying the pmi gene, and emerging shoots were allowed to regenerate on a zeatin-supplemented medium with an initial selection pressure of 20 g l(-1) mannose. Rooting was induced in the selected shoots on an indole-3-butyric acid (IBA)-supplemented medium with a selection pressure of 15 g l(-1) mannose. PCR with marker gene-specific primers and chlorophenol red (CPR) assay of the shoots indicated that shoots had been transformed. RT-PCR and Southern analysis of selected regenerated plants further confirmed integration of the transgene into the chickpea genome. These positive results suggest that the pmi/mannose selection system can be used to produce transgenic plants of chickpea that are free from antibiotic resistance marker genes.

  19. Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein.

    PubMed

    Govender, Nisha; Wong, Mui-Yun

    2017-04-01

    A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

  20. Transients in chloroplast gene transcription

    SciTech Connect

    Puthiyaveetil, Sujith; Allen, John F.

    2008-04-18

    Transcriptional regulation of chloroplast genes is demonstrated by Quantitative Polymerase Chain Reaction (qPCR). These genes encode apoproteins of the reaction centres of photosystem I and photosystem II. Their transcription is regulated by changes in wavelength of light selectively absorbed by photosystem I and photosystem II, and therefore by the redox state of an electron carrier located between the two photosystems. Chloroplast transcriptional redox regulation is shown to have greater amplitude, and the kinetics of transcriptional changes are more complex, than suggested by previous experiments using only DNA probes in Northern blot experiments. Redox effects on chloroplast transcription appear to be superimposed on an endogenous rhythm of mRNA abundance. The functional significance of these transients in chloroplast gene transcription is discussed.

  1. Agrobacterium-mediated transformation of Ruta graveolens L.

    PubMed

    Lièvre, Karine; Tran, Thi Lê Minh; Doerper, Sébastien; Hehn, Alain; Lacoste, Paul; Thomasset, Brigitte; Bourgaud, Frédéric; Gontier, Eric

    2009-01-01

    Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- -to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1Rif containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and beta-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding beta-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings.

  2. Development of Transgenic Papaya through Agrobacterium-Mediated Transformation

    PubMed Central

    Azad, Md. Abul Kalam; Rabbani, Md. Golam; Amin, Latifah; Sidik, Nik Marzuki

    2013-01-01

    Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and β-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi. PMID:24066284

  3. Agrobacterium-mediated transformation of Euphorbia tirucalli callus.

    PubMed

    Uchida, Hidenobu; Yamashita, Hirofumi; Anai, Toyoaki; Muranaka, Toshiya; Ohyama, Kanji

    2010-01-01

    In order to establish a basis for transformation technology in the petroleum plant Euphorbia tirucalli, the callus of the plant was infected with Agrobacterium, washed with distilled water, sterilized with distilled water containing 100 mg/l of carbenicillin, selected on solidified B5 medium containing 13 mg/l of G418 and 100 mg/l of carbenicillin, and then on solidified B5 medium containing 25 mg/l of G418 and 100 mg/l of carbenicillin for the transgenic calli, and then the callus lines were subcultured successively on solidified B5 medium containing 50 mg/l of G418. We performed PCR analysis of sterilized G418-resistant callus line DNA and concluded that the gene introduced was integrated into the callus genome.

  4. Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. x P. maximowiczii A. Henry.

    PubMed

    Yevtushenko, Dmytro P; Misra, Santosh

    2010-03-01

    Many economically important species of Populus, especially those in sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. In this study, a simple and reliable protocol was developed for the efficient Agrobacterium-mediated transformation of a difficult-to-transform, but commercially viable, hybrid poplar Populus nigra L. x P. maximowiczii A. Henry (NM6). A plant transformation vector designed to express the beta-glucuronidase (GUS) gene was used to detect transformation events at early stages of plant regeneration and to optimize parameters affecting poplar transformation. The use of zeatin riboside in shoot-induction medium, regeneration of shoots via indirect organogenesis, and early selection pressure were the major modifications that drastically improved the efficiency of poplar transformation and minimized the number of untransformed regenerants. Transgenic shoots were routinely obtained 4-10 weeks after co-culture with A. tumefaciens, with a greater than 90% rate of plant recovery. Stable transgene integration, ranging from a single insertion to ten copies per genome, was confirmed by Southern blot analysis. The mean transformation frequency was 36.3% and about two-thirds of the lines had 1-2 transgene copies. Among the explants, petioles and leaves had a higher transformation frequency than did stem segments. Growth characteristics and the morphology of transgenic poplar plants were identical to untransformed controls. These findings will accelerate the development of P. nigra x P. maximowiczii plants with novel traits, and may also be useful to improve transformation procedures for other Populus species.

  5. Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi.

    PubMed

    Lin, Xinping; Liu, Sasa; Bao, Ruiqi; Gao, Ning; Zhang, Sufang; Zhu, Rongqian; Zhao, Zongbao Kent

    2017-04-06

    Oleaginous yeast Lipomyces starkeyi, a promising strain of great biotechnical importance, is able to accumulate over 60% of its cell biomass as triacylglycerols (TAGs). It is promising to directly produce the derivatives of TAGs, such as long-chain fatty acid methyl esters and alkanes, in L. starkeyi. However, techniques for genetic modification of this oleaginous yeast are lacking, thus, further research is needed to develop genetic tools and functional elements. Here, we used two exogenous promoters (pGPD and pPGK) from oleaginous yeast Rhodosporidium toruloides to establish a simpler Agrobacterium-mediated transformation (AMT) method for L. starkeyi. Hygromycin-resistant transformants were obtained on antibiotic-contained plate. Mitotic stability test, genotype verification by PCR, and protein expression confirmation all demonstrated the success of this method. Furthermore, the strength of these two promoters was evaluated at the phenotypic level on a hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. The PGK promoter strength was 2.2-fold as that of GPD promoter to initiate the expression of the hygromycin-resistance gene. This study provided an easy and efficient genetic manipulation method and elements of the oleaginous yeast L. starkeyi for constructing superior strains to produce advanced biofuels.

  6. Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation

    PubMed Central

    Wang, Gen-Ping; Yu, Xiu-Dao; Sun, Yong-Wei; Jones, Huw D.; Xia, Lan-Qin

    2016-01-01

    Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of “clean” GM wheat containing only the foreign genes of agronomic importance. PMID:27708648

  7. Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.).

    PubMed

    Zhang, C L; Chen, D F; McCormac, A C; Scott, N W; Elliott, M C; Slater, A

    2001-02-01

    Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate "escapes" and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3-11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2-5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.

  8. Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava.

    PubMed

    Bull, S E; Owiti, J A; Niklaus, M; Beeching, J R; Gruissem, W; Vanderschuren, H

    2009-01-01

    Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop.

  9. Genomic regions responsible for amenability to Agrobacterium-mediated transformation in barley

    PubMed Central

    Hisano, Hiroshi; Sato, Kazuhiro

    2016-01-01

    Different plant cultivars of the same genus and species can exhibit vastly different genetic transformation efficiencies. However, the genetic factors underlying these differences in transformation rate remain largely unknown. In barley, ‘Golden Promise’ is one of a few cultivars reliable for Agrobacterium-mediated transformation. By contrast, cultivar ‘Haruna Nijo’ is recalcitrant to genetic transformation. We identified genomic regions of barley important for successful transformation with Agrobacterium, utilizing the ‘Haruna Nijo’ × ‘Golden Promise’ F2 generation and genotyping by 124 genome-wide SNP markers. We observed significant segregation distortions of these markers from the expected 1:2:1 ratio toward the ‘Golden Promise’-type in regions of chromosomes 2H and 3H, indicating that the alleles of ‘Golden Promise’ in these regions might contribute to transformation efficiency. The same regions, which we termed Transformation Amenability (TFA) regions, were also conserved in transgenic F2 plants generated from a ‘Morex’ × ‘Golden Promise’ cross. The genomic regions identified herein likely include necessary factors for Agrobacterium-mediated transformation in barley. The potential to introduce these loci into any haplotype of barley opens the door to increasing the efficiency of transformation for target alleles into any haplotype of barley by the TFA-based methods proposed in this report. PMID:27874056

  10. Development of Agrobacterium-mediated transformation of highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease.

    PubMed

    Elayabalan, Sivalingam; Kalaiponmani, Kalaimughilan; Subramaniam, Sreeramanan; Selvarajan, Ramasamy; Panchanathan, Radha; Muthuvelayoutham, Ramlatha; Kumar, Krish K; Balasubramanian, Ponnuswami

    2013-04-01

    One of the most severe viral diseases of hill banana is caused by banana bunchy top virus (BBTV), a nanovirus transmitted by the aphid Pentalonia nigronervosa. In this study, we reported the Agrobacterium-mediated transformation on a highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease. The target of the RNA interference (RNAi) is the rep gene, encoded by the BBTV-DNA1. In order to develop RNAi construct targeting the BBTV rep gene, the full-length rep gene of 870 bp was polymerase chain reaction amplified from BBTV infected hill banana sample DNA, cloned and confirmed by DNA sequencing. The partial rep gene fragment was cloned in sense and anti sense orientation in the RNAi intermediate vector, pSTARLING-A. After cloning in pSTARLING-A, the cloned RNAi gene cassette was released by NotI enzyme digestion and cloned into the NotI site of binary vector, pART27. Two different explants, embryogenic cells and embryogenic cell suspension derived microcalli were used for co-cultivation. Selection was done in presence of 100 mg/L kanamycin. In total, 143 putative transgenic hill banana lines were generated and established in green house condition. The presence of the transgenes was confirmed in the selected putative transgenic hill banana lines by PCR and reverse transcription PCR analyses. Transgenic hill banana plants expressing RNAi-BBTV rep were obtained and shown to resist infection by BBTV. The transformed plants are symptomless, and the replication of challenge BBTV almost completely suppressed. Hence, the RNAi mediating resistances were shown to be effective management of BBTV in hill banana.

  11. Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications.

    PubMed

    Krenek, Pavel; Samajova, Olga; Luptovciak, Ivan; Doskocilova, Anna; Komis, George; Samaj, Jozef

    2015-11-01

    Agrobacterium tumefaciens is widely used as a versatile tool for development of stably transformed model plants and crops. However, the development of Agrobacterium based transient plant transformation methods attracted substantial attention in recent years. Transient transformation methods offer several applications advancing stable transformations such as rapid and scalable recombinant protein production and in planta functional genomics studies. Herein, we highlight Agrobacterium and plant genetics factors affecting transfer of T-DNA from Agrobacterium into the plant cell nucleus and subsequent transient transgene expression. We also review recent methods concerning Agrobacterium mediated transient transformation of model plants and crops and outline key physical, physiological and genetic factors leading to their successful establishment. Of interest are especially Agrobacterium based reverse genetics studies in economically important crops relying on use of RNA interference (RNAi) or virus-induced gene silencing (VIGS) technology. The applications of Agrobacterium based transient plant transformation technology in biotech industry are presented in thorough detail. These involve production of recombinant proteins (plantibodies, vaccines and therapeutics) and effectoromics-assisted breeding of late blight resistance in potato. In addition, we also discuss biotechnological potential of recombinant GFP technology and present own examples of successful Agrobacterium mediated transient plant transformations. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

    PubMed Central

    2011-01-01

    Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated

  13. A simple and efficient Agrobacterium-mediated procedure for transformation of tomato.

    PubMed

    Sharma, Manoj K; Solanke, Amolkumar U; Jani, Dewal; Singh, Yogendra; Sharma, Arun K

    2009-09-01

    We describe a highly efficient and reproducible Agrobacterium-mediated transformation protocol applicable to several varieties of tomato (Solanum lycopersicum, earlier known as Lycopersicum esculentum). Conditions such as co-cultivation period, bacterial concentration, concentration of benzyl amino purine (BAP), zeatin and indole acetic acid (IAA) were optimized. Co-cultivation of explants with a bacterial concentration of 108 cells/ml for three days on 2 mg/l BAP, followed by regeneration on a medium containing 1 mg/ml zeatin resulted in a transformation frequency of 41.4%. Transformation of tomato plants was confirmed by Southern blot analysis and beta-glucuronidase (GUS) assay. The protocol developed showed very high efficiency of transformation for tomato varieties Pusa Ruby, Arka Vikas and Sioux. The optimized transformation procedure is simple, efficient and does not require tobacco, Petunia, tomato suspension feeder layer or acetosyringone.

  14. Agrobacterium-mediated transformation in Alpinia galanga (Linn.) Willd. for enhanced acetoxychavicol acetate production.

    PubMed

    Rao, Kiranmayee; Chodisetti, Bhuvaneswari; Mangamoori, Lakshmi Narasu; Giri, Archana

    2012-09-01

    Agrobacterium-mediated transformations ensure elevated amounts of secondary metabolite accumulation with genetic and biosynthetic stability. In the present study, Alpinia galanga rich in bioactive compounds was genetically transformed using different strains of Agrobacterium rhizogenes viz. LBA 9402, A(4), 532, 2364 and PRTGus. Even though a higher growth rate was obtained with the LBA 9402 strain, maximum acetoxychavicol acetate accumulation (ACA) was seen in the PRTGus transformant. PRTGus root line has shown 10.1 fold higher ACA content in comparison to the control roots. The lowest ACA production was shown by the A(4) transformant (4.9 fold). The quantification of ACA in the transformed roots was carried out by using HPLC, which was found to be in the order of PRTGus > LBA 9402 > 2364 > 532 > A(4). The fast growth rate of hairy roots, genetic stability and their ability to synthesize more than one metabolite offer a promising system for the production of valuable secondary metabolites.

  15. Agrobacterium-mediated transformation of apricot (Prunus armeniaca L.) leaf explants.

    PubMed

    Petri, César; Wang, Hong; Alburquerque, Nuria; Faize, Mohamed; Burgos, Lorenzo

    2008-08-01

    A protocol for Agrobacterium-mediated stable transformation for scored, whole leaf explants of the apricot (Prunus armeniaca) cultivar Helena was developed. Regenerated shoots were selected using a two-step increased concentrations of paromomycin sulphate. Different factors affecting survival of transformed buds, including possible toxicity of green fluorescent protein (GFP) and time of exposure to high cytokine concentration in the regeneration medium, were examined. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines was 5.6%, when optimal conditions for bud survival were provided. Southern blot analysis on four randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene. This is the first time that stable transformation of an apricot cultivar is reported and constitutes also one of the few reports on the transformation of Prunus cultivars.

  16. Setaria viridis floral-dip: A simple and rapid Agrobacterium-mediated transformation method.

    PubMed

    Martins, Polyana Kelly; Nakayama, Thiago Jonas; Ribeiro, Ana Paula; Cunha, Bárbara Andrade Dias Brito da; Nepomuceno, Alexandre Lima; Harmon, Frank G; Kobayashi, Adilson Kenji; Molinari, Hugo Bruno Correa

    2015-06-01

    Setaria viridis was recently described as a new monocotyledonous model species for C4 photosynthesis research and genetic transformation. It has biological attributes (rapid life cycle, small genome, diploid, short stature and simple growth requirements) that make it suitable for use as a model plant. We report an alternative method of S. viridis transformation using floral dip to circumvent the necessity of tissue culture phase for transgenic plant regeneration. S. viridis spikes at boot stage were selected to be immersed in Agrobacterium suspension. T1 seeds could be identified in 1.5-2 months after floral dipping. We demonstrated through molecular analysis and RFP expression that seeds and resulting plants from dipped inflorescences were transformed. Our results suggest the feasibility of S. viridis floral dip transformation as a time-saving and cost-effective compared with traditional methods. To our knowledge, this is the first report using floral dip in S. viridis as an Agrobacterium-mediated transformation method.

  17. A simple and highly efficient Agrobacterium-mediated transformation protocol for Setaria viridis.

    PubMed

    Martins, Polyana Kelly; Ribeiro, Ana Paula; Cunha, Bárbara Andrade Dias Brito da; Kobayashi, Adilson Kenji; Molinari, Hugo Bruno Correa

    2015-06-01

    The production and use of sugarcane in Brazil is very important for bioenergy production and is recognized as one of the most efficient in the world. In our laboratory, Setaria viridis is being tested as a model plant for sugarcane. S. viridis has biological attributes (rapid life cycle, small genome, diploid, short stature and simple growth requirements) that make it suitable for use as a model system. We report a highly efficient protocol for Agrobacterium-mediated genetic transformation of S. viridis. The optimization of several steps in tissue culture allowed the rapid regeneration of plants and increased the rate of transformation up to 29%. This protocol could become a powerful tool for functional genomics in sugarcane.

  18. Agrobacterium-mediated transformation of Easter lily (Lilium longiflorum cv. Nellie White)

    USDA-ARS?s Scientific Manuscript database

    Conditions were optimized for transient transformation of Lilium longiflorum cv. Nellie White using Agrobacterium tumefaciens. Bulb scale and basal meristem explants were inoculated with A. tumefaciens strain AGL1 containing the binary vector pCAMBIA 2301 which has the uidA gene that codes for ß-gl...

  19. pSiM24 Is a Novel Versatile Gene Expression Vector for Transient Assays As Well As Stable Expression of Foreign Genes in Plants

    PubMed Central

    Sahoo, Dipak Kumar; Dey, Nrisingha; Maiti, Indu Bhushan

    2014-01-01

    We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3′rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP) and β-glucuronidase (GUS) both transiently (agro-infiltration, protoplast electroporation and biolistic) and stably in plant systems (Arabidopsis and tobacco) using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses. PMID:24897541

  20. Optimization of factors affecting Agrobacterium-mediated transformation of Micro-Tom tomatoes.

    PubMed

    Guo, M; Zhang, Y L; Meng, Z J; Jiang, J

    2012-03-16

    Micro-Tom is the smallest known variety of tomatoes. An orthogonal experimental design L(16) (4(5)) was used to optimize Agrobacterium-mediated transformation of cotyledon explants of Lycopersicon esculentum cv. Micro-Tom. Four parameters were investigated to determine their effect on transformation frequency: the concentration of bacterial suspension, time of dip in bacterial suspension, co-cultivation time, and concentration of carbenicillin. We also examined the effect of these parameters on contamination rate, necrosis rate, mortality, cut-surface browning rate, and undamaged explant rate. Both the bacterial and carbenicillin concentrations had a significant influence on the rate of infected explants. The time of co-cultivation also had a significant influence on the transformation parameters. The optimal transformation protocol consisted of an Agrobacterium suspension of 0.5 × 10(8) cells/mL (OD(600) = 0.5) and an infection time of 5 min, one day of co-cultivation and 500 mg/L carbenicillin. Under these conditions, the transformation efficiency of the shoots reached 5.1%; the mean transformation frequency was 3.9% (N = 838).

  1. Establishment of an Agrobacterium-mediated Inoculation System for Cucumber Green Mottle Mosaic Virus

    PubMed Central

    Kang, Minji; Seo, Jang-Kyun; Song, Dami; Choi, Hong-Soo; Kim, Kook-Hyung

    2015-01-01

    The infectious full-length cDNA clones of Cucumber green mottle mosaic virus (CGMMV) isolates KW and KOM, which were isolated from watermelon and oriental melon, respectively, were constructed under the control of the cauliflower mosaic virus 35S promoter. We successfully inoculated Nicotiana benthamiana with the cloned CGMMV isolates KW and KOM by Agrobacterium-mediated infiltration. Virulence and symptomatic characteristics of the cloned CGMMV isolates KW and KOM were tested on several indicator plants. No obvious differences between two cloned isolates in disease development were observed on the tested indicator plants. We also determined full genome sequences of the cloned CGMMV isolates KW and KOM. Sequence comparison revealed that only four amino acids (at positions 228, 699, 1212, and 1238 of the replicase protein region) differ between the cloned isolates KW and KOM. A previous study reported that the isolate KOM could not infect Chenopodium amaranticolor, but the cloned KOM induced chlorotic spots on the inoculated leaves. When compared with the previously reported sequence of the original KOM isolate, the cloned KOM contained one amino acid mutation (Ala to Thr) at position 228 of the replicase protein, suggesting that this mutation might be responsible for induction of chlorotic spots on the inoculated leaves of C. amaranticolor. PMID:26674677

  2. Cell wall biochemical alterations during Agrobacterium-mediated expression of haemagglutinin-based influenza virus-like vaccine particles in tobacco.

    PubMed

    Le Mauff, François; Loutelier-Bourhis, Corinne; Bardor, Muriel; Berard, Caroline; Doucet, Alain; D'Aoust, Marc-André; Vezina, Louis-Philippe; Driouich, Azeddine; Couture, Manon M-J; Lerouge, Patrice

    2017-03-01

    Influenza virus-like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant-based biotechnology allows for the large-scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium-mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post-Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG-I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin-based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.

  3. An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus.

    PubMed

    Dutt, M; Grosser, J W

    2010-11-01

    A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

  4. Agrobacterium-mediated transformation of oat (Avena sativa L.) cultivars via immature embryo and leaf explants.

    PubMed

    Gasparis, Sebastian; Bregier, Cezary; Orczyk, Waclaw; Nadolska-Orczyk, Anna

    2008-11-01

    This paper reports on the successful Agrobacterium-mediated transformation of oat, and on some factors influencing this process. In the first step of the experiments, three cultivars, two types of explant, and three combinations of strain/vectors, which were successfully used for transformation of other cereals were tested. Transgenic plants were obtained from the immature embryos of cvs. Bajka, Slawko and Akt and from leaf base explants of cv. Bajka after transformation with A. thumefaciens strain LBA4404(pTOK233). The highest transformation rate (12.3%) was obtained for immature embryos of cv. Bajka. About 79% of the selected plants proved to be transgenic; however, only 14.3% of the T(0) plants and 27.5% of the T(1) showed GUS expression. Cell competence of both types of explant differed in terms of their transformation ability and transgene expression. The next step of the study was to test the suitability for oat transformation of the pGreen binary vector combined with different selection cassettes: nptII or bar under the nos or 35S promoter. Transgenic plants were selected in combinations transformed with nos::nptII, 35S::nptII and nos::bar. The highest transformation efficiency (5.3%) was obtained for cv. Akt transformed with nos::nptII. A detailed analysis of the T(0) plants selected from a given callus line and their progeny revealed that they were the mixture of transgenic, chimeric-transgenic and non-transgenic individuals. Southern blot analysis of T(0) and T(1) showed simple integration pattern with the low copy number of the introduced transgenes.

  5. Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid.

    PubMed

    Zyprian, E; Kado, C I

    1990-08-01

    A new binary vector system for Agrobacterium-mediated plant transformation was developed. A set of four mini-T vectors comprised of T-DNA border sequences from nopaline-type Ti-plasmid pTiC58 flanking a chimaeric hygromycin-resistance gene for selection of transformants and up to eight unique restriction sites for cloning foreign DNA was constructed on a broad-host replicon containing the oriV of plasmid pSa. In two of the constructs these multiple cloning sites are flanked by a strong promoter to activate transcription of inserted DNA in planta. High-efficiency transformation was prompted by a high-copy, stable virulence helper plasmid pUCD2614, which contains a cloned virulence region of pTiC58 and tandem copies of the par locus of plasmid pTAR. Southern blot hybridization and genetic analyses of the progeny of transformed plants showed that the hygromycin resistance gene was stably inherited.

  6. Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus, Coniothyrium minitans.

    PubMed

    Rogers, Christopher W; Challen, Michael P; Green, Jonathan R; Whipps, John M

    2004-12-15

    Restriction enzyme mediated integration (REMI) and Agrobacterium-mediated transformation (ATMT) were used to transform protoplasts or germinated conidia of the mycoparasite Coniothyrium minitans to hygromycin resistance. Using REMI, up to 32 transformants mug DNA(-1) were obtained, while 37.8 transformants 5 x 10(5) germlings(-1) were obtained using ATMT. Single-copy integrations occurred in 8% and 40% of REMI and ATMT transformants, respectively. A novel microtitre plate-based test was developed to expedite screening of 4000 REMI and ATMT C. minitans transformants. Nine pathogenicity mutants that displayed reduced or no pathogenicity on sclerotia of Sclerotinia sclerotiorum were identified.

  7. A new high-frequency Agrobacterium-mediated transformation technique for Sesamum indicum L. using de-embryonated cotyledon as explant.

    PubMed

    Chowdhury, Supriyo; Basu, Arpita; Kundu, Surekha

    2014-09-01

    In spite of the economic importance of sesame (Sesamum indicum L.) and the recent availability of its genome sequence, a high-frequency transformation protocol is still not available. The only two existing Agrobacterium-mediated transformation protocols that are available have poor transformation efficiencies of less than 2%. In the present study, we report a high-frequency, simple, and reproducible transformation protocol for sesame. Transformation was done using de-embryonated cotyledons via somatic embryogenic stages. All the critical parameters of transformation, like incubation period of explants in pre-regeneration medium prior to infection by Agrobacterium tumefaciens, cocultivation period, concentrations of acetosyringone in cocultivation medium, kanamycin concentration, and concentration of plant hormones, including 6-benzylaminopurine, have been optimized. This protocol is superior to the two existing protocols in its high regeneration and transformation efficiencies. The transformed sesame lines have been tested by PCR, RT-PCR for neomycin phosphotransferase II gene expression, and β-glucuronidase (GUS) assay. The regeneration frequency and transformation efficiency are 57.33 and 42.66%, respectively. T0 and T1 generation transgenic plants were analyzed, and several T1 plants homozygous for the transgenes were obtained.

  8. Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43).

    PubMed

    Karthikeyan, Alagarsamy; Pandian, Shunmugiah Karutha; Ramesh, Manikandan

    2011-09-01

    A simple and efficient protocol for the Agrobacterium-mediated transformation of an agronomically useful abiotic sensitive popular indica rice cv. ADT 43 has been developed. Initiation of calli were best achieved from the leaf bases of 4 days old rice seedlings on LS medium supplemented with 2.5mg/L 2,4-D and 1.0mg/L thiamine-HCl. Rice calli immersed in Agrobacterium suspension (strain EHA 105, OD(600)=0.8) were co-cultured on LS30-AsPC medium for 2 days at 25±2°C in the dark. Based on GUS expression analysis, 10min co-cultivation time with 100μM acetosyringone was found optimum for the delivery of gus gene. Calli were proved to be very sensitive to Agrobacterium infection and we found that the level of necrotic response can be minimized after co-cultivation with 30% LS, 10g/L PVP, 10% coconut water and 250mg/L timentin which improved the final transformation efficiency to 9.33%. Molecular and genetic analysis of transgenic plants reveals the integration, expression and inheritance of transgene in the progeny (T(1)) of these plants. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants (T(0) and T(1)). Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  9. A simple shoot multiplication procedure using internode explants, and its application for particle bombardment and Agrobacterium-mediated transformation in watercress.

    PubMed

    Ogita, Shinjiro; Usui, Miki; Shibutani, Nanae; Kato, Yasuo

    2009-07-01

    A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 muM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1-2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 muM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene.

  10. Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties.

    PubMed

    Wang, Ke; Liu, Huiyun; Du, Lipu; Ye, Xingguo

    2016-11-10

    Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker-free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium-mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker-free transgenic wheat plants from various commercial Chinese varieties and their F1 hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T-DNA regions. The average co-integration frequency of the gus and the bar genes located on the two independent T-DNA regions was 49.0% in T0 plants. We further found that the efficiency of generating marker-free plants was related to the number of bar gene copies integrated in the genome. Marker-free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T1 positive plants, but the gus gene was never found to be silenced in T1 plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants.

  11. Application of sonication in combination with vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars.

    PubMed

    Arun, Muthukrishnan; Subramanyam, Kondeti; Mariashibu, Thankaraj Salammal; Theboral, Jeevaraj; Shivanandhan, Ganeshan; Manickavasagam, Markandan; Ganapathi, Andy

    2015-02-01

    Soybean is a recalcitrant crop to Agrobacterium-mediated genetic transformation. Development of highly efficient, reproducible, and genotype-independent transformation protocol is highly desirable for soybean genetic improvement. Hence, an improved Agrobacterium-mediated genetic transformation protocol has been developed for cultivar PK 416 by evaluating various parameters including Agrobacterium tumefaciens strains (LBA4404, EHA101, and EHA105 harboring pCAMBIA1304 plasmid), sonication duration, vacuum infiltration pressure, and vacuum duration using cotyledonary node explants of soybean prepared from 7-day-old seedlings. The transformed plants were successfully developed through direct organogenesis system. Transgene expression was assessed by GUS histochemical and gfp visual assays, and integration was analyzed by PCR and Southern blot hybridization. Among the different combinations and durations evaluated, a maximum transformation efficiency of 18.6 % was achieved when the cotyledonary node explants of cv. PK 416 were sonicated for 20 s and vacuum infiltered for 2 min at 250 mmHg in A. tumefaciens EHA105 suspension. The amenability of the standardized protocol was tested on four more soybean cultivars JS 90-41, Hara Soy, Co 1, and Co 2 in which all the cultivars responded favorably with transformation efficiency ranging from 13.3 to 16.6 %. The transformation protocol developed in the present study would be useful to transform diverse soybean cultivars with desirable traits.

  12. Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line.

    PubMed

    Cho, Myeong-Je; Wu, Emily; Kwan, Jackie; Yu, Maryanne; Banh, Jenny; Linn, Wutt; Anand, Ajith; Li, Zhi; TeRonde, Susan; Register, James C; Jones, Todd J; Zhao, Zuo-Yu

    2014-10-01

    An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds. This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.

  13. Several components of SKP1/Cullin/F-box E3 ubiquitin ligase complex and associated factors play a role in Agrobacterium-mediated plant transformation.

    PubMed

    Anand, Ajith; Rojas, Clemencia M; Tang, Yuhong; Mysore, Kirankumar S

    2012-07-01

    • Successful genetic transformation of plants by Agrobacterium tumefaciens requires the import of bacterial T-DNA and virulence proteins into the plant cell that eventually form a complex (T-complex). The essential components of the T-complex include the single stranded T-DNA, bacterial virulence proteins (VirD2, VirE2, VirE3 and VirF) and associated host proteins that facilitate the transfer and integration of T-DNA. The removal of the proteins from the T-complex is likely achieved by targeted proteolysis mediated by VirF and the plant ubiquitin proteasome complex. • We evaluated the involvement of the host SKP1/culin/F-box (SCF)-E3 ligase complex and its role in plant transformation. Gene silencing, mutant screening and gene expression studies suggested that the Arabidopsis homologs of yeast SKP1 (suppressor of kinetochore protein 1) protein, ASK1 and ASK2, are required for Agrobacterium-mediated plant transformation. • We identified the role for SGT1b (suppressor of the G2 allele of SKP1), an accessory protein that associates with SCF-complex, in plant transformation. We also report the differential expression of many genes that encode F-box motif containing SKP1-interacting proteins (SKIP) upon Agrobacterium infection. • We speculate that these SKIP genes could encode the plant specific F-box proteins that target the T-complex associated proteins for polyubiquitination and subsequent degradation by the 26S proteasome. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  14. The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): role of the binary vector system and selection cassettes.

    PubMed

    Bińka, Agnieszka; Orczyk, Wacław; Nadolska-Orczyk, Anna

    2012-02-01

    The influence of two binary vector systems, pGreen and pCAMBIA, on the Agrobacterium-mediated transformation ability of wheat and triticale was studied. Both vectors carried selection cassettes with bar or nptII driven by different promoters. Two cultivars of wheat, Kontesa and Torka, and one cultivar of triticale, Wanad, were tested. The transformation rates for the wheat cultivars ranged from 0.00 to 3.58% and from 0.00 to 6.79% for triticale. The best values for wheat were 3.58% for Kontesa and 3.14% for Torka, and these were obtained after transformation with the pGreen vector carrying the nptII selection gene under the control of 35S promoter. In the case of the bar selection system, the best transformation rates were, respectively, 1.46 and 1.79%. Such rates were obtained when the 35S::bar cassette was carried by the pCAMBIA vector; they were significantly lower with the pGreen vector. The triticale cultivar Wanad had its highest transformation rate after transformation with nptII driven by 35S in pCAMBIA. The bar selection system for the same triticale cultivar was better when the gene was driven by nos and the selection cassette was carried by pGreen. The integration of the transgenes was confirmed with at least three pairs of specific starters amplifying the fragments of nptII, bar, or gus. The expression of selection genes, measured by reverse transcriptase polymerase chain reaction (RT-PCR) in relation to the actin gene, was low, ranging from 0.00 to 0.63 for nptII and from 0.00 to 0.33 for bar. The highest relative transcript accumulation was observed for nptII driven by 35S and expressed in Kontesa that had been transformed with pGreen.

  15. Highly efficient Agrobacterium-mediated transformation of embryogenic cell suspensions of Musa acuminata cv. Mas (AA) via a liquid co-cultivation system.

    PubMed

    Huang, Xia; Huang, Xue-Lin; Xiao, Wang; Zhao, Jie-Tang; Dai, Xue-Mei; Chen, Yun-Feng; Li, Xiao-Ju

    2007-10-01

    A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0-490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants.

  16. Transient gene expression in electroporated Solanum protoplasts.

    PubMed

    Jones, H; Ooms, G; Jones, M G

    1989-11-01

    Electroporation was used to evaluate parameters important in transient gene expression in potato protoplasts. The protoplasts were from leaves of wild potato Solanum brevidens, and from leaves, tubers and suspension cells of cultivated Solanum tuberosum cv. Désirée. Reporter enzyme activity, chloramphenicol acetyl transferase (CAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, depended on the field strength and the pulse duration used for electroporation. Using field pulses of 85 ms duration, the optimum field strengths for maximum CAT activity were: S. brevidens mesophyll protoplasts--250 V/cm; Désirée mesophyll protoplasts--225 V/cm; Désirée suspension culture protoplasts--225 V/cm; and Désirée tuber protoplasts--150 V/cm. The optimum field strengths correlated inversely with the size of the protoplasts electroporated; this is consistent with biophysical theory. In time courses, maximum CAT activity (in Désirée mesophyll protoplasts) occurred 36-48 h after electroporation. Examination at optimised conditions of a chimaeric gene consisting of a class II patatin promoter linked to the beta-glucuronidase (gus) gene, showed expression (at DNA concentrations between 0-10 pmol/ml) comparable to the CaMV 35S promoter in both tuber and mesophyll protoplasts. At higher DNA concentrations (20-30 pmol/ml) the patatin promoter directed 4-5 times higher levels of gus expression. Implications and potential contributions towards studying gene expression, in particular of homologous genes in potato, are discussed.

  17. Improvement in Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) by the inhibition of polyphenolics released during wounding of cotyledonary node explants.

    PubMed

    Yadav, Reena; Mehrotra, Meenakshi; Singh, Aditya K; Niranjan, Abhishek; Singh, Rani; Sanyal, Indraneel; Lehri, Alok; Pande, Veena; Amla, D V

    2017-01-01

    Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) has been performed using cotyledonary node explants (CNs), which release phenolics upon excision that are detrimental to the viability of Agrobacterium tumefaciens and result in low transformation frequency. Twelve low molecular weight phenolic compounds and salicylic acid were identified in the exudates released upon excision during the preparation of cotyledonary nodes by reverse phase high-performance liquid chromatography (RP-HPLC). Zone inhibition assays performed with the explant exudates released at periodic intervals after excision showed the inhibition of A. tumefaciens. Agroinoculation of freshly excised cotyledonary nodes of chickpea showed 98-99 % inhibition of colony forming units (cfu). Osmium tetraoxide fixation of excised tissues showed enhanced accumulation of phenolics in the sub-epidermal regions causing enzymatic browning, affecting the viability and performance of A. tumefaciens for T-DNA delivery. The periodic analysis of exudates released from excised CNs showed enhanced levels of gallic acid (0.2945 ± 0.014 μg/g), chlorogenic acid (0.0978 ± 0.0046 μg/g), and quercetin (0.0971 ± 0.0046 μg/g) fresh weight, which were detrimental to A. tumefaciens. Quantitative assays and the elution profile showed the maximum leaching of phenolics, flavonoids, and salicylic acid immediately after the excision of explants and continued till 4 to 8 h post-excision. Pre-treatment of excised explants with inhibitors of polyphenol oxidase like L-cysteine, DTT, and sodium thiosulfate before co-cultivation showed the recovery of A. tumefaciens cfu, decreased the accumulation of phenolics, and improved transformation frequency. Our results show the hypersensitive response of excision stress for the expression of defense response-related genes and synthesis of metabolites in grain legume chickpea against pathogen infestation including Agrobacterium.

  18. Transgenic grasspea (Lathyrus sativus L.): factors influencing agrobacterium-mediated transformation and regeneration.

    PubMed

    Barik, D P; Mohapatra, U; Chand, P K

    2005-11-01

    A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD(600) congruent with 0.6 and diluted to a cell density of 10(9) cells ml(-1) for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 mug ml(-1)). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l(-1) indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T(1) seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.

  19. Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors.

    PubMed

    Vega, Juan M; Yu, Weichang; Kennon, Angela R; Chen, Xinlu; Zhang, Zhanyuan J

    2008-02-01

    High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant L-cysteine alone or L-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.

  20. Improved Agrobacterium-mediated transformation and high efficiency of root formation from hypocotyl meristem of spring Brassica napus 'Precocity' cultivar.

    PubMed

    Liu, X X; Lang, S R; Su, L Q; Liu, X; Wang, X F

    2015-12-14

    Rape seed (Brassica napus L.) is one of the most important oil seed crops in the world. Genetic manipulation of rapeseed requires a suitable tissue culture system and an efficient method for plant regeneration, as well as an efficient transformation procedure. However, development of transgenic B. napus has been problematic, and current studies are limited to cultivated varieties. In this study, we report a protocol for regeneration of transgenic rape after Agrobacterium-mediated transformation of hypocotyls from the spring B. napus 'Precocity' cultivar. We analyzed the effects of plant growth regulators in the medium on regeneration. Additionally, factors affecting the transformation efficiency, including seedling age, Agrobacterium concentration, infection time, and co-cultivation time, were assessed by monitoring GUS expression. Results from these experiments revealed that transformation was optimized when the meristematic parts of the hypocotyls were taken from 8 day-old seedlings, cultured on Murashinge and Skoog basal media containing 0.1 mg/L 1-naphthaleneacetic acid and 2.5 mg/L 6-benzylaminopurine, and incubated in Agrobacterium suspension (OD600 = 0.5) for 3 to 5 min, followed by 2 days of co-cultivation. Integration of T-DNA into the plant genome was confirmed by polymerase chain reaction (PCR), b-glucuronidase histochemical staining, and quantitative real-time PCR. The protocols developed for regeneration, transformation, and rooting described in this study could help to accelerate the development of transgenic spring rape varieties with novel features.

  1. Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper).

    PubMed

    Sainger, Manish; Chaudhary, Darshna; Dahiya, Savita; Jaiwal, Ranjana; Jaiwal, Pawan K

    2015-10-01

    An efficient, rapid and direct multiple shoot regeneration system amenable to Agrobacterium-mediated transformation from primary leaf with intact petiole of blackgram (Vigna mungo) is established for the first time. The effect of the explant type and its age, type and concentration of cytokinin and auxin either alone or in combination and genotype on multiple shoot regeneration efficiency and frequency was optimized. The primary leaf explants with petiole excised from 4-day-old seedlings directly developed multiple shoots (an average of 10 shoots/ explant) from the cut ends of the petiole in 95 % of the cultures on MSB (MS salts and B5 vitamins) medium containing 1.0 μM 6-benzylaminopurine. Elongated (2-3 cm) shoots were rooted on MSB medium with 2.5 μM indole-butyric acid and resulted plantlets were hardened and established in soil, where they resumed growth and reached maturity with normal seed set. The regenerated plants were morphologically similar to seed-raised plants and required 8 weeks time from initiation of culture to establish them in soil. The regeneration competent cells present at the cut ends of petiole are fully exposed and are, thus, easily accessible to Agrobacterium, making this plant regeneration protocol amenable for the production of transgenic plants. The protocol was further successfully used to develop fertile transgenic plants of blackgram using Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron. The presence and integration of transgenes in putative T0 plants were confirmed by polymerase chain reaction (PCR) and Southern blot hybridization, respectively. The transgenes were inherited in Mendelian fashion in T1 progeny and a transformation frequency of 1.3 % was obtained. This protocol can be effectively used for transferring new traits in blackgram and other legumes for their

  2. Rapid deletion production in fungi via Agrobacterium mediated transformation of OSCAR deletion contructs.

    USDA-ARS?s Scientific Manuscript database

    Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene’s function in the living organism. In this protocol we describe the OSCAR method of precise and rapid deletion plasmid construction. OSCAR relies on t...

  3. Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N.

    PubMed

    Kemppainen, Minna; Circosta, Ariana; Tagu, Denis; Martin, Francis; Pardo, Alejandro G

    2005-12-01

    The development of an efficient transformation system is required to alter the expression of symbiosis-regulated genes and to develop insertional mutagenesis in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Vegetative mycelium of this fungus was transformed by Agrobacterium tumefaciens-mediated gene transfer. The selection marker was the hygromycin resistance gene of Escherichia coli (hph) under the control of the gpd promoter from Agaricus bisporus and the CaMV 35S terminator as part of the T-DNA. PCR amplification of hph and Southern blot analyses showed that the genome of the hygromycin-resistant transformants contained the cassette. The latter proved mostly single copy and random integration of part of the transgene into the fungal genome. A. tumefaciens-mediated gene transfer should facilitate future development of insertional mutagenesis, targeted gene disruption and RNA interference technology in L. bicolor.

  4. Optimization of transient gene expression system in Gerbera jemosonii petals.

    PubMed

    Hussein, Gihan M; Abu El-Heba, Ghada A; Abdou, Sara M; Abdallah, Naglaa A

    2013-01-01

    Low transformation efficiency and long generation time for production of transgenic Gerbera jemosonii plants leads to vulnerable gene function studies. Thus, transient expression of genes would be an efficient alternative. In this investigation, a transient expression system for gerbera petals based on the Agrobacterium infiltration protocol was developed using the reporter genes β-glucuronidase (gus) and green florescence protein (gfp). Results revealed the incapability of using the gfp gene as a reporter gene for transient expression study in gerbera flowers due to the detection of green fluorescent color in the non-infiltrated gerbera flower petals. However, the gus reporter gene was successfully utilized for optimizing and obtaining the suitable agroinfiltration system in gerbera flowers. The expression of GUS was detectable after three days of agroinfiltration in gerbera cultivars "Express" and "White Grizzly" with dark pink and white flower colors, respectively. The vacuum agroinfiltration protocol has been applied on the cultivar "Express" for evaluating the transient expression of the two genes involved in the anthocyanin pathway (iris-dfr and petunia-f3' 5'h), which is responsible for the color in flowers. In comparison to the control, transient expression results showed change in the anthocyanin pigment in all infiltrated flowers with color genes. Additionally, blue color was detected in the stigma and pollen grains in the infiltrated flowers. Moreover, blue colors with variant intensities were observed in produced calli during the routine work of stable transformation with f3' 5'h gene.

  5. Transient Phenomena in Gene Expression after Induction of Transcription

    PubMed Central

    Deneke, Carlus; Rudorf, Sophia; Valleriani, Angelo

    2012-01-01

    When transcription of a gene is induced by a stimulus, the number of its mRNA molecules changes with time. Here we discuss how this time evolution depends on the shape of the mRNA lifetime distribution. Analysis of the statistical properties of this change reveals transient effects on polysomes, ribosomal profiles, and rate of protein synthesis. Our studies reveal that transient phenomena in gene expression strongly depend on the specific form of the mRNA lifetime distribution. PMID:22558114

  6. Genetic transformation of Metroxylon sagu (Rottb.) cultures via Agrobacterium-mediated and particle bombardment.

    PubMed

    Ibrahim, Evra Raunie; Hossain, Md Anowar; Roslan, Hairul Azman

    2014-01-01

    Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance.

  7. Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment

    PubMed Central

    Ibrahim, Evra Raunie

    2014-01-01

    Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. PMID:25295258

  8. Morphogenetic and chemical stability of long-term maintained Agrobacterium-mediated transgenic Catharanthus roseus plants.

    PubMed

    Verma, Priyanka; Sharma, Abhishek; Khan, Shamshad Ahmad; Mathur, Ajay Kumar; Shanker, Karuna

    2015-01-01

    Transgenic Catharanthus roseus plants (transgenic Dhawal [DT] and transgenic Nirmal [NT]) obtained from the Agrobacterium tumefaciens and Agrobacterium rhizognenes-mediated transformations, respectively, have been maintained in vitro for 5 years. Plants were studied at regular intervals for various parameters such as plant height, leaf size, multiplication rate, alkaloid profile and presence of marker genes. DT plant gradually lost the GUS gene expression and it was not detected in the fifth year while NT plant demonstrated the presence of genes rolA, rolB and rolC even in the fifth year, indicating the more stable nature of Ri transgene. Vindoline content in the DT was two times more than in non-transformed control plants. Alkaloid and tryptophan profiles were almost constant during the 5 years. The cluster analysis revealed that the DT plant is more close to the control Nirmal plant followed by NT plant.

  9. Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones.

    PubMed

    Chateau, S; Sangwan, R S; Sangwan-Norreel, B S

    2000-12-01

    Many plant species and/or genotypes are highly recalcitrant to Agrobacterium-mediated genetic transformation, and yet little is known about this phenomenon. Using several Arabidopsis genotypes/ecotypes, the results of this study indicated that phytohormone pretreatment could overcome this recalcitrance by increasing the transformation rate in the known recalcitrant genotypes. Transient expression of a T-DNA encoded ss-glucuronidase (GUS) gene and stable kanamycin resistance were obtained for the ten Arabidopsis genotypes tested as well as for the mutant uvh1 (up to 69% of petioles with blue spots and up to 42% resistant calli). Cultivation of Arabidopsis tissues on phytohormones for 2-8 d before co-cultivation with Agrobacterium tumefaciens significantly increased transient GUS gene expression by 2-11-fold and stable T-DNA integration with petiole explants. Different Arabidopsis ecotypes revealed differences in their susceptibility to Agrobacterium-mediated transformation and in their type of reaction to pre-cultivation (three types of reactions were defined by gathering ecotypes into three groups). The Arabidopsis uvh1 mutant described as defective in a DNA repair system showed slightly lower competence to transformation than did its progenitor Colombia. This reduced transformation competence, however, could be overcome by 4-d pre-culture with phytohormones. The importance of pre-cultivation with phytohormones for genetic transformation is discussed.

  10. Highly Efficient Agrobacterium-Mediated Transformation of Wheat Via In Planta Inoculation

    NASA Astrophysics Data System (ADS)

    Risacher, Thierry; Craze, Melanie; Bowden, Sarah; Paul, Wyatt; Barsby, Tina

    This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398(1. Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.

  11. Agrobacterium-mediated genetic transformation of the desiccation tolerant resurrection plant Ramonda myconi (L.) Rchb.

    PubMed

    Tóth, Sándor; Kiss, Csaba; Scott, Peter; Kovács, Gabriella; Sorvari, Seppo; Toldi, Ottó

    2006-05-01

    In this paper we describe the first procedure for Agrobacterium tumefaciens-mediated genetic transformation of the desiccation tolerant plant Ramonda myconi (L.) Rchb. Previously, we reported the establishment of a reliable and effective tissue culture system based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. This efficient plant regeneration via callus phase provided a basis for the optimisation of the genetic transformation in R. myconi. For gene delivery, both a standard (method A) and a modified protocol (method B) have been applied. Since the latter has previously resulted in successful transformation of another resurrection plant, Craterostigma plantagineum, an identical protocol was utilized in transformation of R. myconi, as this method may prove general for dicotyledonous resurrection plants. On this basis, physical and biochemical key variables in transformation were evaluated such as mechanical microwounding of plant explants and in vitro preinduction of vir genes. While the physical enhancement of bacterial penetration was proved to be essential for successful genetic transformation of R. myconi, an additional two-fold increase in the transformation frequency was obtained when the above physical and biochemical treatments were applied in combination. All R0 and R1 transgenic plants were fertile, and no morphological abnormalities were observed on the whole-plant level.

  12. An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta.

    PubMed

    Gupta, Vijayta; Ur Rahman, Laiq

    2015-07-01

    Tagetes erecta, L. an asteraceous plant of industrial and medicinal value, contains important compounds like pyrethrins, thiophenes and lutein, possessing immense potential for insecticidal, nematicidal and nutraceutical activities. Considering the importance and demand for these natural compounds, genetic manipulation of this crop for better productivity of secondary metabolites holds great significance. A rapid and reproducible direct regeneration and genetic transformation system is the prerequisite for genetic manipulation of any crop. This paper elucidates the establishment of an efficient direct regeneration and transformation protocol of T. erecta using Agrobacterium tumefaciens. Investigation of the effects of different types of explants (Hypocotyls, cotyledonary leaves, rachis and leaf sections) and different BAP and GA3 combinations on the regeneration frequency of T. erecta suggested that the best regeneration frequency (66 %) with an average of 5.08 ± 0.09 shoot buds/explant was observed from hypocotyl explants cultured on media containing 1.5 mg/l BAP and 5 mg/l GA3. The transformation protocol was established using A. tumefaciens strain LBA4404, containing the binary vector pBI121, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization of kanamycin concentration (200 mg/l) for selection, standardization of cocultivation time (45 min) and acetosyringone concentration (150 μM) for obtaining higher transformation frequency were established using hypocotyl explants. The selected putative transgenic shoots were subsequently rooted on the Murashige and Skoog medium and transferred to the green house successfully. The plants were characterised by analysing the gus expression, amplification of 600 bp npt II fragment and Southern blot

  13. High-level diterpene production by transient expression in Nicotiana benthamiana

    PubMed Central

    2013-01-01

    Background Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis. Results Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2. Conclusion With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants. PMID:24330621

  14. Improved vectors for Agrobacterium mediated genetic manipulation of Hypholoma spp. and other homobasidiomycetes.

    PubMed

    Al-Salihi, Suhad A A; Scott, Thomas A; Bailey, Andy M; Foster, Gary D

    2017-08-24

    The basidiomycete fungi Hypholoma fasciculare and H. sublateritium are both prolific producers of sesquiterpenes and triterpenes, some of which have relevant pharmaceutical properties. Although H. sublateritium has been transformed in the past, the low reported efficiencies highlighted the need for establishing an effective simple transformation system for these valuable species. We have optimized Agrobacterium tumefaciens-mediated transformation through testing various parameters in these two Hypholoma species, showing that a mixture of homogenized mycelia and Agrobacterium (strain LBA4404) co-cultivated for 84h at 25°C is optimal for efficient transformation in these basidiomycetes. This study also reveals the requirements for transgene expression, with the first report of GFP expression in these Hypholoma, the need for an intron for such transgene expression, and further demonstrates the functionality of the expression vector by its use in Clitopilus passeckerianus. This development of transformation system and expression constructs, can facilitate further genetic investigation such as gene functionality in these fungi. Copyright © 2017. Published by Elsevier B.V.

  15. Agrobacterium-Mediated Stable Genetic Transformation of Populus angustifolia and Populus balsamifera

    PubMed Central

    Maheshwari, Priti; Kovalchuk, Igor

    2016-01-01

    The present study demonstrates Agrobacterium tumefaciens-mediated stable genetic transformation of two species of poplar – Populus angustifolia and Populus balsamifera. The binary vector pCAMBIA-Npro-long-Luc containing the luciferase reporter gene was used to transform stem internode and axillary bud explants. Putative transformants were regenerated on selection-free medium using our previously established in vitro regeneration method. Explant type, genotype, effect of pre-culture, Agrobacterium concentration, a time period of infection and varying periods of co-culture with bacteria were tested for the transformation frequency. The highest frequency of transformation was obtained with stem internode explants pre-cultured for 2 days, infected with Agrobacterium culture at the concentration of OD600 = 0.5 for 10 min and co-cultivated with Agrobacterium for 48 h. Out of the two genotypes tested, P. balsamifera exhibited a higher transformation rate in comparison to P. angustifolia. The primary transformants that exhibited luciferase activity in a bioluminescence assay under the CCD camera when subjected to polymerase chain reaction and Southern blot analysis revealed a stable single-copy integration of luc in their genomes. The reported protocol is highly reproducible and can be applied to other species of poplar; it will also be useful for future genetic engineering of one of the most important families of woody plants for sustainable development. PMID:27014319

  16. Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting

    PubMed Central

    2011-01-01

    Background Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T1 progeny. Results An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T1 seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T1 progeny displayed Mendelian inheritance. Conclusions This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications. PMID:21595986

  17. Agrobacterium-mediated transformation of polyploid cereals. The efficiency of selection and transgene expression in wheat.

    PubMed

    Przetakiewicz, Anna; Karaś, Agnieszka; Orczyk, Wacław; Nadolska-Orczyk, Anna

    2004-01-01

    Three combinations of Agrobacterium tumefaciens strains and vectors were used in the transformation of selected Polish wheat cultivars. The combinations were: two hypervirulent strains, AGL1, containing the pDM805 binary plasmid, and EHA101, containing pGAH; and the common Agro strain LBA4404, harboring the super-binary pTOK233 vector. pDM805 contained bar under the control of Ubi1 promoter, pGAH had nptII under nos, and pTOK233 had hpt under 35S. Additionally, pDM805 and pTOK233 carried the gus reporter gene under the Act1 promoter or 35S promoter, respectively. The highest selection rate was 12.6% and was obtained with EHA101(pGAH) on a kanamycin-containing medium. Sixty-five of the plants grown on that medium were PCR positive. The second best combination was LBA4404(pTOK233) and kanamycin selection, which gave an average transformation rate of 2.3%. Phosphinothricin selection gave 1.0% transformation efficiency, while hygromycin, depending on the strain/vector used, gave from 0.2 to 0.4%. PCR tests in T1 revealed that 67% of the lines showed a 3:1 segregation ratio, and 11% a 15:1 ratio, while in 22%, segregation was non-Mendelian. The high number of T0 transgenic plants containing one copy of the transgene was confirmed via Southern blot analysis. Kanamycin resistance in the T1 generation was very low; in some lines, all the progeny were kanamycin sensitive. GUS expression, only tested in young T1 plants, was in agreement with Mendelian segregation in three out of the twelve tested. The factors influencing the efficiency of selection and transgene expression are discussed in this paper.

  18. Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal

    PubMed Central

    Sivanandhan, Ganeshan; Kapil Dev, Gnajothi; Theboral, Jeevaraj; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

    2015-01-01

    In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants. PMID:25927703

  19. Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection.

    PubMed

    Zhao, Ming-Min; An, De-Rong; Zhao, Jian; Huang, Guang-Hua; He, Zu-Hua; Chen, Jiang-Ye

    2006-01-01

    RNA interference (RNAi) silences gene expression by guiding mRNA degradation in a sequence-specific fashion. Small interfering RNA (siRNA), an intermediate of the RNAi pathway, has been shown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells. Here, we report that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) could inhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-associated 126 kDa protein in intact plant tissue. Our results indicate that transiently expressed shRNA efficiently interfered with TMV infection. The interference observed is sequence-specific, and time- and site-dependent. Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumber mosaic virus (CMV), an unrelated tobamovirus. In order to interfere with TMV accumulation in tobacco leaves, it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation. Our results support the view that RNAi opens the door for novel therapeutic procedures against virus diseases. We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expression could be employed as a potent antiviral treatment in plants.nt antiviral treatment in plants.

  20. Transient Silencing of DNA Repair Genes Improves Targeted Gene Integration in the Filamentous Fungus Trichoderma reesei.

    PubMed

    Chum, Pak Yang; Schmidt, Georg; Saloheimo, Markku; Landowski, Christopher P

    2017-08-01

    Trichoderma reesei is a filamentous fungus that is used worldwide to produce industrial enzymes. Industrial strains have traditionally been created though systematic strain improvement using mutagenesis and screening approaches. It is also desirable to specifically manipulate the genes of the organism to further improve and to modify the strain. Targeted integration in filamentous fungi is typically hampered by very low frequencies of homologous recombination. To address this limitation, we have developed a simple transient method for silencing genes in T. reesei Using gene-specific small interfering RNAs (siRNAs) targeted to mus53, we could achieve up to 90% knockdown of mus53 mRNA. As a practical example, we demonstrated that transient silencing of DNA repair genes significantly improved homologous integration of DNA at a specific locus in a standard protoplast transformation. The best transient silencing of mus53 with siRNAs in protoplasts could achieve up to 59% marker gene integration.IMPORTANCE The previous solution for improving targeted integration efficiency has been deleting nonhomologous end joining (NHEJ) DNA repair genes. However, deleting these important repair genes may lead to unintended consequences for genomic stability and could lead to the accumulation of spontaneous mutations. Our method of transiently silencing NHEJ repair pathway genes allows recovery of their important repair functions. Here we report a silencing approach for improving targeted DNA integration in filamentous fungi. Furthermore, our transient silencing method is a truly flexible approach that is capable of knocking down the expression of a target gene in growing mycelial cultures, which could facilitate the broad study of gene functions in T. reesei. Copyright © 2017 American Society for Microbiology.

  1. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora

    PubMed Central

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora. PMID:27337148

  2. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora.

    PubMed

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora.

  3. Improved methods in Agrobacterium-mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols.

    PubMed

    Ramesh, Sunita A; Kaiser, Brent N; Franks, Tricia; Collins, Graham; Sedgley, Margaret

    2006-08-01

    A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 muM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.

  4. Transient expression of glyoxal oxidase from the Chinese wild grape Vitis pseudoreticulata can suppress powdery mildew in a susceptible genotype.

    PubMed

    Guan, Xin; Zhao, Heqing; Xu, Yan; Wang, Yuejin

    2011-04-01

    Vitis pseudoreticulata glyoxal oxidase (VpGLOX) was previously isolated from the Chinese wild vine V. pseudoreticulata accession "Baihe-35-1" during a screen for genes that are upregulated in response to infection with grapevine powdery mildew (Erysiphe necator, PM). In the present study, a possible function of VpGLOX for defense against PM was investigated using Agrobacterium-mediated transient expression. After optimizing agro-infiltration, VpGLOX was transiently overexpressed in leaves of either PM-susceptible (accession "6-12-2") or PM-resistant (accession "6-12-6") plants. The efficiency of transfection was verified using a β-glucuronidase (GUS) reporter and was found to comprise most leaf areas regardless of the initial leaf position. Upon infection with E. necator, clear differences were observed with respect to hyphal development between agro-infiltrated leaves and control groups of both, the susceptible and the resistant, genotypes. The expression of VpGLOX was followed by real-time polymerase chain reaction in both genotypes. Whereas in the susceptible host ("6-12-2") expression was found to increase only in transfected leaves and remained transient, in the resistant host ("6-12-6"), a second peak appeared later in transfected leaves, probably representing the response of the endogenous VpGLOX. The data support the interpretation that VpGLOX is sufficient to confer resistance to E. necator.

  5. Transient Expression of cor Gene in Papaver somniferum

    PubMed Central

    Hosseini, Bahman; Shahriari-Ahmadi, Farajollah; Hashemi, Haleh; Marashi, Mohammad-Hasan; Mohseniazar, Mahdi; Farokhzad, Alireza; Sabokbari, Masoume

    2011-01-01

    Introduction Papaver somniferum is the commercial source of morphine and codeine. The isolation of effective genes involved in the morphine biosynthesis of P. somniferum is very important in the production of specific metabolites achieved using metabolic engi-neering techniques. In this pathway, the key enzyme COR is involved in the conversion of codeinone to codeine and morphinone to morphine. Methods the gene encoding of this enzyme was isolated using primers designed on the base of gene sequence available on (NCBI) for P. somniferum. This gene correct size around (960 bp) was first subcloned into pTZ57RIT vector then cloned into expression vectors (pBI121) between BamHI and SacI sites to allow the expression of cor gene driven by the cauliflower mosaic virus 35S pro-moter. The result was confirmed through different molecular methods e.g. PCR and en-zyme digestion by BamHI and SacI. The recombinant plasmid was transformed into the E. coli strain DH5α using a freeze-thaw method. Having selected positive colones on selection medium, plasmid was extracted by miniprep method and recombinant plasmids were selected based on PCR and digestion. The construct was then mobilized in Agrobacterium tumefaciens C58/pGV3850 (KmR RifR). After gene transformation to P. somniferum plants, the agroinfiltration method was also used for transient expression of COR enzyme. Results evaluation results showed that morphine and codeine were detectable in the leaves of transgenic plants containing cor transgene and there was significant difference in the final production. After completing this experiment for three times, results showed that in 11 sets from 15 sets of leaves experiment tested, main alkaloids (codeine, morphine, papaverin, noscapine and thebaine) were detectable. Conclusion Whereas no signal was detected in non-infiltrated control leaves or in leaves infiltrated with non-recombinant bacteria for morphine and codeine, others such as thebaine and papaverine were detectable

  6. An in vivo transient expression system can be applied for rapid and effective selection of artificial microRNA constructs for plant stable genetic transformation.

    PubMed

    Bhagwat, Basdeo; Chi, Ming; Su, Li; Tang, Haifeng; Tang, Guiliang; Xiang, Yu

    2013-05-20

    The utility of artificial microRNAs (amiRNAs) to induce loss of gene function has been reported for many plant species, but expression efficiency of the different amiRNA constructs in different transgenic plants was less predictable. In this study, expressions of amiRNAs through the gene backbone of Arabidopsis miR168a were examined by both Agrobacterium-mediated transient expression and stable plant genetic transformation. A corresponding trend in expression of amiRNAs by the same amiRNA constructs between the transient and the stable expression systems was observed in the experiments. Plant genetic transformation of the constructs that were highly expressible in amiRNAs in the transient agro-infiltration assays resulted in generation of transgenic lines with high level of amiRNAs. This provides a simple method for rapid and effective selection of amiRNA constructs used for a time-consuming genetic transformation in plants. Copyright © 2013. Published by Elsevier Ltd.

  7. An efficient method of agrobacterium-mediated genetic transformation and regeneration in local Indian cultivar of groundnut (Arachis hypogaea) using grafting.

    PubMed

    Tiwari, Vivekanand; Chaturvedi, Amit Kumar; Mishra, Avinash; Jha, Bhavanath

    2015-01-01

    Groundnut (Arachis hypogaea L.) is an industrial crop used as a source of edible oil and nutrients. In this study, an efficient method of regeneration and Agrobacterium-mediated genetic transformation is reported for a local cultivar GG-20 using de-embryonated cotyledon explant. A high regeneration 52.69 ± 2.32 % was achieved by this method with 66.6 μM 6-benzylaminopurine (BAP), while the highest number of shoot buds per explant, 17.67 ± 3.51, was found with 20 μM BAP and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The bacterial culture OD, acetosyringone and L-cysteine concentration were optimized as 1.8, 200 μM and 50 mg L(-1), respectively, in co-cultivation media. It was observed that the addition of 2,4-D in co-cultivation media induced accumulation of endogenous indole-3-acetic acid (IAA). The optimized protocol exhibited 85 % transformation efficiency followed by 14.65 ± 1.06 % regeneration, of which 3.82 ± 0.6 % explants were survived on hygromycin after selection. Finally, 14.58 ± 2.95 % shoots (regenerated on survived explants) were rooted on rooting media (RM3). In grafting method, regenerated shoots (after hygromycin selection) were grafted on the non-transformed stocks with 100 % survival and new leaves emerged in 3 weeks. The putative transgenic plants were then confirmed by PCR, Southern hybridization, reverse transcriptase PCR (RT-PCR) and β-glucuronidase (GUS) histochemical assay. The reported method is efficient and rapid and can also be applied to other crops which are recalcitrant and difficult in rooting.

  8. Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation.

    PubMed

    Tanaka, Hidenori; Toyama, Jun; Hashiguchi, Masatsugu; Kutsuna, Yasuyo; Tsuruta, Shin-ichi; Akashi, Ryo; Hoffmann, Franz

    2008-08-25

    Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for non-hairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5 mg/L benzylamino purine (BAP), 100 mg/L kanamycin and 250 mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5 mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR.

  9. Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14.

    PubMed

    Nyaboga, Evans N; Njiru, Joshua M; Tripathi, Leena

    2015-01-01

    Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1-2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70-80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava.

  10. Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14

    PubMed Central

    Nyaboga, Evans N.; Njiru, Joshua M.; Tripathi, Leena

    2015-01-01

    Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1–2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70–80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava. PMID:26113851

  11. Transient Expression of Chimeric Genes Delivered into Pollen by Microprojectile Bombardment 1

    PubMed Central

    Twell, David; Klein, Theodore M.; Fromm, Michael E.; McCormick, Sheila

    1989-01-01

    Chimeric genes containing a pollen-specific promoter from tomato (Lycopersicon esculentum) or the CaMV35S promoter were transiently expressed following their introduction into tobacco (Nicotiana tabacum) pollen using high velocity microprojectiles. Transient expression of the microprojectile-introduced genes in leaves and pollen was similar to that observed for these genes in stably transformed tobacco plants. Images Figure 2 PMID:16667175

  12. High-efficiency Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) and regeneration of insect-resistant transgenic plants.

    PubMed

    Mehrotra, Meenakshi; Sanyal, Indraneel; Amla, D V

    2011-09-01

    To develop an efficient genetic transformation system of chickpea (Cicer arietinum L.), callus derived from mature embryonic axes of variety P-362 was transformed with Agrobacterium tumefaciens strain LBA4404 harboring p35SGUS-INT plasmid containing the uidA gene encoding β-glucuronidase (GUS) and the nptII gene for kanamycin selection. Various factors affecting transformation efficiency were optimized; as Agrobacterium suspension at OD(600) 0.3 with 48 h of co-cultivation period at 20°C was found optimal for transforming 10-day-old MEA-derived callus. Inclusion of 200 μM acetosyringone, sonication for 4 s with vacuum infiltration for 6 min improved the number of GUS foci per responding explant from 1.0 to 38.6, as determined by histochemical GUS assay. For introducing the insect-resistant trait into chickpea, binary vector pRD400-cry1Ac was also transformed under optimized conditions and 18 T(0) transgenic plants were generated, representing 3.6% transformation frequency. T(0) transgenic plants reflected Mendelian inheritance pattern of transgene segregation in T(1) progeny. PCR, RT-PCR, and Southern hybridization analysis of T(0) and T(1) transgenic plants confirmed stable integration of transgenes into the chickpea genome. The expression level of Bt-Cry protein in T(0) and T(1) transgenic chickpea plants was achieved maximum up to 116 ng mg(-1) of soluble protein, which efficiently causes 100% mortality to second instar larvae of Helicoverpa armigera as analyzed by an insect mortality bioassay. Our results demonstrate an efficient and rapid transformation system of chickpea for producing non-chimeric transgenic plants with high frequency. These findings will certainly accelerate the development of chickpea plants with novel traits.

  13. Assessment of factors influencing the Agrobacterium-mediated in planta seed transformation of brinjal (Solanum melongena L.).

    PubMed

    Subramanyam, Kondeti; Rajesh, Manoharan; Jaganath, Balusamy; Vasuki, Amirthalingam; Theboral, Jeevaraj; Elayaraja, Dhandapani; Karthik, Sivabalan; Manickavasagam, Markandan; Ganapathi, Andy

    2013-09-01

    An efficient and reproducible in planta transformation method was developed for brinjal using seed as an explant. The brinjal seeds were infected with Agrobacterium tumefaciens EHA 105 harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA®. Several parameters influencing the in planta seed transformation such as pre-culture duration, acetosyringone concentration, surfactants, duration of sonication, vacuum pressure and vacuum duration have been evaluated. The putatively transformed (T 0) brinjal plants were screened by GUS histochemical analysis. Among the different combinations and concentrations tested, when the 18-h pre-cultured brinjal seeds were sonicated for 20 min and vacuum infiltered for 3 min at 500 mm of Hg in Agrobacterium suspension containing 100 μM acetosyringone, 0.2 % Silwett L-77 favoured the Agrobacterium infection and showed maximum transformation efficiency. Among the five brinjal varieties evaluated, Arka Samhitha showed maximum transformation efficiency at 45.66 %. The transgene was successfully transmitted to progeny plants (T 1) which was evidenced by GUS histochemical analysis, polymerase chain reaction and Southern hybridisation. The in planta protocol developed in the present study would be beneficial to transfer the economically and nutritionally important genes into different varieties of brinjal, and the transgenic brinjal plants can be produced in less time (approximately 27 days).

  14. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

    NASA Technical Reports Server (NTRS)

    Cardoza, V.; Stewart, C. N.

    2003-01-01

    An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

  15. (Analysis of proteins essential for Agrobacterium mediated DNA transfer to plant cells). [Single-stranded DNA binding proteins

    SciTech Connect

    Not Available

    1989-12-14

    The tumor inducing (Ti) plasmid of Agrobacterium contains two regions important for infection and transformation of plant cells. One region, the T-DNA, is transferred as a single strand into the plant cell, while the virulence (vir) region is responsible for recognition of susceptible cells, synthesis of the T-DNA single strand (T-strand), formation of a T-strand protein complex and transfer of this complex into susceptible cells. A DNA binding protein, VirE2, was identified as a product of the vir region. Sequencing of the 9000 kilobase pair virB region has been completed. Expression of 10 of the predicted 11 open reading frames (ORFs) was demonstrated in Escherichia coli. Translational coupling was demonstrated for 5 ORFs. Hydropathy analysis indicates that 9 of 11 ORFs have hydrophobic regions that could permit membrane channel formation. In related work, analysis of protons that potentiate movement of plant viruses was discussed, with indications that the tobacco mosaic virus (TMV) protein P30 may mediate transfer of TMV RNA through plasmadesmata. Also, using the T-DNA element as a marker, genes responsible for abnormal flower development are being cloned and isolated. 3 refs. (MHB)

  16. Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum.

    PubMed

    Zhang, Tao; Ren, Ping; Chaturvedi, Vishnu; Chaturvedi, Sudha

    2015-08-01

    The mechanisms of cold adaptation by fungi remain unknown. This topic is of high interest due to the emergence of white-nose syndrome (WNS), a skin infection of hibernating bats caused by Pseudogymnoascus destructans (Pd). Recent studies indicated that apart from Pd, there is an abundance of other Pseudogymnoascus species in the hibernacula soil. We developed an Agrobacterium tumefaciens-mediated transformation (ATMT) system for Pd and a related fungus Pseudogymnoascus pannorum (Pp) to advance experimental studies. URE1 gene encoding the enzyme urease was used as an easy to screen marker to facilitate molecular genetic analyses. A Uracil-Specific Excision Reagent (USER) Friendly pRF-HU2 vector containing Pd or Pp ure1::hygromycin (HYG) disruption cassette was introduced into A. tumefaciens AGL-1 cells by electroporation and the resulting strains were co-cultivated with conidia of Pd or Pp for various durations and temperatures to optimize the ATMT system. Overall, 680 Pd (0.006%) and 1800 Pp (0.018%) transformants were obtained from plating of 10(7) conidia; their recoveries were strongly correlated with the length of the incubation period (96h for Pd; 72h for Pp) and with temperature (15-18°C for Pd; 25°C for Pp). The homologous recombination in transformants was 3.1% for Pd and 16.7% for Pp. The availability of a standardized ATMT system would allow future molecular genetic analyses of Pd and related cold-adapted fungi. Copyright © 2015. Published by Elsevier Inc.

  17. Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato

    PubMed Central

    Tai, Thomas H.; Dahlbeck, Douglas; Clark, Eszter T.; Gajiwala, Paresh; Pasion, Romela; Whalen, Maureen C.; Stall, Robert E.; Staskawicz, Brian J.

    1999-01-01

    The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site–leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species. PMID:10570214

  18. Overexpression of several Arabidopsis histone genes increases Agrobacterium-medicated transformation and transgene expression in plants

    USDA-ARS?s Scientific Manuscript database

    The Arabidopsis histone H2A-1 is important for Agrobacterium-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, in the rat5 mutant results in decreased T-(transferred) DNA integration into the plant genome, whereas over-expression of HTA1 increases transformation freq...

  19. Transformation of somatic embryos of Prunus incisa ‘February Pink’ with a visible reporter gene

    USDA-ARS?s Scientific Manuscript database

    An Agrobacterium-mediated transformation system was developed for the ornamental cherry species Prunus incisa. This system uses both an antibiotic resistance gene (NPTII) and a visible selectable marker, the green fluorescent protein (GFP), to select plants. Cells from leaf and root explants were tr...

  20. Rationale for developing new virus vectors to analyze gene function in grasses through virus-induced gene silencing.

    PubMed

    Ramanna, Hema; Ding, Xin Shun; Nelson, Richard S

    2013-01-01

    The exploding availability of genome and EST-based sequences from grasses requires a technology that allows rapid functional analysis of the multitude of genes that these resources provide. There are several techniques available to determine a gene's function. For gene knockdown studies, silencing through RNAi is a powerful tool. Gene silencing can be accomplished through stable transformation or transient expression of a fragment of a target gene sequence. Stable transformation in rice, maize, and a few other species, although routine, remains a relatively low-throughput process. Transformation in other grass species is difficult and labor-intensive. Therefore, transient gene silencing methods including Agrobacterium-mediated and virus-induced gene silencing (VIGS) have great potential for researchers studying gene function in grasses. VIGS in grasses already has been used to determine the function of genes during pathogen challenge and plant development. It also can be used in moderate-throughput reverse genetics screens to determine gene function. However, the number of viruses modified to serve as silencing vectors in grasses is limited, and the silencing phenotype induced by these vectors is not optimal: the phenotype being transient and with moderate penetration throughout the tissue. Here, we review the most recent information available for VIGS in grasses and summarize the strengths and weaknesses in current virus-grass host systems. We describe ways to improve current virus vectors and the potential of other grass-infecting viruses for VIGS studies. This work is necessary because VIGS for the foreseeable future remains a higher throughput and more rapid system to evaluate gene function than stable transformation.

  1. Transient expression systems for plant-derived biopharmaceuticals.

    PubMed

    Komarova, Tatiana V; Baschieri, Selene; Donini, Marcello; Marusic, Carla; Benvenuto, Eugenio; Dorokhov, Yuri L

    2010-08-01

    In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of proteins. In addition, Agrobacterium-mediated expression also allows the production in plants of complex proteins assembled from subunits. Currently, the transient expression methods are preferential over any other transgenic system for the exploitation of large and unrestricted numbers of plants in a contained environment. By designing optimal constructs and related means of delivery into plant cells, the overall technology plan considers scenarios that envisage high yield of bioproducts and ease in monitoring the whole spectrum of upstream production, before entering good manufacturing practice facilities. In this way, plant-derived bioproducts show promise of high competitiveness towards classical eukaryotic cell factory systems.

  2. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells.

    PubMed

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-10-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants.

  3. Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

    PubMed

    Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

    2013-01-01

    Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

  4. Identification and deletion analysis of the promoter of the pepper SAR8.2 gene activated by bacterial infection and abiotic stresses.

    PubMed

    Lee, Sung Chul; Hwang, Byung Kook

    2006-07-01

    The pepper SAR8.2 gene, CASAR82A, was locally and systemically induced in pepper plants which had been infected by Xanthomonas campestris pv. vesicatoria or by Pseudomonas fluorescens. The DNA 1,283 bp sequence upstream of the CASAR82A gene was assessed with regard to the activity of the CASAR82A promoter fused to the beta-glucuronidase (GUS) reporter gene, via an Agrobacterium-mediated transient expression assay. In tobacco leaves which transiently expressed the -831 bp CASAR82A promoter, GUS activity was locally and systemically induced by Pseudomonas syringae pv. tabaci. GUS activity, which was driven by the -831 promoter, was also differentially activated in the leaves as the result of treatment with salicylic acid, ethylene, methyl jasmonate, abscisic acid, NaCl, and low temperatures. The -831 bp sequence upstream of the CASAR82A gene elicited full promoter activity in response to pathogen infection, abiotic elicitors, and environmental stresses. The expression of the pepper transcription factor, CARAV1, was shown to activate the CASAR82A promoter. Analyses of a series of 5'-deletions of the CASAR82A promoter revealed that novel cis-acting elements necessary for the induction of gene expression as the result of exposure to pathogen and abiotic elicitors appear to be localized in the promoter region between -831 and -759 bp.

  5. Sequencing, genomic organization, and preliminary promoter analysis of a black cherry (R)-(+)-mandelonitrile lyase gene.

    PubMed

    Hu, Z; Poulton, J E

    1997-12-01

    The flavoprotein (R)-(+)-mandelonitrile lyase (MDL; EC 4.1.2.10) plays a key role in cyanogenesis in rosaceous stone fruits. An MDL gene (mdl3) and its corresponding cDNA (MDL3) were isolated from black cherry (Prunus serotina) and characterized. The mdl3 gene contains 2292 bp of the 5' flanking region, the entire coding region, and 300 bp of the 3' flanking region. The coding region is interrupted by three short introns, of which one possesses the usual GC-AG splice junction dinucleotides. This gene encodes a polypeptide of 573 amino acids that includes a putative signal sequence, 13 potential N-glycosylation sites, and a presumptive flavin adenine dinucleotide-binding site. To determine whether the 5' flanking region of the mdl3 gene is capable of driving MDL expression, it was fused to the beta-glucuronidase reporter gene for Agrobacterium-mediated transformation into tobacco. Matching endogenous MDL expression patterns, beta-glucuronidase staining was observed in maturing embryos and seeds; it also occurred in postembryonic tissues, especially in association with vascular tissues. After developing a homologous transient transformation system to facilitate identification of putative regulatory sequences, we demonstrated that 125 bp (-107 to +18) of the 5' flanking sequence of the mdl3 gene is sufficient for MDL expression in protoplasts derived from immature black cherry embryos.

  6. Transient expression of exogenous gus gene in Porphyra yezoensis (Rhodophyta)

    NASA Astrophysics Data System (ADS)

    Kuang, Mei; Wang, Su-Juan; Li, Yao; Shen, Da-Leng; Zeng, Cheng-Kui

    1998-03-01

    Electroporation, PEC, PEG plus electroporation and Biolistics methods were tested in gene transformation of P. yezoensis. The exogenous gus was from plasmid of pBI121 and pCAMBIA1301, both contain the CaMV35S promoter. The receptors included the protoplasts, tissues and free-living conchocelis filaments of P. yezoensis. Several factors, for example, the voltage, capacitance and bivalent cations, etc., were studied. Results show that these four methods are all efficient for gene transformation in P. yezoensis; and that PEG is the best one, with transformation efficiency of up to 4×10-5. GUS activity was detected 26 days after transformation by using PEG method.

  7. Transient Gene Expression in Maize, Rice, and Wheat Cells Using an Airgun Apparatus 1

    PubMed Central

    Oard, James H.; Paige, David F.; Simmonds, John A.; Gradziel, Thomas M.

    1990-01-01

    An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNA-coated tungsten particles. The β-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 × 10−3. In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals. Images Figure 7 Figure 8 Figure 9 PMID:16667278

  8. Agrobacterium-mediated disruption of a nonribosomal peptide synthetase gene in the invertebrate pathogen Metarhizium anisopliae reveals a peptide spore factor

    USDA-ARS?s Scientific Manuscript database

    Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative n...

  9. Transient gene expression system established in Porphyra yezoensis is widely applicable in Bangiophycean algae.

    PubMed

    Hirata, Ryo; Takahashi, Megumu; Saga, Naotsune; Mikami, Koji

    2011-10-01

    The establishment of transient gene expression systems in the marine red macroalga Porphyra yezoensis has been useful for the molecular analysis of cellular processes in this species. However, there has been no successful report about the expression of foreign genes in other red macroalgae, which has impeded the broader understanding of the molecular biology of these species. We therefore examined whether the P. yezoensis transient gene expression system was applicable to other red macroalgae. The results indicated that a codon-optimized GUS, designated PyGUS, and plant-adapted sGFP(S65T) were successfully expressed under the control of the P. yezoensis PyAct1 promoter in gametophytic cells of six Porphyra species and also in Bangia fuscopurpurea, all of which are classified as Bangiophyceae. In contrast, there were no reporter-expressing cells in the Florideophycean algae examined. These results indicate the availability of PyGUS and sGFP as reporters and the 5' upstream region of the PyAct1 gene as a heterologous promoter for transient gene expression in Bangiophycean algae, which could provide a clue to the efficient expression of foreign genes and transformation in marine red macroalgae.

  10. Transient GUS Gene Expression in Date Palm Fruit Using Agroinjection Transformation Technique.

    PubMed

    Solliman, Mohei El-Din M; Mohasseb, Hebaallah A; Al-Khateeb, Abdullatif A; Al-Khayri, Jameel M; Al-Khateeb, Suliman A

    2017-01-01

    Transient expression of foreign genes in plant tissue is a valuable tool for testing the efficacy of transformation methods. In this work, we present, for the first time, the utilization of agroinjection as an efficient transformation system for gene delivery in date palm fruit. The research utilized Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pRI201-AN-GUS carrying the beta-glucuronidase (GUS) gene, under the control of a CaMV 35S and kanamycin (NPTII) as an antibiotic gene under the control of a NOS promoter. Based on histochemical assay of agroinjected fruit for the GUS gene expressions, this protocol has proved to be an efficient and reliable tool for transgene expression in date palm. PCR for plasmid DNA, extracted from the transformed Agrobacterium, demonstrated the generation of the expected amplicon, corresponding to the GUS gene using GUS primers.

  11. Closely spaced multiple mutations as potential signatures of transient hypermutability in human genes.

    PubMed

    Chen, Jian-Min; Férec, Claude; Cooper, David N

    2009-10-01

    Data from diverse organisms suggests that transient hypermutability is a general mutational mechanism with the potential to generate multiple synchronous mutations, a phenomenon probably best exemplified by closely spaced multiple mutations (CSMMs). Here we have attempted to extend the concept of transient hypermutability from somatic cells to the germline, using human inherited disease-causing multiple mutations as a model system. Employing stringent criteria for data inclusion, we have retrospectively identified numerous potential examples of pathogenic CSMMs that exhibit marked similarities to the CSMMs reported in other systems. These examples include (1) eight multiple mutations, each comprising three or more components within a sequence tract of <100 bp; (2) three possible instances of "mutation showers"; and (3) numerous highly informative "homocoordinate" mutations. Using the proportion of CpG substitution as a crude indicator of the relative likelihood of transient hypermutability, we present evidence to suggest that CSMMs comprising at least one pair of mutations separated by < or =100 bp may constitute signatures of transient hypermutability in human genes. Although this analysis extends the generality of the concept of transient hypermutability and provides new insights into what may be considered a novel mechanism of mutagenesis underlying human inherited disease, it has raised serious concerns regarding current practices in mutation screening.

  12. Natural Variation in the Pto Pathogen Resistance Gene Within Species of Wild Tomato (Lycopersicon). I. Functional Analysis of Pto Alleles

    PubMed Central

    Rose, Laura E.; Langley, Charles H.; Bernal, Adriana J.; Michelmore, Richard W.

    2005-01-01

    Disease resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) in the cultivated tomato, Lycopersicon esculentum, and the closely related L. pimpinellifolium is triggered by the physical interaction between plant disease resistance protein, Pto, and the pathogen avirulence protein, AvrPto. To investigate the extent to which variation in the Pto gene is responsible for naturally occurring variation in resistance to Pst, we determined the resistance phenotype of 51 accessions from seven species of Lycopersicon to isogenic strains of Pst differing in the presence of avrPto. One-third of the plants displayed resistance specifically when the pathogen expressed AvrPto, consistent with a gene-for-gene interaction. To test whether this resistance in these species was conferred specifically by the Pto gene, alleles of Pto were amplified and sequenced from 49 individuals and a subset (16) of these alleles was tested in planta using Agrobacterium-mediated transient assays. Eleven alleles conferred a hypersensitive resistance response (HR) in the presence of AvrPto, while 5 did not. Ten amino acid substitutions associated with the absence of AvrPto recognition and HR were identified, none of which had been identified in previous structure-function studies. Additionally, 3 alleles encoding putative pseudogenes of Pto were isolated from two species of Lycopersicon. Therefore, a large proportion, but not all, of the natural variation in the reaction to strains of Pst expressing AvrPto can be attributed to sequence variation in the Pto gene. PMID:15944360

  13. Natural variation in the Pto pathogen resistance gene within species of wild tomato (Lycopersicon). I. Functional analysis of Pto alleles.

    PubMed

    Rose, Laura E; Langley, Charles H; Bernal, Adriana J; Michelmore, Richard W

    2005-09-01

    Disease resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) in the cultivated tomato, Lycopersicon esculentum, and the closely related L. pimpinellifolium is triggered by the physical interaction between plant disease resistance protein, Pto, and the pathogen avirulence protein, AvrPto. To investigate the extent to which variation in the Pto gene is responsible for naturally occurring variation in resistance to Pst, we determined the resistance phenotype of 51 accessions from seven species of Lycopersicon to isogenic strains of Pst differing in the presence of avrPto. One-third of the plants displayed resistance specifically when the pathogen expressed AvrPto, consistent with a gene-for-gene interaction. To test whether this resistance in these species was conferred specifically by the Pto gene, alleles of Pto were amplified and sequenced from 49 individuals and a subset (16) of these alleles was tested in planta using Agrobacterium-mediated transient assays. Eleven alleles conferred a hypersensitive resistance response (HR) in the presence of AvrPto, while 5 did not. Ten amino acid substitutions associated with the absence of AvrPto recognition and HR were identified, none of which had been identified in previous structure-function studies. Additionally, 3 alleles encoding putative pseudogenes of Pto were isolated from two species of Lycopersicon. Therefore, a large proportion, but not all, of the natural variation in the reaction to strains of Pst expressing AvrPto can be attributed to sequence variation in the Pto gene.

  14. Host haemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene.

    PubMed

    Asgari, S; Hellers, M; Schmidt, O

    1996-10-01

    Polydnaviruses produced by the hymenopteran endoparasitoid Cotesia rubecula are deposited together with the egg into the lepidopteran host Pieris rapae and are apparently involved in the suppression of the host's defence system. Around 6 h post-parasitization host haemocytes change their surface properties, actin cytoskeleton structure and adhesion properties. Here we show that a single polydnavirus gene is expressed inside the caterpillar haemocytes in a transient fashion between 4 to 8 h post-parasitization.

  15. [Transient congenital hypothyroidism due to biallelic defects of DUOX2 gene. Two clinical cases].

    PubMed

    Enacán, Rosa E; Masnata, María E; Belforte, Fiorella; Papendieck, Patricia; Olcese, María C; Siffo, Sofía; Gruñeiro-Papendieck, Laura; Targovnik, Héctor; Rivolta, Carina M; Chiesa, Ana E

    2017-06-01

    Congenital hypothyroidism affects 1:2000-3000 newborns detected by neonatal screening programs. Dual oxidases, DUOX1 and 2, generate hydrogen peroxide needed for the thyroid hormone synthesis. Hipotiroidismo congénito transitorio por defectos bialélicos del gen DUOX2. Dos casos clínicos Transient congenital hypothyroidism due to biallelic defects of DUOX2 gene. Two clinical cases Mutations in the DUOX2 gene have been described in transient and permanent congenital hypothyroidism. Two brothers with congenital hypothyroidism detected by neonatal screening with eutopic gland and elevated thyroglobulin are described. They were treated with levothyroxine until it could be suspended in both during childhood, assuming the picture as transient. Organification disorder was confirmed. Both patients were compounds heterozygous for a mutation in exon 9 of the paternal allele (c.1057_1058delTT, p.F353PfsX36 or p.F353fsX388) and in exon 11 of the maternal allele (c.1271T > G, p.Y425X) of DUOX2 gene. Our finding confirms that the magnitude of the defect of DUOX2 is not related to the number of inactivated alleles, suggesting compensatory mechanisms in the peroxide supply. Sociedad Argentina de Pediatría.

  16. Study on transient expression of gus gene in Chlorelia ellipsoidea (Chlorophyta) by using biolistic particle delivery system

    NASA Astrophysics Data System (ADS)

    Chen, Ying; Li, Wen-Bin; Bai, Qin-Hua; Sun, Yong-Ru

    1998-03-01

    Study on the transient expression of GUS gene at different growing stage of Chlorella ellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between microprojectile and target cell, bombardment times, are reported in this paper. The results showed that C. ellipsoidea in exponential phase has higer level of transient expression and that treatment with osmosis can improve the GUS transient expression notably. The effect of distance or bombardment times was not observed.

  17. Increased gene targeting in Ku70 and Xrcc4 transiently deficient human somatic cells.

    PubMed

    Bertolini, Luciana R; Bertolini, Marcelo; Maga, Elizabeth A; Madden, Knut R; Murray, James D

    2009-02-01

    The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell's genome is important to advance strategies for biotechnology and genetic medicine.

  18. Selenite transiently represses transcription of photosynthesis-related genes in potato leaves.

    PubMed

    Poggi, Valeria; Del Vescovo, Valerio; Di Sanza, Claudio; Negri, Rodolfo; Hochkoeppler, Alejandro

    2008-01-01

    A striking response of potato leaves to aspersion with selenite was observed at the transcriptional level by means of cDNA microarrays analysis. This response is characterized by a general transient repression of genes coding for components of photosynthetic systems and of other light-regulated genes. In particular, maximal repression was observed 8 h after selenite aspersion, while 24 h after the treatment a complete recovery of normal transcriptional levels was detected. Another general feature of the transcriptional response to selenite is represented by the transcriptional induction of genes related to amino acid metabolism, and to stress defense; interestingly, two genes coding for glutathione S-transferases were found early-induced upon selenite treatment.

  19. Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses.

    PubMed

    Kuate, Seraphin; Stefanou, Daniela; Hoffmann, Dennis; Wildner, Oliver; Uberla, Klaus

    2004-11-01

    The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.

  20. Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia

    PubMed Central

    El-Osta, Assam; Brasacchio, Daniella; Yao, Dachun; Pocai, Alessandro; Jones, Peter L.; Roeder, Robert G.; Cooper, Mark E.; Brownlee, Michael

    2008-01-01

    The current goal of diabetes therapy is to reduce time-averaged mean levels of glycemia, measured as HbA1c, to prevent diabetic complications. However, HbA1c only explains <25% of the variation in risk of developing complications. Because HbA1c does not correlate with glycemic variability when adjusted for mean blood glucose, we hypothesized that transient spikes of hyperglycemia may be an HbA1c–independent risk factor for diabetic complications. We show that transient hyperglycemia induces long-lasting activating epigenetic changes in the promoter of the nuclear factor κB (NF-κB) subunit p65 in aortic endothelial cells both in vitro and in nondiabetic mice, which cause increased p65 gene expression. Both the epigenetic changes and the gene expression changes persist for at least 6 d of subsequent normal glycemia, as do NF-κB–induced increases in monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 expression. Hyperglycemia-induced epigenetic changes and increased p65 expression are prevented by reducing mitochondrial superoxide production or superoxide-induced α-oxoaldehydes. These results highlight the dramatic and long-lasting effects that short-term hyperglycemic spikes can have on vascular cells and suggest that transient spikes of hyperglycemia may be an HbA1c–independent risk factor for diabetic complications. PMID:18809715

  1. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector

    PubMed Central

    Condreay, J. Patrick; Witherspoon, Sam M.; Clay, William C.; Kost, Thomas A.

    1999-01-01

    Recombinant baculoviruses can serve as gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types. Furthermore, by inclusion of a dominant selectable marker in the viral vector, cell lines can be derived that stably express recombinant genes. A virus was constructed containing two expression cassettes controlled by constitutive mammalian promoters: the cytomegalovirus immediate early promoter/enhancer directing expression of green fluorescent protein and the simian virus 40 (SV40) early promoter controlling neomycin phosphotransferase II. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. In addition to commonly used transformed cell lines such as HeLa, CHO, Cos-7, and 293, this list includes primary human keratinocytes and bone marrow fibroblasts. In all cases, addition of butyrate or trichostatin A (a selective histone deacetylase inhibitor) to transduced cells markedly enhanced the levels of reporter protein expression observed. When transduced cells are put under selection with the antibiotic G418, cell lines can be obtained at high frequency that stably maintain the expression cassettes of the vector DNA and exhibit stable, high-level expression of the reporter gene. Stably transduced derivatives have been selected from a substantial number of different cell types, suggesting that stable lines can be derived from any cell type that exhibits transient expression. PMID:9874783

  2. Tyrosine triple mutated AAV2-BDNF gene therapy in a rat model of transient IOP elevation

    PubMed Central

    Igarashi, Tsutomu; Kobayashi, Maika; Kameya, Shuhei; Fujimoto, Chiaki; Nakamoto, Kenji; Takahashi, Hisatomo; Igarashi, Toru; Miyake, Noriko; Iijima, Osamu; Hirai, Yukihiko; Shimada, Takashi; Okada, Takashi; Takahashi, Hiroshi

    2016-01-01

    Purpose We examined the neuroprotective effects of exogenous brain-derived neurotrophic factor (BDNF), which provides protection to retinal ganglion cells (RGCs) in rodents, in a model of transient intraocular pressure (IOP) elevation using a mutant (triple Y-F) self-complementary adeno-associated virus type 2 vector encoding BDNF (tm-scAAV2-BDNF). Methods The tm-scAAV2-BDNF or control vector encoding green fluorescent protein (GFP; tm-scAAV2-GFP) was intravitreally administered to rats, which were then divided into four groups: control, ischemia/reperfusion (I/R) injury only, I/R injury with tm-scAAV2-GFP, and tm-scAAV2-BDNF. I/R injury was then induced by transiently increasing IOP, after which the rats were euthanized to measure the inner retinal thickness and cell counts in the RGC layer. Results Intravitreous injection of tm-scAAV2-BDNF resulted in high levels of BDNF expression in the neural retina. Histological analysis showed that the inner retinal thickness and cell numbers in the RGC layer were preserved after transient IOP elevation in eyes treated with tm-scAAV2-BDNF but not in the other I/R groups. Significantly reduced glial fibrillary acidic protein (GFAP) immunostaining after I/R injury in the rats that received tm-scAAV2-BDNF indicated reduced retinal stress, and electroretinogram (ERG) analysis confirmed preservation of retinal function in the tm-scAAV2-BDNF group. Conclusions These results demonstrate the feasibility and effectiveness of neuroprotective gene therapy using tm-scAAV2-BDNF to protect the inner retina from transiently high intraocular pressure. An in vivo gene therapeutic approach to the clinical management of retinal diseases in conditions such as glaucoma, retinal artery occlusion, hypertensive retinopathy, and diabetic retinopathy thus appears feasible. PMID:27440998

  3. Molecular cloning and functional characterization of Catharanthus roseus hydroxymethylbutenyl 4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway.

    PubMed

    Ginis, Olivia; Courdavault, Vincent; Melin, Céline; Lanoue, Arnaud; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Courtois, Martine; Oudin, Audrey

    2012-05-01

    The Madagascar periwinkle produces monoterpenoid indole alkaloids (MIA) of high interest due to their therapeutical values. The terpenoid moiety of MIA is derived from the methyl erythritol phosphate (MEP) and seco-iridoid pathways. These pathways are regarded as the limiting branch for MIA biosynthesis in C. roseus cell and tissue cultures. In previous studies, we demonstrated a coordinated regulation at the transcriptional and spatial levels of genes from both pathways. We report here on the isolation of the 5'-flanking region (1,049 bp) of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene from the MEP pathway. To investigate promoter transcriptional activities, the HDS promoter was fused to GUS reporter gene. Agrobacterium-mediated transformation of young tobacco leaves revealed that the cloned HDS promoter displays a tissue-specific GUS staining restricted to the vascular region of the leaves and limited to a part of the vein that encompasses the phloem in agreement with the previous localization of HDS transcripts in C. roseus aerial organs. Further functional characterizations in stably or transiently transformed C. roseus cells allowed us to identify the region that can be consider as the minimal promoter and to demonstrate the induction of HDS promoter by several hormonal signals (auxin, cytokinin, methyljasmonate and ethylene) leading to MIA production. These results, and the bioinformatic analysis of the HDS 5'-region, suggest that the HDS promoter harbours a number of cis-elements binding specific transcription factors that would regulate the flux of terpenoid precursors involved in MIA biosynthesis.

  4. Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

    PubMed Central

    Sohaskey, C D; Arnold, C; Barbour, A G

    1997-01-01

    A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system. PMID:9352937

  5. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

    PubMed

    Cervera, Laura; Gutiérrez-Granados, Sonia; Berrow, Nicholas Simon; Segura, Maria Mercedes; Gòdia, Francesc

    2015-05-01

    Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered. © 2014 Wiley Periodicals, Inc.

  6. Overexpression of barley hva1 gene in creeping bentgrass for improving drought tolerance.

    PubMed

    Fu, Daolin; Huang, Bingru; Xiao, Yanmei; Muthukrishnan, Subbaratnam; Liang, George H

    2007-04-01

    The objectives of this study were to test the feasibility of introducing barley hva1 gene, a LEA3 member, into perennial grass species using the Agrobacterium-mediated transformation technique and to determine whether heterologous expression of hva1 would alleviate water-deficit injury in grass species. Creeping bentgrass (Agrostis stolonifera var. palustris), a drought-intolerant grass species, was transformed transiently or stably using three different promoters in conjunction with the downstream report/target genes. Two abscisic acid (ABA)-inducible promoters, ABA1 and ABA2 derived from ABA-response complex (ABRC3) were used to examine stress-responsive expression of the green fluorescent protein (GFP). Transient expression of GFP demonstrated the inducibility of ABA1 and ABA2 promoters in response to exogenous ABA application. The ABA2 promoter was further studied for stress-responsive expression of hva1 and a maize Ubi-1 promoter was tested for constitutive expression of the gene. In the T(0) generation, the Ubi-1::hva1 transformants displayed variable expression levels of HVA1 protein under normal growth conditions. The hva1 gene in the ABA2::hva1 transformants maintained low expression under well-watered conditions, but was upregulated under water-deficit conditions. The tolerance to water deficit of T(0) transgenic lines was assessed by measuring leaf relative water content and visually rating the severity of leaf wilting during to water stress. Under water-stressed conditions, some transgenic lines maintained high water content in leaves and showed significantly less extent of leaf wilting compared with non-transgenic control plants. These results indicated that the introduction of barley hva1 gene using constitutive or stress-inducible promoters lessened water-deficit injury in creeping bentgrass, suggesting that heterologous expression of LEA3 protein genes may enhance the survival ability of creeping bentgrass in water limiting environments.

  7. Development of molecular resistance in potato against potato leaf roll virus and potato virus Y through Agrobacterium-mediated double transgenesis

    USDA-ARS?s Scientific Manuscript database

    Potato leafroll virus (PLRV) and potato virus Y (PVY) are the two major viral problems for the potato production all over the world. Transgenic approaches involving the expression of viral genes are being developed to provide protection for plants against viral diseases. The purpose of this study w...

  8. Establishment of transient gene expression systems in protoplasts from Liriodendron hybrid mesophyll cells.

    PubMed

    Huo, Ailing; Chen, Zhenyu; Wang, Pengkai; Yang, Liming; Wang, Guangping; Wang, Dandan; Liao, Suchan; Cheng, Tielong; Chen, Jinhui; Shi, Jisen

    2017-01-01

    Liriodendron is a genus of the magnolia family comprised of two flowering tree species that produce hardwoods of great ecological and economic value. However, only a limited amount of genetic research has been performed on the Liriodendron genus partly because transient or stable transgenic trees have been difficult to produce. In general, transient expression systems are indispensable for rapid, high-throughput screening and systematic characterization of gene functions at a low cost; therefore, development of such a system for Liriodendron would provide a necessary step forward for research on Magnoliaceae and other woody trees. Herein, we describe an efficient and rapid protocol for preparing protoplasts from the leaf mesophyll tissue of a Liriodendron hybrid and an optimized system for polyethylene glycol-mediated transient transfection of the protoplasts. Because the leaves of the Liriodendron hybrid are waxy, we formulated an enzyme mix containing 1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10, and 0.1% (w/v) Pectolyase Y-23 to efficiently isolate protoplasts from the Liriodendron hybrid leaf mesophyll tissue in 3 h. We optimized Liriodendron protoplast transfection efficiency by including 20 μg plasmid DNA per 104 protoplasts, a transformation time of 20 min, and inclusion of 20% (w/v) polyethylene glycol 4000. After integrating the Liriodendron WOX1 gene into pJIT166-GFP to produce a WOX1-GFP fusion product and transfecting it into isolated protoplasts, LhWOX1-GFP was found to localize to the nucleus according to its green fluorescence.

  9. Establishment of transient gene expression systems in protoplasts from Liriodendron hybrid mesophyll cells

    PubMed Central

    Huo, Ailing; Chen, Zhenyu; Wang, Pengkai; Yang, Liming; Wang, Guangping; Wang, Dandan; Liao, Suchan; Cheng, Tielong; Chen, Jinhui; Shi, Jisen

    2017-01-01

    Liriodendron is a genus of the magnolia family comprised of two flowering tree species that produce hardwoods of great ecological and economic value. However, only a limited amount of genetic research has been performed on the Liriodendron genus partly because transient or stable transgenic trees have been difficult to produce. In general, transient expression systems are indispensable for rapid, high-throughput screening and systematic characterization of gene functions at a low cost; therefore, development of such a system for Liriodendron would provide a necessary step forward for research on Magnoliaceae and other woody trees. Herein, we describe an efficient and rapid protocol for preparing protoplasts from the leaf mesophyll tissue of a Liriodendron hybrid and an optimized system for polyethylene glycol–mediated transient transfection of the protoplasts. Because the leaves of the Liriodendron hybrid are waxy, we formulated an enzyme mix containing 1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10, and 0.1% (w/v) Pectolyase Y-23 to efficiently isolate protoplasts from the Liriodendron hybrid leaf mesophyll tissue in 3 h. We optimized Liriodendron protoplast transfection efficiency by including 20 μg plasmid DNA per 104 protoplasts, a transformation time of 20 min, and inclusion of 20% (w/v) polyethylene glycol 4000. After integrating the Liriodendron WOX1 gene into pJIT166-GFP to produce a WOX1-GFP fusion product and transfecting it into isolated protoplasts, LhWOX1-GFP was found to localize to the nucleus according to its green fluorescence. PMID:28323890

  10. Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.

    PubMed

    Cheng, Yun-Qing; Yang, Jun; Xu, Feng-Ping; An, Li-Jia; Liu, Jian-Feng; Chen, Zhi-Wen

    2009-12-01

    A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.

  11. Hypoxia drives transient site-specific copy gain and drug-resistant gene expression

    PubMed Central

    Black, Joshua C.; Atabakhsh, Elnaz; Kim, Jaegil; Biette, Kelly M.; Van Rechem, Capucine; Ladd, Brendon; Burrowes, Paul d.; Donado, Carlos; Mattoo, Hamid; Kleinstiver, Benjamin P.; Song, Bing; Andriani, Grasiella; Joung, J. Keith; Iliopoulos, Othon; Montagna, Cristina; Pillai, Shiv; Getz, Gad

    2015-01-01

    Copy number heterogeneity is a prominent feature within tumors. The molecular basis for this heterogeneity remains poorly characterized. Here, we demonstrate that hypoxia induces transient site-specific copy gains (TSSGs) in primary, nontransformed, and transformed human cells. Hypoxia-driven copy gains are not dependent on HIF1α or HIF2α; however, they are dependent on the KDM4A histone demethylase and are blocked by inhibition of KDM4A with a small molecule or the natural metabolite succinate. Furthermore, this response is conserved at a syntenic region in zebrafish cells. Regions with site-specific copy gain are also enriched for amplifications in hypoxic primary tumors. These tumors exhibited amplification and overexpression of the drug resistance gene CKS1B, which we recapitulated in hypoxic breast cancer cells. Our results demonstrate that hypoxia provides a biological stimulus to create transient site-specific copy alterations that could result in heterogeneity within tumors and cell populations. These findings have major implications in our understanding of copy number heterogeneity and the emergence of drug resistance genes in cancer. PMID:25995187

  12. Hypoxia drives transient site-specific copy gain and drug-resistant gene expression.

    PubMed

    Black, Joshua C; Atabakhsh, Elnaz; Kim, Jaegil; Biette, Kelly M; Van Rechem, Capucine; Ladd, Brendon; Burrowes, Paul D; Donado, Carlos; Mattoo, Hamid; Kleinstiver, Benjamin P; Song, Bing; Andriani, Grasiella; Joung, J Keith; Iliopoulos, Othon; Montagna, Cristina; Pillai, Shiv; Getz, Gad; Whetstine, Johnathan R

    2015-05-15

    Copy number heterogeneity is a prominent feature within tumors. The molecular basis for this heterogeneity remains poorly characterized. Here, we demonstrate that hypoxia induces transient site-specific copy gains (TSSGs) in primary, nontransformed, and transformed human cells. Hypoxia-driven copy gains are not dependent on HIF1α or HIF2α; however, they are dependent on the KDM4A histone demethylase and are blocked by inhibition of KDM4A with a small molecule or the natural metabolite succinate. Furthermore, this response is conserved at a syntenic region in zebrafish cells. Regions with site-specific copy gain are also enriched for amplifications in hypoxic primary tumors. These tumors exhibited amplification and overexpression of the drug resistance gene CKS1B, which we recapitulated in hypoxic breast cancer cells. Our results demonstrate that hypoxia provides a biological stimulus to create transient site-specific copy alterations that could result in heterogeneity within tumors and cell populations. These findings have major implications in our understanding of copy number heterogeneity and the emergence of drug resistance genes in cancer. © 2015 Black et al.; Published by Cold Spring Harbor Laboratory Press.

  13. Plant–Agrobacterium interaction mediated by ethylene and super-Agrobacterium conferring efficient gene transfer

    PubMed Central

    Nonaka, Satoko; Ezura, Hiroshi

    2014-01-01

    Agrobacterium tumefaciens has a unique ability to transfer genes into plant genomes. This ability has been utilized for plant genetic engineering. However, the efficiency is not sufficient for all plant species. Several studies have shown that ethylene decreased the Agrobacterium-mediated transformation frequency. Thus, A. tumefaciens with an ability to suppress ethylene evolution would increase the efficiency of Agrobacterium-mediated transformation. Some studies showed that plant growth-promoting rhizobacteria (PGPR) can reduce ethylene levels in plants through 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which cleaves the ethylene precursor ACC into α-ketobutyrate and ammonia, resulting in reduced ethylene production. The whole genome sequence data showed that A. tumefaciens does not possess an ACC deaminase gene in its genome. Therefore, providing ACC deaminase activity to the bacteria would improve gene transfer. As expected, A. tumefaciens with ACC deaminase activity, designated as super-Agrobacterium, could suppress ethylene evolution and increase the gene transfer efficiency in several plant species. In this review, we summarize plant–Agrobacterium interactions and their applications for improving Agrobacterium-mediated genetic engineering techniques via super-Agrobacterium. PMID:25520733

  14. Transient gene expression in tobacco using Gibson assembly and the Gene Gun.

    PubMed

    Mattozzi, Matthew D; Voges, Mathias J; Silver, Pamela A; Way, Jeffrey C

    2014-04-18

    In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5' mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work(11), and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

  15. Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

    PubMed Central

    Mattozzi, Matthew D.; Voges, Mathias J.; Silver, Pamela A.; Way, Jeffrey C.

    2014-01-01

    In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5’ mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy. PMID:24796418

  16. Exercise induces a transient increase in transcription of the GLUT-4 gene in skeletal muscle.

    PubMed

    Neufer, P D; Dohm, G L

    1993-12-01

    Endurance exercise training elicits an increase in mitochondrial density as well as GLUT-4 glucose transporter protein content in skeletal muscle. Corresponding increases in mRNA for respiratory enzymes and GLUT-4 indicate that pretranslational control mechanisms are involved in this adaptive process. To directly test whether transcription of the GLUT-4 gene is activated in response to exercise training, nuclei were isolated from red hindlimb skeletal muscle of rats after 1 wk of exercise training (8% grade, 32 m/min, 40 min, twice/day). Rats were killed either 30 min, 3 h, or 24 h after the last training session. GLUT-4 transcription, determined by nuclear run-on analysis, was unaltered after 30 min, increased by 1.8-fold after 3 h, but was no longer different from controls 24 h after exercise. A similar transient increase in GLUT-4 transcription was evident, but less pronounced (1.4-fold), in untrained rats after a single bout of exercise, suggesting that the postexercise induction in GLUT-4 gene transcription is enhanced by exercise training. GLUT-4 protein content was increased 1.7-fold after 1 wk of training in the absence of any corresponding change in GLUT-4 mRNA, providing evidence that the initial increase in GLUT-4 expression involves translational and/or posttranslational control mechanisms. These findings demonstrate that muscle GLUT-4 expression in response to exercise training is subject to both transcriptional and posttranscriptional regulation. We propose that the increase in GLUT-4 mRNA evident with extended periods of training may result from a shift to pretranslational control and is the cumulative effect of repeated postexercise transient increases in GLUT-4 gene transcription.

  17. Mungbean yellow mosaic virus (MYMV) AC4 suppresses post-transcriptional gene silencing and an AC4 hairpin RNA gene reduces MYMV DNA accumulation in transgenic tobacco.

    PubMed

    Sunitha, Sukumaran; Shanmugapriya, Gnanasekaran; Balamani, Veluthambi; Veluthambi, Karuppannan

    2013-06-01

    Mungbean yellow mosaic virus (MYMV) is a legume-infecting geminivirus that causes yellow mosaic disease in blackgram, mungbean, soybean, Frenchbean and mothbean. AC4/C4, which is nested completely within the Rep gene, is less conserved among geminiviruses. Much less is known about its role in viral pathogenesis other than its known role in the suppression of host-mediated gene silencing. Transient expression of MYMV AC4 by agroinfiltration suppressed post-transcriptional gene silencing in Nicotiana benthamiana 16c expressing green fluorescence protein, at a level comparable to MYMV TrAP expression. AC4 full-length gene and an inverted repeat of AC4 (comprising the full-length AC4 sequence in sense and antisense orientations with an intervening intron) which makes a hairpin RNA (hpRNA) upon transcription were introduced into tobacco by Agrobacterium-mediated leaf disc transformation. Leaf discs of the transgenic plants were agroinoculated with partial dimers of MYMV and used to study the effect of the AC4-sense and AC4 hpRNA genes on MYMV DNA accumulation. Leaf discs of two transgenic plants that express the AC4-sense gene displayed an increase in MYMV DNA accumulation. Leaf discs of six transgenic plants containing the AC4 hpRNA gene accumulated small-interfering RNAs (siRNAs) specific to AC4, and upon agroinoculation with MYMV they exhibited a severe reduction in the accumulation of MYMV DNA. Thus, the MYMV AC4 hpRNA gene has emerged as a good candidate to engineer resistance against MYMV in susceptible plants.

  18. Transient gene therapy to treat cutaneous radiation syndrome: development in a minipig model.

    PubMed

    Riccobono, Diane; Forcheron, Fabien; Agay, Diane; Scherthan, Harry; Meineke, Viktor; Drouet, Michel

    2014-06-01

    Cutaneous radiation syndrome is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Adipocyte derived stem cells injection may improve tissue regeneration through secreted factors. Thus mesenchymal stem cells secretome optimization, using transient transfection, may represent a new strategy to treat this syndrome. Sonic hedgehog, a secreted protein involved in cell proliferation and angiogenesis, has been chosen as a first candidate. Here preliminary results are reported of the therapeutic potential of transient gene therapy to cure cutaneous radiation syndrome in a minipig model. Adipocyte derived stem cells were transiently transfected by electroporation with a plasmid coding for Sonic Hedgehog. Göttingen minipigs were locally irradiated using a (60)Co gamma source at the dose of 50 Gy and received Phosphate Buffer Salin (controls: n = 8), stem cells (50 × 10⁶ each time, n = 5) or transfected stem cells (25±7 × 10⁶ each time, n = 1). All controls exhibited a homogeneous clinical evolution of cutaneous radiation syndrome with final necrosis (day 91). In stem cell injected minipigs, an ultimate wound healing was observed in four out of five grafted animals (day 130 ± 28, complete in two of them) (historical results). The Sonic hedgehog animal, albeit injected with a lower number of transfected stem cells, presented a very similar evolution of skin healing without necrosis or uncontrolble pain. Globally this preliminary report suggests that local injection of Sonic Hedgehog transfected adipocyte derived stem cells may improve wound healing. Thus work is ongoing to evaluate this therapeutic strategy on a larger number of animals.

  19. Transient genotype-by-environment interactions following environmental shock provide a source of expression variation for essential genes.

    PubMed

    Eng, Kevin H; Kvitek, Daniel J; Keles, Sündüz; Gasch, Audrey P

    2010-02-01

    Understanding complex genotype-by-environment interactions (GEIs) is crucial for understanding phenotypic variation. An important factor often overlooked in GEI studies is time. We measured the contribution of GEIs to expression variation in four nonlaboratory Saccharomyces cerevisiae strains responding dynamically to a 25 degrees -37 degrees heat shock. GEI was a major force explaining expression variation, affecting 55% of the genes analyzed. Importantly, almost half of these expression patterns showed GEI influence only during the transition between environments, but not in acclimated cells. This class reveals a genotype-by-environment-by-time interaction that affected expression of a large fraction of yeast genes. Strikingly, although transcripts subject to persistent GEI effects were enriched for nonessential genes with upstream TATA elements, those displaying transient GEIs were enriched for essential genes regardless of TATA regulation. Genes subject to persistent GEI influences showed relaxed constraint on acclimated gene expression compared to the average yeast gene, whereas genes restricted to transient GEIs did not. We propose that transient GEI during the transition between environments provides a previously unappreciated source of expression variation, particularly for essential genes.

  20. Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells.

    PubMed

    Meissner, P; Pick, H; Kulangara, A; Chatellard, P; Friedrich, K; Wurm, F M

    2001-10-20

    Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.

  1. Transient and stable transfections of mouse myoblasts with genes coding for pro-angiogenic factors.

    PubMed

    Bialas, M; Krupka, M; Janeczek, A; Rozwadowska, N; Fraczek, M; Kotlinowski, J; Kucharzewska, P; Lackowska, B; Kurpisz, M

    2011-04-01

    Cardiomyocyte loss in the ischaemic heart can be the reason of many complications, eventually being even the cause of patient's death. Despite many promises, cell therapy with the use of skeletal muscle stem cells (SMSC) still remains to be modified and improved. Combined cell and gene therapy seems to be a promising strategy to heal damaged myocardium. In the present study we have investigated the influence of a simultaneous overexpression of two potent pro-angiogenic genes encoding the fibroblast growth factor-4 (FGF-4) and the vascular endothelial growth factor-A (VEGF-A) on a myogenic murine C2C12 cell line. We have demonstrated in in vitro conditions that myoblasts which overexpressed these factors exhibited significant changes in the cell cycle and pro-angiogenic potential with only slight differences in the expression of the myogenic genes. There was not observed the influence of transient or stable overexpression of FGF-4 and VEGF on cell apoptosis/necrosis in standard or oxidative stress conditions comparing to non transfected controls. Overall, our results suggest that the possible transplantation of myoblasts overexpressing pro-angiogenic factors may potentially improve the functionality of the injured myocardium although the definite proof must originate from in situ conducted pre-clinical studies.

  2. Characterization of the sporophyte-preferential gene promoter from the red alga Porphyra yezoensis using transient gene expression.

    PubMed

    Uji, Toshiki; Mizuta, Hiroyuki; Saga, Naotsune

    2013-04-01

    The life cycle of plants entails an alternation of generations, the diploid sporophyte and haploid gametophyte stages. There is little information about the characteristics of gene expression during each phase of marine macroalgae. Promoter analysis is a useful method for understanding transcriptional regulation; however, there is no report of promoter analyses in marine macroalgae. In this study, with the aim of elucidating the differences in the transcriptional regulatory mechanisms between the gametophyte and sporophyte stages in the marine red alga Porphyra yezoensis, we isolated the promoter from the sporophyte preferentially expressed gene PyKPA1, which encodes a sodium pump, and analyzed its promoter using a transient gene expression system with a synthetic β-glucuronidase (PyGUS) reporter. The deletion of -1432 to -768 relative to the transcription start site resulted in decreased GUS activity in sporophytes. In contrast, deletion from -767 to -527 increased GUS activity in gametophytes. Gain-of-function analyses showed that the -1432 to -760 region enhanced the GUS activity of a heterologous promoter in sporophytes, whereas the -767 to -510 region repressed it in gametophytes. Further mutation and gain-of-function analyses of the -767 to -510 region revealed that a 20-bp GC-rich sequence (-633 to -614) is responsible for the gametophyte-specific repressed expression. These results showed that the sporophyte-specific positive regulatory region and gametophyte-specific negative regulatory sequence play a crucial role in the preferential expression of PyKPA1 in P. yezoensis sporophytes.

  3. Transient early wheeze and lung function in early childhood associated with chronic obstructive pulmonary disease genes.

    PubMed

    Kerkhof, Marjan; Boezen, H Marike; Granell, Raquel; Wijga, Alet H; Brunekreef, Bert; Smit, Henriëtte A; de Jongste, Johan C; Thijs, Carel; Mommers, Monique; Penders, John; Henderson, John; Koppelman, Gerard H; Postma, Dirkje S

    2014-01-01

    It has been hypothesized that a disturbed early lung development underlies the susceptibility to chronic obstructive pulmonary disease (COPD). Little is known about whether subjects genetically predisposed to COPD show their first symptoms or reduced lung function in childhood. We investigated whether replicated genes for COPD associate with transient early wheeze (TEW) and lung function levels in 6- to 8-year-old children and whether cigarette smoke exposure in utero and after birth (environmental tobacco smoke [ETS]) modifies these effects. The association of COPD-related genotypes of 20 single nucleotide polymorphisms in 15 genes with TEW, FEV1, forced vital capacity (FVC), and FEV1/FVC ratio was studied in the Prevention and Incidence of Asthma and Mite Allergy (PIAMA) birth cohort (n = 1996) and replicated in the Child, parents and health: lifestyle and genetic constitution (KOALA) and Avon Longitudinal Study of Parents and Children (ALSPAC) cohorts. AGER showed replicated association with FEV1/FVC ratio. TNS1 associated with more TEW in PIAMA and lower FEV1 in ALSPAC. TNS1 interacted with ETS in PIAMA, showing lower FEV1 in exposed children. HHIP rs1828591 interacted with cigarette smoke exposure in utero in PIAMA and with ETS in ALSPAC, with lower lung function in nonexposed children. SERPINE2, FAM13A, and MMP12 associated with higher FEV1 and FVC, and SERPINE2, HHIP, and TGFB1 interacted with cigarette smoke exposure in utero in PIAMA only, showing adverse effects of exposure on FEV1 being limited to children with genotypes conferring the lowest risk of COPD. Our findings indicate relevant involvement of at least 3 COPD genes in lung development and lung growth by demonstrating associations pointing toward reduced airway caliber in early childhood. Furthermore, our results suggest that COPD genes are involved in the infant's lung response to smoke exposure in utero and in early life. Copyright © 2013 The Authors. Published by Mosby, Inc. All rights

  4. Identification of novel pepper genes involved in Bax- or INF1-mediated cell death responses by high-throughput virus-induced gene silencing.

    PubMed

    Lee, Jeong Hee; Kim, Young Cheol; Choi, Doil; Park, Jeong Mee

    2013-11-19

    Hot pepper is one of the economically important crops in Asia. A large number of gene sequences, including expressed sequence tag (EST) and genomic sequences are publicly available. However, it is still a daunting task to determine gene function due to difficulties in genetic modification of a pepper plants. Here, we show the application of the virus-induced gene silencing (VIGS) repression for the study of 459 pepper ESTs selected as non-host pathogen-induced cell death responsive genes from pepper microarray experiments in Nicotiana benthamiana. Developmental abnormalities in N. benthamiana plants are observed in the 32 (7%) pepper ESTs-silenced plants. Aberrant morphological phenotypes largely comprised of three groups: stunted, abnormal leaf, and dead. In addition, by employing the combination of VIGS and Agrobacterium-mediated transient assays, we identified novel pepper ESTs that involved in Bax or INF1-mediated cell death responses. Silencing of seven pepper ESTs homologs suppressed Bax or INF1-induced cell death, five of which suppressed both cell death responses in N. benthamiana. The genes represented by these five ESTs encode putative proteins with functions in endoplasmic reticulum (ER) stress and lipid signaling. The genes represented by the other two pepper ESTs showing only Bax-mediated cell death inhibition encode a CCCH-type zinc finger protein containing an ankyrin-repeat domain and a probable calcium-binding protein, CML30-like. Taken together, we effectively isolated novel pepper clones that are involved in hypersensitive response (HR)-like cell death using VIGS, and identified silenced clones that have different responses to Bax and INF1 exposure, indicating separate signaling pathways for Bax- and INF1-mediated cell death.

  5. Identification of Novel Pepper Genes Involved in Bax- or INF1-Mediated Cell Death Responses by High-Throughput Virus-Induced Gene Silencing

    PubMed Central

    Lee, Jeong Hee; Kim, Young Cheol; Choi, Doil; Park, Jeong Mee

    2013-01-01

    Hot pepper is one of the economically important crops in Asia. A large number of gene sequences, including expressed sequence tag (EST) and genomic sequences are publicly available. However, it is still a daunting task to determine gene function due to difficulties in genetic modification of a pepper plants. Here, we show the application of the virus-induced gene silencing (VIGS) repression for the study of 459 pepper ESTs selected as non-host pathogen-induced cell death responsive genes from pepper microarray experiments in Nicotiana benthamiana. Developmental abnormalities in N. benthamiana plants are observed in the 32 (7%) pepper ESTs-silenced plants. Aberrant morphological phenotypes largely comprised of three groups: stunted, abnormal leaf, and dead. In addition, by employing the combination of VIGS and Agrobacterium-mediated transient assays, we identified novel pepper ESTs that involved in Bax or INF1-mediated cell death responses. Silencing of seven pepper ESTs homologs suppressed Bax or INF1-induced cell death, five of which suppressed both cell death responses in N. benthamiana. The genes represented by these five ESTs encode putative proteins with functions in endoplasmic reticulum (ER) stress and lipid signaling. The genes represented by the other two pepper ESTs showing only Bax-mediated cell death inhibition encode a CCCH-type zinc finger protein containing an ankyrin-repeat domain and a probable calcium-binding protein, CML30-like. Taken together, we effectively isolated novel pepper clones that are involved in hypersensitive response (HR)-like cell death using VIGS, and identified silenced clones that have different responses to Bax and INF1 exposure, indicating separate signaling pathways for Bax- and INF1-mediated cell death. PMID:24256816

  6. Transient Nod factor-dependent gene expression in the nodulation-competent zone of soybean (Glycine max [L.] Merr.) roots.

    PubMed

    Hayashi, Satomi; Reid, Dugald E; Lorenc, Michał T; Stiller, Jiri; Edwards, David; Gresshoff, Peter M; Ferguson, Brett J

    2012-10-01

    All lateral organ development in plants, such as nodulation in legumes, requires the temporal and spatial regulation of genes and gene networks. A total mRNA profiling approach using RNA-seq to target the specific soybean (Glycine max) root tissues responding to compatible rhizobia [i.e. the Zone Of Nodulation (ZON)] revealed a large number of novel, often transient, mRNA changes occurring during the early stages of nodulation. Focusing on the ZON enabled us to discard the majority of root tissues and their developmentally diverse gene transcripts, thereby highlighting the lowly and transiently expressed nodulation-specific genes. It also enabled us to concentrate on a precise moment in early nodule development at each sampling time. We focused on discovering genes regulated specifically by the Bradyrhizobium-produced Nod factor signal, by inoculating roots with either a competent wild-type or incompetent mutant (nodC(-) ) strain of Bradyrhizobium japonicum. Collectively, 2915 genes were identified as being differentially expressed, including many known soybean nodulation genes. A number of unknown nodulation gene candidates and soybean orthologues of nodulation genes previously reported in other legume species were also identified. The differential expression of several candidates was confirmed and further characterized via inoculation time-course studies and qRT-PCR. The expression of many genes, including an endo-1,4-β-glucanase, a cytochrome P450 and a TIR-LRR-NBS receptor kinase, was transient, peaking quickly during the initiation of nodule ontogeny. Additional genes were found to be down-regulated. Significantly, a set of differentially regulated genes acting in the gibberellic acid (GA) biosynthesis pathway was discovered, suggesting a novel role of GAs in nodulation. © 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  7. Transformation of radish (Raphanus sativus L.) via sonication and vacuum infiltration of germinated seeds with Agrobacterium harboring a group 3 LEA gene from B. napus.

    PubMed

    Park, Byong-Jin; Liu, Zaochang; Kanno, Akira; Kameya, Toshiaki

    2005-10-01

    A protocol for producing transgenic radish (Raphanus sativus) was obtained by using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation. The Agrobacterium strain LBA4404 contained the binary vector pBI121-LEA (late embyogenesis abundant), which carried a Group 3 LEA gene, from Brassica napus. Among six combinations, Agrobacterium-mediated transformation assisted by a combination of 5-min sonication with 5-min vacuum infiltration resulted in the highest transformation frequency. The existence, integration and expression of transferred LEA gene in transgenic T(1) plants were confirmed by PCR, genomic Southern and Western blot analysis. Transgenic radish demonstrated better growth performance than non-transformed control plants under osmotic and salt stress conditions. Accumulation of Group 3 LEA protein in the vegetative tissue of transgenic radish conferred increased tolerance to water deficit and salt stress.

  8. Exercise induces transient transcriptional activation of the PGC-1α gene in human skeletal muscle

    PubMed Central

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P Darrell

    2003-01-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1α (PGC-1α) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell culture and rodent skeletal muscle. To determine whether PGC-1α transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl-CoA dehydrogenase mRNA, were higher in the trained than the untrained leg prior to exercise. Exercise induced a marked transient increase (P < 0.05) in PGC-1α transcription (10- to > 40-fold) and mRNA content (7- to 10-fold), peaking within 2 h after exercise. Activation of PGC-1α was greater in the trained leg despite the lower relative workload. Interestingly, exercise did not affect nuclear respiratory factor 1 (NRF-1) mRNA, a gene induced by PGC-1α in cell culture. HKII, mitochondrial transcription factor A, peroxisome proliferator activated receptor α, and calcineurin Aα and Aβ mRNA were elevated (≈2- to 6-fold; P < 0.05) at 6 h of recovery in the untrained leg but did not change in the trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC-1α transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator, these findings suggest that PGC-1α may coordinate the activation of metabolic genes in human muscle in response to exercise. PMID:12563009

  9. Exercise induces transient transcriptional activation of the PGC-1alpha gene in human skeletal muscle.

    PubMed

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P Darrell

    2003-02-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1alpha (PGC-1alpha) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell culture and rodent skeletal muscle. To determine whether PGC-1alpha transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl-CoA dehydrogenase mRNA, were higher in the trained than the untrained leg prior to exercise. Exercise induced a marked transient increase (P < 0.05) in PGC-1alpha transcription (10- to > 40-fold) and mRNA content (7- to 10-fold), peaking within 2 h after exercise. Activation of PGC-1alpha was greater in the trained leg despite the lower relative workload. Interestingly, exercise did not affect nuclear respiratory factor 1 (NRF-1) mRNA, a gene induced by PGC-1alpha in cell culture. HKII, mitochondrial transcription factor A, peroxisome proliferator activated receptor alpha, and calcineurin Aalpha and Abeta mRNA were elevated (approximately 2- to 6-fold; P < 0.05) at 6 h of recovery in the untrained leg but did not change in the trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC-1alpha transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator, these findings suggest that PGC-1alpha may coordinate the activation of metabolic genes in human muscle in response to exercise.

  10. Bcmimp1, a Botrytis cinerea Gene Transiently Expressed in planta, Encodes a Mitochondrial Protein

    PubMed Central

    Benito-Pescador, David; Santander, Daniela; Arranz, M.; Díaz-Mínguez, José M.; Eslava, Arturo P.; van Kan, Jan A. L.; Benito, Ernesto P.

    2016-01-01

    Botrytis cinerea is a widespread necrotrophic fungus which infects more than 200 plant species. In an attempt to characterize the physiological status of the fungus in planta and to identify genetic factors contributing to its ability to infect the host cells, a differential gene expression analysis during the interaction B. cinerea-tomato was carried out. Gene Bcmimp1 codes for a mRNA detected by differential display in the course of this analysis. During the interaction with the host, it shows a transient expression pattern with maximal expression levels during the colonization and maceration of the infected tissues. Bioinformatic analysis suggested that BCMIMP1 is an integral membrane protein located in the mitochondrial inner membrane. Co-localization experiments with a BCMIMP1-GFP fusion protein confirmed that the protein is targeted to the mitochondria. ΔBcmimp1 mutants do not show obvious phenotypic differences during saprophytic growth and their infection ability was unaltered as compared to the wild-type. Interestingly, the mutants produced increased levels of reactive oxygen species, likely as a consequence of disturbed mitochondrial function. Although Bcmimp1 expression is enhanced in planta it cannot be considered a pathogenicity factor. PMID:26952144

  11. Bcmimp1, a Botrytis cinerea Gene Transiently Expressed in planta, Encodes a Mitochondrial Protein.

    PubMed

    Benito-Pescador, David; Santander, Daniela; Arranz, M; Díaz-Mínguez, José M; Eslava, Arturo P; van Kan, Jan A L; Benito, Ernesto P

    2016-01-01

    Botrytis cinerea is a widespread necrotrophic fungus which infects more than 200 plant species. In an attempt to characterize the physiological status of the fungus in planta and to identify genetic factors contributing to its ability to infect the host cells, a differential gene expression analysis during the interaction B. cinerea-tomato was carried out. Gene Bcmimp1 codes for a mRNA detected by differential display in the course of this analysis. During the interaction with the host, it shows a transient expression pattern with maximal expression levels during the colonization and maceration of the infected tissues. Bioinformatic analysis suggested that BCMIMP1 is an integral membrane protein located in the mitochondrial inner membrane. Co-localization experiments with a BCMIMP1-GFP fusion protein confirmed that the protein is targeted to the mitochondria. ΔBcmimp1 mutants do not show obvious phenotypic differences during saprophytic growth and their infection ability was unaltered as compared to the wild-type. Interestingly, the mutants produced increased levels of reactive oxygen species, likely as a consequence of disturbed mitochondrial function. Although Bcmimp1 expression is enhanced in planta it cannot be considered a pathogenicity factor.

  12. ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle*

    PubMed Central

    Buvinic, Sonja; Almarza, Gonzalo; Bustamante, Mario; Casas, Mariana; López, Javiera; Riquelme, Manuel; Sáez, Juan Carlos; Huidobro-Toro, Juan Pablo; Jaimovich, Enrique

    2009-01-01

    ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca2+ concentration, with an EC50 value of 7.8 ± 3.1 μm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μm suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y2 receptor and pannexin-1. As reported previously for electrical stimulation, 500 μm ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca2+ homeostasis and muscle physiology. PMID:19822518

  13. Application of a Scalable Plant Transient Gene Expression Platform for Malaria Vaccine Development.

    PubMed

    Spiegel, Holger; Boes, Alexander; Voepel, Nadja; Beiss, Veronique; Edgue, Gueven; Rademacher, Thomas; Sack, Markus; Schillberg, Stefan; Reimann, Andreas; Fischer, Rainer

    2015-01-01

    Despite decades of intensive research efforts there is currently no vaccine that provides sustained sterile immunity against malaria. In this context, a large number of targets from the different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates. None of these candidates has fulfilled expectations, and as long as we lack a single target that induces strain-transcending protective immune responses, combining key antigens from different life cycle stages seems to be the most promising route toward the development of efficacious malaria vaccines. After the identification of potential targets using approaches such as omics-based technology and reverse immunology, the rapid expression, purification, and characterization of these proteins, as well as the generation and analysis of fusion constructs combining different promising antigens or antigen domains before committing to expensive and time consuming clinical development, represents one of the bottlenecks in the vaccine development pipeline. The production of recombinant proteins by transient gene expression in plants is a robust and versatile alternative to cell-based microbial and eukaryotic production platforms. The transfection of plant tissues and/or whole plants using Agrobacterium tumefaciens offers a low technical entry barrier, low costs, and a high degree of flexibility embedded within a rapid and scalable workflow. Recombinant proteins can easily be targeted to different subcellular compartments according to their physicochemical requirements, including post-translational modifications, to ensure optimal yields of high quality product, and to support simple and economical downstream processing. Here, we demonstrate the use of a plant transient expression platform based on transfection with A. tumefaciens as essential component of a malaria vaccine development workflow involving screens for expression, solubility, and stability using fluorescent fusion proteins. Our

  14. Application of a Scalable Plant Transient Gene Expression Platform for Malaria Vaccine Development

    PubMed Central

    Spiegel, Holger; Boes, Alexander; Voepel, Nadja; Beiss, Veronique; Edgue, Gueven; Rademacher, Thomas; Sack, Markus; Schillberg, Stefan; Reimann, Andreas; Fischer, Rainer

    2015-01-01

    Despite decades of intensive research efforts there is currently no vaccine that provides sustained sterile immunity against malaria. In this context, a large number of targets from the different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates. None of these candidates has fulfilled expectations, and as long as we lack a single target that induces strain-transcending protective immune responses, combining key antigens from different life cycle stages seems to be the most promising route toward the development of efficacious malaria vaccines. After the identification of potential targets using approaches such as omics-based technology and reverse immunology, the rapid expression, purification, and characterization of these proteins, as well as the generation and analysis of fusion constructs combining different promising antigens or antigen domains before committing to expensive and time consuming clinical development, represents one of the bottlenecks in the vaccine development pipeline. The production of recombinant proteins by transient gene expression in plants is a robust and versatile alternative to cell-based microbial and eukaryotic production platforms. The transfection of plant tissues and/or whole plants using Agrobacterium tumefaciens offers a low technical entry barrier, low costs, and a high degree of flexibility embedded within a rapid and scalable workflow. Recombinant proteins can easily be targeted to different subcellular compartments according to their physicochemical requirements, including post-translational modifications, to ensure optimal yields of high quality product, and to support simple and economical downstream processing. Here, we demonstrate the use of a plant transient expression platform based on transfection with A. tumefaciens as essential component of a malaria vaccine development workflow involving screens for expression, solubility, and stability using fluorescent fusion proteins. Our

  15. Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).

    PubMed

    Wu, Jun-Zheng; Liu, Qin; Geng, Xiao-Shan; Li, Kai-Mian; Luo, Li-Juan; Liu, Jin-Ping

    2017-03-14

    Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced. Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization. In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 10(7) protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H(+)/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system. We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

  16. Transient Hypermutagenesis Accelerates the Evolution of Legume Endosymbionts following Horizontal Gene Transfer

    PubMed Central

    Remigi, Philippe; Capela, Delphine; Clerissi, Camille; Tasse, Léna; Torchet, Rachel; Bouchez, Olivier; Batut, Jacques; Cruveiller, Stéphane; Rocha, Eduardo P. C.; Masson-Boivin, Catherine

    2014-01-01

    Horizontal gene transfer (HGT) is an important mode of adaptation and diversification of prokaryotes and eukaryotes and a major event underlying the emergence of bacterial pathogens and mutualists. Yet it remains unclear how complex phenotypic traits such as the ability to fix nitrogen with legumes have successfully spread over large phylogenetic distances. Here we show, using experimental evolution coupled with whole genome sequencing, that co-transfer of imuABC error-prone DNA polymerase genes with key symbiotic genes accelerates the evolution of a soil bacterium into a legume symbiont. Following introduction of the symbiotic plasmid of Cupriavidus taiwanensis, the Mimosa symbiont, into pathogenic Ralstonia solanacearum we challenged transconjugants to become Mimosa symbionts through serial plant-bacteria co-cultures. We demonstrate that a mutagenesis imuABC cassette encoded on the C. taiwanensis symbiotic plasmid triggered a transient hypermutability stage in R. solanacearum transconjugants that occurred before the cells entered the plant. The generated burst in genetic diversity accelerated symbiotic adaptation of the recipient genome under plant selection pressure, presumably by improving the exploration of the fitness landscape. Finally, we show that plasmid imuABC cassettes are over-represented in rhizobial lineages harboring symbiotic plasmids. Our findings shed light on a mechanism that may have facilitated the dissemination of symbiotic competency among α- and β-proteobacteria in natura and provide evidence for the positive role of environment-induced mutagenesis in the acquisition of a complex lifestyle trait. We speculate that co-transfer of complex phenotypic traits with mutagenesis determinants might frequently enhance the ecological success of HGT. PMID:25181317

  17. RNA-Dependent RNA Polymerase (NIb) of the Potyviruses Is an Avirulence Factor for the Broad-Spectrum Resistance Gene Pvr4 in Capsicum annuum cv. CM334

    PubMed Central

    Seo, Seungyeon; Lee, Joo Hyun; Choi, Doil

    2015-01-01

    Potyviruses are one of the most destructive viral pathogens of Solanaceae plants. In Capsicum annuum landrace CM334, a broad-spectrum gene, Pvr4 is known to be involved in resistance against multiple potyviruses, including Pepper mottle virus (PepMoV), Pepper severe mosaic virus (PepSMV), and Potato virus Y (PVY). However, a potyvirus avirulence factor against Pvr4 has not been identified. To identify the avirulence factor corresponding to Pvr4 in potyviruses, we performed Agrobacterium-mediated transient expressions of potyvirus protein coding regions in potyvirus-resistant (Pvr4) and -susceptible (pvr4) pepper plants. Hypersensitive response (HR) was observed only when a RNA-dependent RNA polymerase (NIb) of PepMoV, PepSMV, or PVY was expressed in Pvr4-bearing pepper leaves in a genotype-specific manner. In contrast, HR was not observed when the NIb of Tobacco etch virus (TEV), a virulent potyvirus, was expressed in Pvr4-bearing pepper leaves. Our results clearly demonstrate that NIbs of PepMoV, PepSMV, and PVY serve as avirulence factors for Pvr4 in pepper plants. PMID:25760376

  18. Nodular fasciitis: a novel model of transient neoplasia induced by MYH9-USP6 gene fusion.

    PubMed

    Erickson-Johnson, Michele R; Chou, Margaret M; Evers, Barbara R; Roth, Christopher W; Seys, Amber R; Jin, Long; Ye, Ying; Lau, Alan W; Wang, Xiaoke; Oliveira, Andre M

    2011-10-01

    Nodular fasciitis (NF) is a relatively common mass-forming and self-limited subcutaneous pseudosarcomatous myofibroblastic proliferation of unknown pathogenesis. Due to its rapid growth and high mitotic activity, NF is often misdiagnosed as a sarcoma. While studying the USP6 biology in aneurysmal bone cyst and other mesenchymal tumors, we identified high expression levels of USP6 mRNA in two examples of NF. This finding led us to further examine the mechanisms underlying USP6 overexpression in these lesions. Upon subsequent investigation, genomic rearrangements of the USP6 locus were found in 92% (44 of 48) of NF. Rapid amplification of 5'-cDNA ends identified MYH9 as the translocation partner. RT-PCR and direct sequencing revealed the fusion of the MYH9 promoter region to the entire coding region of USP6. Control tumors and tissues were negative for this fusion. Xenografts of cells overexpressing USP6 in nude mice exhibited clinical and histological features similar to human NF. The identification of a sensitive and specific abnormality in NF holds the potential to be used diagnostically. Considering the self-limited nature of the lesion, NF may represent a model of 'transient neoplasia', as it is, to our knowledge, the first example of a self-limited human disease characterized by a recurrent somatic gene fusion event. © 2011 USCAP, Inc All rights reserved

  19. Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease.

    PubMed

    Sendín, Lorena Noelia; Orce, Ingrid Georgina; Gómez, Rocío Liliana; Enrique, Ramón; Grellet Bournonville, Carlos Froilán; Noguera, Aldo Sergio; Vojnov, Adrián Alberto; Marano, María Rosa; Castagnaro, Atilio Pedro; Filippone, María Paula

    2017-04-01

    Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

  20. AAV-mediated gene targeting is significantly enhanced by transient inhibition of nonhomologous end joining or the proteasome in vivo.

    PubMed

    Paulk, Nicole K; Loza, Laura Marquez; Finegold, Milton J; Grompe, Markus

    2012-06-01

    Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah(-/-)Ku70(-/-) double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy.

  1. Transient silencing of the KASII genes is feasible in Nicotiana benthamiana for metabolic engineering of wax ester composition

    PubMed Central

    Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sitbon, Folke; Sun, Chuanxin

    2015-01-01

    The beta-ketoacyl-ACP synthase II (KASII) is an enzyme in fatty acid biosynthesis, catalyzing the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. Mutations in KASII genes in higher plants can lead to lethality, which makes it difficult to utilize the gene for lipid metabolic engineering. We demonstrated previously that transient expression of plastid-directed fatty acyl reductases and wax ester synthases could result in different compositions of wax esters. We hypothesized that changing the ratio between C16 (palmitoyl-compounds) and C18 (stearoyl-compounds) in the plastidic acyl-ACP pool by inhibition of KASII expression would change the yield and composition of wax esters via substrate preference of the introduced enzymes. Here, we report that transient inhibition of KASII expression by three different RNAi constructs in leaves of N. benthamiana results in almost complete inhibition of KASII expression. The transient RNAi approach led to a shift of carbon flux from a pool of C18 fatty acids to C16, which significantly increased wax ester production in AtFAR6-containing combinations. The results demonstrate that transient inhibition of KASII in vegetative tissues of higher plants enables metabolic studies towards industrial production of lipids such as wax esters with specific quality and composition. PMID:26063537

  2. Transient silencing of the KASII genes is feasible in Nicotiana benthamiana for metabolic engineering of wax ester composition.

    PubMed

    Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sitbon, Folke; Sun, Chuanxin

    2015-06-11

    The beta-ketoacyl-ACP synthase II (KASII) is an enzyme in fatty acid biosynthesis, catalyzing the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. Mutations in KASII genes in higher plants can lead to lethality, which makes it difficult to utilize the gene for lipid metabolic engineering. We demonstrated previously that transient expression of plastid-directed fatty acyl reductases and wax ester synthases could result in different compositions of wax esters. We hypothesized that changing the ratio between C16 (palmitoyl-compounds) and C18 (stearoyl-compounds) in the plastidic acyl-ACP pool by inhibition of KASII expression would change the yield and composition of wax esters via substrate preference of the introduced enzymes. Here, we report that transient inhibition of KASII expression by three different RNAi constructs in leaves of N. benthamiana results in almost complete inhibition of KASII expression. The transient RNAi approach led to a shift of carbon flux from a pool of C18 fatty acids to C16, which significantly increased wax ester production in AtFAR6-containing combinations. The results demonstrate that transient inhibition of KASII in vegetative tissues of higher plants enables metabolic studies towards industrial production of lipids such as wax esters with specific quality and composition.

  3. Transient receptor potential genes, smoking, occupational exposures and cough in adults

    PubMed Central

    2012-01-01

    Background Transient receptor potential (TRP) vanilloid and ankyrin cation channels are activated by various noxious chemicals and may play an important role in the pathogenesis of cough. The aim was to study the influence of single nucleotide polymorphisms (SNPs) in TRP genes and irritant exposures on cough. Methods Nocturnal, usual, and chronic cough, smoking, and job history were obtained by questionnaire in 844 asthmatic and 2046 non-asthmatic adults from the Epidemiological study on the Genetics and Environment of Asthma (EGEA) and the European Community Respiratory Health Survey (ECRHS). Occupational exposures to vapors, gases, dusts, and/or fumes were assessed by a job-exposure matrix. Fifty-eight tagging SNPs in TRPV1, TRPV4, and TRPA1 were tested under an additive model. Results Statistically significant associations of 6 TRPV1 SNPs with cough symptoms were found in non-asthmatics after correction for multiple comparisons. Results were consistent across the eight countries examined. Haplotype-based association analysis confirmed the single SNP analyses for nocturnal cough (7-SNP haplotype: p-global = 4.8 × 10-6) and usual cough (9-SNP haplotype: p-global = 4.5 × 10-6). Cough symptoms were associated with exposure to irritants such as cigarette smoke and occupational exposures (p < 0.05). Four polymorphisms in TRPV1 further increased the risk of cough symptoms from irritant exposures in asthmatics and non-asthmatics (interaction p < 0.05). Conclusions TRPV1 SNPs were associated with cough among subjects without asthma from two independent studies in eight European countries. TRPV1 SNPs may enhance susceptibility to cough in current smokers and in subjects with a history of workplace exposures. PMID:22443337

  4. Dominant mutations in the cation channel gene transient receptor potential vanilloid 4 cause an unusual spectrum of neuropathies

    PubMed Central

    Zimoń, Magdalena; Baets, Jonathan; Auer-Grumbach, Michaela; Berciano, José; Garcia, Antonio; Lopez-Laso, Eduardo; Merlini, Luciano; Hilton-Jones, David; McEntagart, Meriel; Crosby, Andrew H.; Barisic, Nina; Boltshauser, Eugen; Shaw, Christopher E.; Landouré, Guida; Ludlow, Christy L.; Gaudet, Rachelle; Houlden, Henry; Reilly, Mary M.; Fischbeck, Kenneth H.; Sumner, Charlotte J.; Timmerman, Vincent; Jordanova, Albena

    2010-01-01

    Hereditary neuropathies form a heterogeneous group of disorders for which over 40 causal genes have been identified to date. Recently, dominant mutations in the transient receptor potential vanilloid 4 gene were found to be associated with three distinct neuromuscular phenotypes: hereditary motor and sensory neuropathy 2C, scapuloperoneal spinal muscular atrophy and congenital distal spinal muscular atrophy. Transient receptor potential vanilloid 4 encodes a cation channel previously implicated in several types of dominantly inherited bone dysplasia syndromes. We performed DNA sequencing of the coding regions of transient receptor potential vanilloid 4 in a cohort of 145 patients with various types of hereditary neuropathy and identified five different heterozygous missense mutations in eight unrelated families. One mutation arose de novo in an isolated patient, and the remainder segregated in families. Two of the mutations were recurrent in unrelated families. Four mutations in transient receptor potential vanilloid 4 targeted conserved arginine residues in the ankyrin repeat domain, which is believed to be important in protein–protein interactions. Striking phenotypic variability between and within families was observed. The majority of patients displayed a predominantly, or pure, motor neuropathy with axonal characteristics observed on electrophysiological testing. The age of onset varied widely, ranging from congenital to late adulthood onset. Various combinations of additional features were present in most patients including vocal fold paralysis, scapular weakness, contractures and hearing loss. We identified six asymptomatic mutation carriers, indicating reduced penetrance of the transient receptor potential vanilloid 4 defects. This finding is relatively unusual in the context of hereditary neuropathies and has important implications for diagnostic testing and genetic counselling. PMID:20460441

  5. Transient overexpression of exogenous APOBEC3A causes C-to-U RNA editing of thousands of genes.

    PubMed

    Sharma, Shraddha; Patnaik, Santosh K; Kemer, Zeynep; Baysal, Bora E

    2016-05-05

    APOBEC3A cytidine deaminase induces site-specific C-to-U RNA editing of hundreds of genes in monocytes exposed to hypoxia and/or interferons and in pro-inflammatory macrophages. To examine the impact of APOBEC3A overexpression, we transiently expressed APOBEC3A in HEK293T cell line and performed RNA sequencing. APOBEC3A overexpression induces C-to-U editing at more than 4,200 sites in transcripts of 3,078 genes resulting in protein recoding of 1,110 genes. We validate recoding RNA editing of genes associated with breast cancer, hematologic neoplasms, amyotrophic lateral sclerosis, Alzheimer disease and primary pulmonary hypertension. These results highlight the fundamental impact of APOBEC3A overexpression on human transcriptome by widespread RNA editing.

  6. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    NASA Technical Reports Server (NTRS)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

    Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

  7. Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

    NASA Technical Reports Server (NTRS)

    Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

    1998-01-01

    Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

  8. A transient assay system for the assessment of cell-autonomous gene function in dehydration-stressed barley

    PubMed Central

    Marzin, Stephan; Mihaly, Robert; Pauk, Janos; Schweizer, Patrick

    2008-01-01

    Drought is a serious, worldwide problem for crop production and also affects yields of barley and wheat, together with other stressors such as frost, viral diseases, or fungal pathogens. Although a number of candidate genes have been identified by transcriptome approaches in recent years, only very few have been tested in functional assays for a beneficial effect on drought tolerance. Here, a transient assay system in microprojectile-bombarded barley leaves is described that allows the functional testing of dehydration stress-related candidate genes by RNA interference (RNAi) or overexpression. Cellular stress or damage in dedydrated leaves is reported by a reduced accumulation of slowly maturing, native red-fluorescing protein DsRed that is known to be sensitive to denaturing conditions. After a dehydration-stress period of 4 d during which the relative fresh weight of leaves was kept at 60–66% of initial fresh weight, a reproducible reduction of normalized DsRed fluorescence was observed. In order to obtain proof of concept, a number of barley mRNAs homologous to drought response genes were selected and targeted by transient induced gene silencing (TIGS). TIGS of four tested genes resulted in a significantly stronger decrease of normalized DsRed fluorescence in dehydration-stressed leaves, whereas they had no effect in fully turgescent control leaves. These genes encode barley drought-responsive factor HvDRF1 (DREB2-like), dehydrin 6, late embryogenesis-abundant protein HVA1, and the vacuolar sodium/proton antiporter HvHNX1. The four targeted transcripts were also found to accumulate rapidly in dehydration-stressed barley leaf segments. The results suggest a value of the TIGS system for functional pre-screening of larger numbers of drought or dehydration stress-related candidate genes in barley. PMID:18641397

  9. Poly(ethyleneimine)-mediated large-scale transient gene expression: influence of molecular weight, polydispersity and N-propionyl groups.

    PubMed

    Kadlecova, Zuzana; Nallet, Sophie; Hacker, David L; Baldi, Lucia; Klok, Harm-Anton; Wurm, Florian M

    2012-05-01

    Three synthesis lots of linear poly(ethyleneimine) (PEI) are compared to a fully hydrolyzed linear PEI (commercially available as PEI "Max") regarding structure, polyplex formation with plasmid DNA, and transfection of suspension-adapted HEK-293E cells. PEI "Max" binds DNA more efficiently than the other PEIs, but it is the least effective in terms of transient recombinant protein yield. One PEI lot is fractionated by means of SEC. The fractions of high-M(n) PEI are the most efficient for complex formation and transfection. Nevertheless, the highest transient recombinant protein yields are achieved with unfractionated PEI. The results demonstrate that the polydispersity and charge density of linear PEI are important parameters for gene delivery to suspension-adapted HEK-293E cells.

  10. The transient outward current in mice lacking the potassium channel gene Kv1.4

    PubMed Central

    London, Barry; Wang, Dao W; Hill, Joseph A; Bennett, Paul B

    1998-01-01

    The transient outward current (Ito) plays a prominent role in the repolarization phase of the cardiac action potential. Several K+ channel genes, including Kv1.4, are expressed in the heart, produce rapidly inactivating currents when heterologously expressed, and may be the molecular basis of Ito.We engineered mice homozygous for a targeted disruption of the K+ channel gene Kv1.4 and compared Ito in wild-type (Kv1.4+/+), heterozygous (Kv1.4+/-) and homozygous ‘knockout’ (Kv1.4−/−) mice. Kv1.4 RNA was truncated in Kv1.4−/− mice and protein expression was absent.Adult myocytes isolated from Kv1.4+/+, Kv1.4+/− and Kv1.4−/− mice had large rapidly inactivating outward currents. The peak current densities at 60 mV (normalized by cellular capacitance, in pA pF−1; means ± s.e.m.) were 53.8 ± 5.3, 45.3 ± 2.2 and 44.4 ± 2.8 in cells from Kv1.4+/+, Kv1.4+/− and Kv1.4−/− mice, respectively (P < 0.02 for Kv1.4+/+ vs. Kv1.4−/−). The steady-state values (800 ms after the voltage clamp step) were 30.9 ± 2.9, 26.9 ± 3.8 and 23.5 ± 2.2, respectively (P < 0.02 for Kv1.4+/+ vs. Kv1.4−/−). The inactivating portion of the current was unchanged in the targeted mice.The voltage dependence and time course of inactivation were not changed by targeted disruption of Kv1.4. The mean best-fitting V½ (membrane potential at 50 % inactivation) values for myocytes from Kv1.4 +/+, Kv1.4+/− and Kv1.4−/− mice were -53.5 ± 3.7, -51.1 ± 2.6 and -54.2 ± 2.4 mV, respectively. The slope factors (k) were -10.1 ± 1.4, -8.8 ± 1.4 and -9.5 ± 1.2 mV, respectively. The fast time constants for development of inactivation at -30 mV were 27.8 ± 2.2, 26.2 ± 5.1 and 19.6 ± 2.1 ms in Kv1.4+/+, Kv1.4+/− and Kv1.4−/− myocytes, respectively. At +30 mV, they were 35.5 ± 2.6, 30.0 ± 2.1 and 28.7 ± 1.6 ms, respectively. The time constants for the rapid phase of recovery from inactivation at -80 mV were 32.5 ± 8.2, 23.3 ± 1.8 and 39.0 ± 3.7 ms, respectively

  11. Optimatization of transient transformation methods to study gene expression in Musa acuminata (AAA group) cultivar Ambon Lumut

    NASA Astrophysics Data System (ADS)

    Prayuni, Kinasih; Dwivany, Fenny M.

    2015-09-01

    Banana is classified as a climateric fruit, whose ripening is regulated by ethylene. Ethylene is synthesized from ACC (1-aminocyclopropane-1-carboxylic acid) by ACC oxidase enzyme which is encoded by ACO gene. Controling an important gene expression in ethylene biosynthesis pathway has became a target to delay the ripening process. Therefore in the previous study we have designed a MaACO-RNAi construct to control MaACO gene expression. In this research, we study the effectiveness of different transient transformation methods to deliver the construct. Direct injection, with or no vaccum infiltration methods were used to deliver MaACO-RNAi construct. All of the methods succesfully deliver the construct into banana fruits based on RT-PCR result.

  12. The two α-dox genes of Nicotiana attenuata: overlapping but distinct functions in development and stress responses

    PubMed Central

    2010-01-01

    Background Plant fatty acid α-dioxygenases (α-DOX) are oxylipin-forming enzymes induced by biotic and abiotic stresses, which also participate in developmental processes. In Nicotiana attenuata, herbivory strongly induces the expression of an α-dox1 gene. To determine its role, we silenced its expression using Agrobacterium-mediated plant transformation with an inverted repeat construct. More than half of the transformed lines showed a severe dwarf growth phenotype that was very similar to the phenotype of tomato plants mutated at a second α-dox isoform. This led us to identify the corresponding α-dox2 gene in N. attenuata and examine the regulation of both α-dox genes as well as the consequences of their silencing in plant development and anti-herbivore defense. Results The transformed lines exhibiting a dwarf growth phenotype are co-silenced for both α-dox genes resulting in a nearly complete suppression of α-DOX activity, which is associated with increases in ABA, JA and anthocyanin levels, all metabolic signatures of oxidative stress. The other lines, only silenced for α-dox1, developed similarly to wild-type plants, exhibited a 40% reduction of α-DOX activity resulting in a 50% reduction of its main product in planta (2-HOT) and showed no signs of oxidative stress. In contrast to α-dox1, the expression of α-dox2 gene is not induced by wounding or elicitors in the oral secretions of Manduca sexta. Instead, α-dox2 is expressed in roots and flowers which lack α-dox1 expression, but both genes are equally regulated during leaf maturation. We transiently silenced α-dox gene copies with gene-specific constructs using virus induced gene silencing and determined the consequences for plant development and phytohormone and 2-HOT levels. While individual silencing of α-dox1 or α-dox2 had no effects on plant growth, the co-suppression of both α-dox genes decreased plant growth. Plants transiently silenced for both α-dox genes had increased constitutive

  13. A fungal conserved gene from the basidiomycete Hebeloma cylindrosporum is essential for efficient ectomycorrhiza formation.

    PubMed

    Doré, Jeanne; Marmeisse, Roland; Combier, Jean-Philippe; Gay, Gilles

    2014-10-01

    We used Agrobacterium-mediated insertional mutagenesis to identify genes in the ectomycorrhizal fungus Hebeloma cylindrosporum that are essential for efficient mycorrhiza formation. One of the mutants presented a dramatically reduced ability to form ectomycorrhizas when grown in the presence of Pinus pinaster. It failed to form mycorrhizas in the presence of glucose at 0.5 g liter(-1), a condition favorable for mycorrhiza formation by the wild-type strain. However, it formed few mycorrhizas when glucose was replaced by fructose or when glucose concentration was increased to 1 g liter(-1). Scanning electron microscopy examination of these mycorrhizas revealed that this mutant was unable to differentiate true fungal sheath and Hartig net. Molecular analyses showed that the single-copy disrupting T-DNA was integrated 6,884 bp downstream from the start codon, of an open reading frame potentially encoding a 3,096-amino-acid-long protein. This gene, which we named HcMycE1, has orthologs in numerous fungi as well as different other eukaryotic microorganisms. RNAi inactivation of HcMycE1 in the wild-type strain also led to a mycorrhizal defect, demonstrating that the nonmycorrhizal phenotype of the mutant was due to mutagenic T-DNA integration in HcMycE1. In the wild-type strain colonizing P. pinaster roots, HcMycE1 was transiently upregulated before symbiotic structure differentiation. Together with the inability of the mutant to differentiate these structures, this suggests that HcMycE1 plays a crucial role upstream of the fungal sheath and Hartig net differentiation. This study provides the first characterization of a fungal mutant altered in mycorrhizal ability.

  14. Comparative analysis of the locus control region of the rabbit beta-like gene cluster: HS3 increases transient expression of an embryonic epsilon-globin gene.

    PubMed Central

    Hardison, R; Xu, J; Jackson, J; Mansberger, J; Selifonova, O; Grotch, B; Biesecker, J; Petrykowska, H; Miller, W

    1993-01-01

    The rabbit homolog to the locus control region (LCR) of the human beta-like globin gene cluster was isolated, and long segments containing the DNase I hypersensitive sites (HS) were sequenced. The order and spacing of HS4, HS3, HS2 and HS1 are conserved between rabbit and human. Alignment of these sequences with their homologs from human, goat, and mouse shows that very long segments of DNA match between species, for over a thousand base pairs on either side of the previously identified functional cores, indicating that some important functions are found outside the cores. The activity of rabbit HS2 and HS3 was tested by attaching each to a novel reporter gene constructed by inserting the luciferase coding region into the rabbit epsilon-globin gene. In contrast to previous reports showing no effect of human or mouse HS3 on transient expression, both the rabbit HS2 and HS3 DNA fragments separately increased transient expression from the epsilon-luciferase hybrid gene and expression from stably integrated constructs in K562 erythroleukemia cells. PMID:8464710

  15. Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2).

    PubMed

    Kobayashi, Michie; Yamamoto-Katou, Ayako; Katou, Shinpei; Hirai, Katsuyuki; Meshi, Tetsuo; Ohashi, Yuko; Mitsuhara, Ichiro

    2011-07-01

    The Tm-2 gene of tomato and its allelic gene, Tm-2(2), confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2(2), Tm-2(2) confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2(2). Although resistance induced by Tm-2 and Tm-2(2) is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2(2) induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2(2) but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2(2) is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2(2) are involved in HR cell death.

  16. Virus-induced gene silencing and transient gene expression in soybean using Bean pod mottle virus infectious clones

    USDA-ARS?s Scientific Manuscript database

    Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200-300 base pair size range. The recombinant viruses are used to ...

  17. Characterization of novel gene expression related to glyoxal oxidase by agro-infiltration of the leaves of accession Baihe-35-1 of Vitis pseudoreticulata involved in production of H2O2 for resistance to Erysiphe necator.

    PubMed

    Zhao, Heqing; Guan, Xin; Xu, Yan; Wang, Yuejin

    2013-06-01

    Glyoxal oxidase (GLOX), an extracellular H(2)O(2)-producing enzyme, has been reported in Phanerochaete chrysosporium and Ustilago maydis. We previously isolated a grapevine GLOX gene from the highly resistant to Erysiphe necator Chinese wild Vitis pseudoreticulata accession Baihe-35-1 and designated it as VpGLOX (GenBank accession no. DQ201181). Transient expression of VpGLOX can suppress Powdery Mildew in susceptible genotype were studied. To further investigate the function of the VpGLOX gene, real-time PCR and Western blot analysis were performed to examine expression patterns at transcriptional and translational levels, respectively. The results showed that VpGLOX expression at the transcriptional level increased significantly in the disease-resistant accession Baihe-35-1 after Erysiphe necator inoculation, but no significant changes in the susceptible accession, V. pseudoreticulata accession Guangxi-2 could be observed. As evident from a Western blot analysis, VpGLOX protein increased slightly in Baihe-35-1 after E. necator inoculation, but not statistical significant difference changes in Guangxi-2. The immunolocalization via immunogold electron microscopy showed that VpGLOX was mainly located in the adaxial epidermal cell wall of E. necator-inoculated leaves of both Baihe-35-1 and Guangxi-2. Agrobacterium-mediated transient expression assays revealed that VpGLOX expression could produce H(2)O(2), which may directly play a role in defense mechanism during plant-pathogen interactions. Our results could provide further insight into the biological role of VpGLOX in the defense response against E. necator in V. pseudoreticulata.

  18. Use of the VvMybA1 gene for non-destructive quantification of promoter activity via color histogram analysis in grapevine (Vitis vinifera) and tobacco.

    PubMed

    Li, Zhijian T; Dhekney, Sadanand A; Gray, Dennis J

    2011-10-01

    We report the development of a convenient plant-based reporter system to analyze promoters and facilitate selection of genetically engineered plants. The VvMybA1 gene of grapevine (Vitis vinifera L.) regulates the last metabolic step of anthocyanin biosynthesis and its ectopic expression leads to anthocyanin production in otherwise non-pigmented cells. To develop an anthocyanin-based quantitative reporter system, the VvMybA1 gene was isolated from V. vinifera 'Merlot' and placed under control of three promoters to test its ability to distinguish different activity levels. Promoters included a double enhanced CaMV35S (d35S) promoter, a double enhanced CsVMV (dCsVMV) promoter or a bi-directional dual promoter (BDDP), resulting in transformation vectors DAT, CAT and DEAT, respectively. These vectors were introduced into grapevine and tobacco via Agrobacterium-mediated transformation for transient and stable expression analysis. A linear relationship between the mean red brightness (MRB) and optical density (OD) values with a 0.99 regression coefficient was identified in a dilution series of anthocyanin, thus allowing the use of histogram data for non-destructive and real-time assessment of transcriptional activity. Results of histogram-based analysis of color images from transformed grapevine somatic embryos (SE) and various tissues of transgenic tobacco showed a consistent six to sevenfold promoter activity increase of DEAT over DAT. This expression increase was verified by spectroscopic measurement of anthocyanin concentrations in sepal tissue of transgenic tobacco plants. These results were congruent with previously findings of promoter activity derived from GUS fluorometric assay, thus demonstrating for the first time that the VvMybA1 gene could offer a simple, versatile and reliable plant-based alternative for quantitative promoter analysis in plants.

  19. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes

    PubMed Central

    2011-01-01

    Background Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue. Results Here, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts. Conclusions We show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes. PMID:21961694

  20. Local and transient gene expression primes the liver to resist cancer metastasis.

    PubMed

    Goodwin, Tyler J; Zhou, Yingqiu; Musetti, Sara N; Liu, Rihe; Huang, Leaf

    2016-11-09

    The liver is the primary site of metastasis for gastrointestinal cancers and is a location highly susceptible to the establishment of metastasis in numerous other primary cancers, including breast, lung, and pancreatic cancers. The current standard of care typically consists of primary tumor resection and systemic administration of potent but toxic chemotherapeutics, yielding a minimal improvement in the median survival rate. CXCL12, a chemokine, is a key factor for activating the migration/survival pathways of CXCR4(+) cancer cells and for recruiting immunosuppressive cells to areas of inflammation. Therefore, reducing CXCL12 concentrations within the liver has the potential to decrease tumor and immunosuppressive cell activation/migration within the liver. However, because of off-target toxicities associated with systemic administration of anti-CXCL12 therapies, transient and liver-specific expression of a CXCL12 trap is necessary. To address this challenge, we developed a lipid calcium phosphate nanoparticle optimized for delivering plasmid DNA, encoding an engineered CXCL12 protein trap, to the nucleus of liver hepatocytes. This pCXCL12-trap formulation yielded transient (4 days) liver-specific expression, which greatly decreased the occurrence of liver metastasis in two aggressive liver metastasis models, including colorectal [CT-26(FL3)] and breast (4T1) cancers. Subsequent studies in an aggressive human colorectal liver metastasis model (HT-29) decreased the establishment of liver metastasis more effectively than did systemic administration of the CXCL12 protein trap and to a level comparable to a high-dose regimen of a potent CXCR4 antagonist (AMD3100). Copyright © 2016, American Association for the Advancement of Science.

  1. Hyperglycemia and hypercapnia suppress BDNF gene expression in vulnerable regions after transient forebrain ischemia in the rat.

    PubMed

    Uchino, H; Lindvall, O; Siesjö, B K; Kokaia, Z

    1997-12-01

    Preischemic hyperglycemia or superimposed hypercapnia exaggerates brain damage caused by transient forebrain ischemia. Because high regional levels of brain-derived neurotrophic factor (BDNF) protein correlate with resistance to ischemic damage, we studied the expression of BDNF mRNA using in situ hybridization in rats subjected to 10 minutes of forebrain ischemia under normoglycemic, hyperglycemic, or hypercapnic conditions. Compared with normoglycemic animals, the increase of BDNF mRNA using in situ hybridization in rats subjected to 10 minutes of forebrain ischemia under normoglycemic, or hypercapnic conditions. Compared with normoglycemic animals, the increase of BDNF mRNA in dentate granule cells was attenuated and that in CA3 pyramidal neurons completely prevented in hyperglycemic rats. No ischemia-induced increases of BDNF mRNA levels in the hippocampal formation were detected in hypercapnic animals. Hyperglycemic and hypercapnic rats showed transiently decreased expression of BDNF mRNA levels in the cingulate cortex, which was not observed in normoglycemic animals. The results suggest that suppression of the BDNF gene might contribute to the increased vulnerability of the CA3 region and cingulate cortex in hyperglycemic and hypercapnic animals.

  2. How transient alterations of organelles in mammalian cells submitted to electric field may explain some aspects of gene electrotransfer process.

    PubMed

    Phez, Emilie; Gibot, Laure; Rols, Marie-Pierre

    2016-12-01

    Electric pulses can be used to transiently permeabilize the cell plasma membrane. This method is nowadays employed as a safe and efficient means to deliver therapeutic molecules into target cells and tissues. Despite the large bulk of literature on this topic, there is a lack of knowledge about the mechanism(s) of molecule delivery. The behavior of the cells both while the field is on and after its application is indeed not well described. Questions about cell organelle alterations remain unanswered. We report here evidence for a number of ultrastructural alterations in mammalian cells exposed to electric pulses. Specifically, CHO cells were subjected to trains of 10 pulses lasting 5ms using an electric field of 800V/cm, i.e. under conditions leading both to membrane permeabilization, gene transfer and expression. Cells were observed to undergo morphological alterations of the mitochondria and nucleus. These modifications, detected in the minutes following pulse delivery, were transient. They may have direct consequences on molecule delivery and therefore may explain various aspects of the mechanisms of DNA electrotransfer. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Transient middle cerebral artery occlusion in mice induces neuronal expression of RNF213, a susceptibility gene for moyamoya disease.

    PubMed

    Sato-Maeda, Mika; Fujimura, Miki; Kanoke, Atsushi; Morita-Fujimura, Yuiko; Niizuma, Kuniyasu; Tominaga, Teiji

    2016-01-01

    Although recent genome-wide and locus-specific association studies revealed that the RING finger protein 213 (RNF213) gene is an important susceptibility gene for moyamoya disease (MMD), the exact mechanism by which the genetic alteration of RNF213 contributes to the development of MMD has not yet been elucidated. A quantitative reverse transcription polymerase chain reaction (PCR) analysis revealed that the constitutive expression of the RNF213 gene was very low in adult and embryonic brain tissue. However, information regarding the temporal and spatial expression patterns of the RNF213 gene under the condition of cerebral ischemia, which is one of characteristic pathologies associated with MMD, is currently limited. In order to address this critical issue, Rnf213 mRNA expression was investigated in mouse brains subjected to 60 min of transient middle cerebral artery occlusion (tMCAO). Male C57BL6/j mice underwent tMCAO through the intraluminal blockade of MCA. Expression of the Rnf213 gene in the tMCAO brain was investigated with in situ RNA hybridization and a real-time PCR analysis from 1 to 72 h after tMCAO. In situ RNA hybridization revealed a significant increase in Rnf213 mRNA levels in the cerebral cortex supplied by the affected MCA, especially at the penumbra area, as early as 6h after tMCAO, and these levels had increased further by 24 h. Rnf213 gene expression remained unchanged in the non-ischemic hemisphere or control specimens. Double staining of Rnf213 mRNA with NeuN immunohistochemistry revealed Rnf213 hybridization signal expression mostly in neurons. The real-time PCR analysis confirmed induction of the Rnf213 gene after tMCAO. Therefore, the Rnf213 gene was up-regulated in the ischemic brain, especially at the penumbra area, 6 h after tMCAO. Early increases in RNF213 gene expression in neurons after tMCAO indicate its involvement in cerebral ischemia, which is an underlying pathology of MMD. Further investigation is required to clarify its

  4. Expression of Terminal Effector Genes in Mammalian Neurons Is Maintained by a Dynamic Relay of Transient Enhancers.

    PubMed

    Rhee, Ho Sung; Closser, Michael; Guo, Yuchun; Bashkirova, Elizaveta V; Tan, G Christopher; Gifford, David K; Wichterle, Hynek

    2016-12-21

    Generic spinal motor neuron identity is established by cooperative binding of programming transcription factors (TFs), Isl1 and Lhx3, to motor-neuron-specific enhancers. How expression of effector genes is maintained following downregulation of programming TFs in maturing neurons remains unknown. High-resolution exonuclease (ChIP-exo) mapping revealed that the majority of enhancers established by programming TFs are rapidly deactivated following Lhx3 downregulation in stem-cell-derived hypaxial motor neurons. Isl1 is released from nascent motor neuron enhancers and recruited to new enhancers bound by clusters of Onecut1 in maturing neurons. Synthetic enhancer reporter assays revealed that Isl1 operates as an integrator factor, translating the density of Lhx3 or Onecut1 binding sites into transient enhancer activity. Importantly, independent Isl1/Lhx3- and Isl1/Onecut1-bound enhancers contribute to sustained expression of motor neuron effector genes, demonstrating that outwardly stable expression of terminal effector genes in postmitotic neurons is controlled by a dynamic relay of stage-specific enhancers. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Transient gene and microRNA expression profile changes of confluent human fibroblast cells in space

    NASA Astrophysics Data System (ADS)

    Wu, Honglu; Story, Michael; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Lu, Tao

    2016-07-01

    Microgravity, or an altered gravity environment from the Earth1g, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of these cells. Whether non-proliferating cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted onboard the International Space Station (ISS), confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days, respectively, for investigations of gene and miRNA expression profile changes in these cells. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67 positive cells. Gene and miRNA expression data indicated activation of NFkB and other growth related pathways involving HGF and Vegf along with down regulation of the Let-7 miRNA family. On Day 14 when the cells were mostly non-proliferating, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples with respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for αa-tubulin and fibronectin showed no difference between flown and ground samples. Taken together, our study suggests that in true non-dividing human fibroblast cells in culture, microgravity experienced in space has little effect on the gene and miRNA expression profiles.

  6. Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene

    SciTech Connect

    Grison, R.; Grezes-Besset, B.; Lucante, N.

    1996-05-01

    Constitutive overexpression of a protein involved in plant defense mechanisms to disease is one of the strategies proposed to increase plant tolerance to fungal pathogens. A hybrid endochitinase gene under a constitutive promoter was introduced by Agrobacterium-mediated transformation into a winter-type oilseed rape (Brassica napus var. oleifera) inbred line. Progeny from transformed plants was challenged using three different fungal pathogens (Cylindrosporium concentricum, Phoma lingam, Sclerotinia sclerotiorum) in field trials at two different geographical locations. These plants exhibited an increased tolerance to disease as compared with the nontransgenic parental plants. 31 refs., 1 fig., 2 tabs.

  7. Transient expression of βC1 protein differentially regulates host genes related to stress response, chloroplast and mitochondrial functions

    PubMed Central

    2010-01-01

    Background Geminiviruses are emerging plant pathogens that infect a wide variety of crops including cotton, cassava, vegetables, ornamental plants and cereals. The geminivirus disease complex consists of monopartite begomoviruses that require betasatellites for the expression of disease symptoms. These complexes are widespread throughout the Old World and cause economically important diseases on several crops. A single protein encoded by betasatellites, termed βC1, is a suppressor of gene silencing, inducer of disease symptoms and is possibly involved in virus movement. Studies of the interaction of βC1 with hosts can provide useful insight into virus-host interactions and aid in the development of novel control strategies. We have used the differential display technique to isolate host genes which are differentially regulated upon transient expression of the βC1 protein of chili leaf curl betasatellite (ChLCB) in Nicotiana tabacum. Results Through differential display analysis, eight genes were isolated from Nicotiana tabacum, at two and four days after infitration with βC1 of ChLCB, expressed under the control of the Cauliflower mosaic virus 35S promoter. Cloning and sequence analysis of differentially amplified products suggested that these genes were involved in ATP synthesis, and acted as electron carriers for respiration and photosynthesis processes. These differentially expressed genes (DEGs) play an important role in plant growth and development, cell protection, defence processes, replication mechanisms and detoxification responses. Kegg orthology based annotation system analysis of these DEGs demonstrated that one of the genes, coding for polynucleotide nucleotidyl transferase, is involved in purine and pyrimidine metabolic pathways and is an RNA binding protein which is involved in RNA degradation. Conclusion βC1 differentially regulated genes are mostly involved in chloroplast and mitochondrial functions. βC1 also increases the expression of those

  8. cis and trans activation of globin gene transcription in transient assays.

    PubMed

    Treisman, R; Green, M R; Maniatis, T

    1983-12-01

    We examined the effects of the simian virus 40 enhancer sequence on transcription of cloned human alpha- and beta-globin genes shortly after their introduction into cultured mammalian cells. We find that (i) detectable transcription of the beta-globin gene but not the alpha-globin gene requires linkage to the enhancer; (ii) the enhancer increases the amount of beta-globin RNA at least 100-fold but results in only a 5- to 10-fold increase in the amount of alpha-globin RNA; (iii) plasmid replication does not increase the level of beta-globin RNA, regardless of linkage to the enhancer, but does result in an approximately equal to 50-fold increase in the level of alpha-globin RNA; (iv) the enhancer is not required for and does not increase transcription of either gene in 293 cells, an adenovirus 5-transformed human kidney cell line. We also show that an enhancer sequence is not required for activity of the normally enhancer-dependent simian virus 40 early promoter in 293 cells, indicating that these cells contain a trans-acting factor(s) that circumvents the requirement for the enhancer sequence.

  9. Transient Gene and miRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Lu, Tao; Wong, Michael; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Wang, Xiaoyu; Wu, Honglu

    2015-01-01

    Microgravity or an altered gravity environment from the static 1 gravitational constant has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of the cells. Whether non-dividing cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted on the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days for investigations of gene and miRNA (microRNA) expression profile changes in these cells. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly even though they were confluent, as measured by the expression of the protein Ki-67 positive cells, and the cells in space grew slightly faster. Gene and miRNA expression data indicated activation of NF(sub kappa)B (nuclear factor kappa-light-chain-enhancer of activated B cells) and other growth related pathways involving HGF and VEGF in the flown cells. On Day 14 when the cells were mostly non-dividing, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples in respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeleton changes by immunohistochemistry staining of the cells with antibodies for alpha-tubulin showed no difference between the flight and ground samples. Results of our study suggest that in true non-dividing human fibroblast cells, microgravity in

  10. The transient receptor potential channel TRPA1: from gene to pathophysiology.

    PubMed

    Nilius, Bernd; Appendino, Giovanni; Owsianik, Grzegorz

    2012-11-01

    The Transient Receptor Potential Ankyrin 1 channel (TRPA1), is a member of the large TRP family of ion channels, and functions as a Ca(2+) permeable non-selective cation channel in many different cell processes, ranging from sensory to homeostatic tasks. TRPA1 is highly conserved across the animal kingdom. The only mammalian TRPA subfamily member, TRPA1, is widely expressed in neuronal (e.g. sensory dorsal root and trigeminal ganglia neurons)- and in non-neuronal cells (e.g. epithelial cells, hair cells). It exhibits 14-19 amino-(N-)terminal ankyrin repeats, an unusual structural feature. The TRPA1 channel is activated by noxious cold (<17 °C) as well as by a plethora of chemical compounds that includes not only electrophilic compounds and oxidants that can modify, in an alkylative or oxidative fashion, nucleophilic cysteine residues in the channel's N-terminus, but also compounds that do not covalently bind to the channel proteins (e.g. menthol, nifedipin). Based on localization and functional properties, TRPA1 is considered a key player in acute and chronic (neuropathic) pain and inflammation. Moreover, its role in the (patho)physiology of nearly all organ systems is anticipated, and will be discussed along with the potential of TRPA1 as a drug target for the management of various pathological conditions.

  11. Transcriptional 'memory' of a stress: transient chromatin and memory (epigenetic) marks at stress-response genes.

    PubMed

    Avramova, Zoya

    2015-07-01

    Drought, salinity, extreme temperature variations, pathogen and herbivory attacks are recurring environmental stresses experienced by plants throughout their life. To survive repeated stresses, plants provide responses that may be different from their response during the first encounter with the stress. A different response to a similar stress represents the concept of 'stress memory'. A coordinated reaction at the organismal, cellular and gene/genome levels is thought to increase survival chances by improving the plant's tolerance/avoidance abilities. Ultimately, stress memory may provide a mechanism for acclimation and adaptation. At the molecular level, the concept of stress memory indicates that the mechanisms responsible for memory-type transcription during repeated stresses are not based on repetitive activation of the same response pathways activated by the first stress. Some recent advances in the search for transcription 'memory factors' are discussed with an emphasis on super-induced dehydration stress memory response genes in Arabidopsis.

  12. Site-Specific Recombination-Based Genetic System for Reporting Transient or Low-Level Gene Expression†

    PubMed Central

    Casavant, N. Carol; Beattie, Gwyn A.; Phillips, Gregory J.; Halverson, Larry J.

    2002-01-01

    We report here the construction, characterization, and application of a plasmid-based genetic system that reports the expression of a target promoter by effecting an irreversible, heritable change in a bacterial cell. This system confers strong repression of the reporter gene gfp in the absence of target promoter expression and utilizes the site-specific recombination machinery of bacteriophage P22 to trigger high-level reporter gene expression in the original cell and its progeny after target gene induction. We demonstrate the effectiveness of this genetic system by tailoring it to indicate the availability of arabinose to the biological control agent Enterobacter cloacae JL1157 in culture and in the barley rhizosphere. The presence of bioavailable arabinose triggered the production of P22 excisionase and integrase from the reporter plasmid pAraLHB in JL1157, and this led to excision of the cI repressor gene, which is flanked by att sites, and the subsequent irreversible expression of gfp in the original cell and in its progeny. In culture, nearly 100% of an E. cloacae JL1157(pAraLHB) population expressed gfp after exposure to 6.5 to 65 μM arabinose for 3 h. We used this biosensor to demonstrate that arabinose was released from the seeds of several legumes and grass species during germination and from roots of barley seedlings grown hydroponically or in soil. When introduced into microcosms containing barley, the biosensor permitted the localization of arabinose along the roots. Arabinose was present near the root-seed junction and on the seminal roots but was not detected at the root tips. This recombination-based reporter system should be useful for monitoring bacterial exposure to transient or low levels of specific molecules directly in the environment. PMID:12089047

  13. Phenylpropanoid pathway intermediates regulate transient expression of a chalcone synthase gene promoter.

    PubMed Central

    Loake, G J; Choudhary, A D; Harrison, M J; Mavandad, M; Lamb, C J; Dixon, R A

    1991-01-01

    A chimeric gene construct containing a bean chalcone synthase (CHS) promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed when electroporated into alfalfa protoplasts that were then exposed to a fungal elicitor. Low concentrations (5 x 10(-6) to 10(-4) M) of exogenously applied trans-cinnamic acid (CA), the first intermediate of the phenylpropanoid pathway, slightly stimulated elicitor-induced CAT expression, whereas high concentrations (greater than 10(-4) M) severely reduced expression to below the levels observed in the absence of elicitor. In contrast, trans-p-coumaric acid (4-CA, the second intermediate in the pathway) stimulated expression from the CHS promoter up to 4.5-fold at 5 x 10(-4) M. Expression of CAT driven by the promoters of other elicitor-inducible defense response genes was not markedly affected by CA or 4-CA. Stimulation of CHS promoter expression by low concentrations of CA and 4-CA was completely abolished by 5' deletion to position -130, but not -174. When the -180 to -130 region of the CHS15 promoter was coelectroporated into elicited protoplasts on a separate plasmid along with the intact -326 CHS-CAT construct, the decreased CAT expression as a function of CA or 4-CA concentration was consistent with the coelectroporated sequence competing in trans with the intact promoter for the binding of a factor(s) involved in the up regulation of CHS transcription by 4-CA and low concentrations of CA. Our data support the hypothesis that phenylpropanoid compounds may act as natural and specific regulators of plant gene expression and define the location of a cis-acting element in the CHS15 promoter involved in the induction by phenylpropanoid pathway intermediates. PMID:1820822

  14. Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements

    PubMed Central

    Secco, David; Wang, Chuang; Shou, Huixia; Schultz, Matthew D; Chiarenza, Serge; Nussaume, Laurent; Ecker, Joseph R; Whelan, James; Lister, Ryan

    2015-01-01

    Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress. DOI: http://dx.doi.org/10.7554/eLife.09343.001 PMID:26196146

  15. Generation of marker-free plastid transformants using a transiently cointegrated selection gene.

    PubMed

    Klaus, Sebastian M J; Huang, Fong-Chin; Golds, Timothy J; Koop, Hans-Ulrich

    2004-02-01

    Genetic engineering of higher plant plastids typically involves stable introduction of antibiotic resistance genes as selection markers. Even though chloroplast genes are maternally inherited in most crops, the possibility of marker transfer to wild relatives or microorganisms cannot be completely excluded. Furthermore, marker expression can be a substantial metabolic drain. Therefore, efficient methods for complete marker removal from plastid transformants are necessary. One method to remove the selection gene from higher plant plastids is based on loop-out recombination, a process difficult to control because selection of homoplastomic transformants is unpredictable. Another method uses the CRE/lox system, but requires additional retransformation and sexual crossing for introduction and subsequent removal of the CRE recombinase. Here we describe the generation of marker-free chloroplast transformants in tobacco using the reconstitution of wild-type pigmentation in combination with plastid transformation vectors, which prevent stable integration of the kanamycin selection marker. One benefit of a procedure using mutants is that marker-free plastid transformants can be produced directly in the first generation (T0) without retransformation or crossing.

  16. Evaluation of genes encoding for the transient outward current (Ito) identifies the KCND2 gene as a cause of J-wave syndrome associated with sudden cardiac death.

    PubMed

    Perrin, Mark J; Adler, Arnon; Green, Sharon; Al-Zoughool, Foad; Doroshenko, Petro; Orr, Nathan; Uppal, Shaheen; Healey, Jeff S; Birnie, David; Sanatani, Shubhayan; Gardner, Martin; Champagne, Jean; Simpson, Chris; Ahmad, Kamran; van den Berg, Maarten P; Chauhan, Vijay; Backx, Peter H; van Tintelen, J Peter; Krahn, Andrew D; Gollob, Michael H

    2014-12-01

    J-wave ECG patterns are associated with an increased risk of sudden arrhythmic death, and experimental evidence supports a transient outward current (I(to))-mediated mechanism of J-wave formation. This study aimed to determine the frequency of genetic mutations in genes encoding the I(to) in patients with J waves on ECG. Comprehensive mutational analysis was performed on I(to)-encoding KCNA4, KCND2, and KCND3 genes, as well as the previously described J-wave-associated KCNJ8 gene, in 51 unrelated patients with ECG evidence defining a J-wave syndrome. Only patients with a resuscitated cardiac arrest or type 1 Brugada ECG pattern were included for analysis. A rare genetic mutation of the KCND2 gene, p.D612N, was identified in a single patient. Co-expression of mutant and wild-type KCND2 with KChIP2 in HEK293 cells demonstrated a gain-of-function phenotype, including an increase in peak I(to) density of 48% (P<0.05) in the heterozygous state. Using computer modeling, this increase in Ito resulted in loss of the epicardial action potential dome, predicting an increased ventricular transmural Ito gradient. The previously described KCNJ8-S422L mutation was not identified in this cohort of patients with ECG evidence of J-wave syndrome. These findings are the first to implicate the KCND2 gene as a novel cause of J-wave syndrome associated with sudden cardiac arrest. However, genetic defects in I(to)-encoding genes seem to be an uncommon cause of sudden cardiac arrest in patients with apparent J-wave syndromes. © 2014 American Heart Association, Inc.

  17. Cloning and characterization of TaVIP2 gene from Triticum aestivum and functional analysis in Nicotiana tabacum

    PubMed Central

    Zhao, Pei; Wang, Ke; Lin, Zhishan; Zhang, Wei; Du, Lipu; Zhang, Yunlong; Ye, Xingguo

    2016-01-01

    Wheat is recalcitrant to genetic transformation. A potential solution is to manipulate the expression of some host proteins involved in T-DNA integration process. VirE2 interacting protein 2 (VIP2) plays an important role in T-DNA transport and integration. In this study, a TaVIP2 gene was cloned from common wheat. Southern blot and allele-specific polymerase chain reaction (AS-PCR) combined with an online chromosomal location software tool revealed that three TaVIP2 genes were located on wheat chromosomes 1AL, 1BL, and 1DL. These three homoeoallelic TaVIP2 genes all contained 13 exons and 12 introns, and their coding sequences were the same; there were a few single nucleotide polymorphisms (SNPs) among the three genes. The heterologous expression of the TaVIP2 gene in tobacco led to enhancement of the Agrobacterium-mediated transformation efficiency up to 2.5-fold. Transgenic tobacco plants expressing TaVIP2 showed enhanced resistance to powdery mildew. Further quantitative real-time PCR (qRT-PCR) revealed that overexpression of TaVIP2 in transgenic tobacco up-regulated the expression of an endogenous gene, NtPR-1, which likely contributed to powdery mildew resistance in transgenic tobacco. Our study indicates that the TaVIP2 gene may be highly useful in efforts to improve Agrobacterium-mediated transformation efficiency and to enhance powdery mildew resistance in wheat. PMID:27857194

  18. Evaluation of TRPM (transient receptor potential melastatin) genes expressions in myocardial ischemia and reperfusion.

    PubMed

    Demir, Tuncer; Yumrutas, Onder; Cengiz, Beyhan; Demiryurek, Seniz; Unverdi, Hatice; Kaplan, Davut Sinan; Bayraktar, Recep; Ozkul, Nadide; Bagcı, Cahit

    2014-05-01

    In the present study, the expression levels of TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, and TRPM8 genes were evaluated in heart tissues after ischemia/reperfusion (IR). For this study, 30 albino male Wistar rats were equally divided into three groups as follows: Group 1: control group (n:10), Group II: ischemia group (ischemia for 60 min) (n:10) and Group III: IR (reperfusion 48 h after ischemia for 60 min and reperfusion for 48 h). The expression levels of the TRPM genes were analyzed by semi-quantitative reverse transcriptase-PCR. When compared to the ischemia control, the expression levels of TRPM2, TRPM4, and TRPM6 did not change, whereas that of TRPM7 increased. However, TRPM1, TRPM3, TRPM5, and TRPM8 were not expressed in heart tissue. Histopathological analysis of the myocardial tissues showed that the structures that were most damaged were those exposed to IR. The findings showed that there is a positive relationship between TRPM7 expression and myocardial IR injury.

  19. Enhanced salt tolerance of alfalfa (Medicago sativa) by rstB gene transformation.

    PubMed

    Zhang, Wan-Jun; Wang, Tao

    2015-05-01

    Generating salt tolerance forage plant is essential for use of the land affected by high salinity. A salt tolerance gene rstB was used as a selectable marker gene in Agrobacterium-mediated transformation of tobacco under a selective regime of 170mM NaCl. The transgenic plants showed clear improvement in salt tolerance. To improve salt tolerance of alfalfa (Medicago sativa L.), rstB gene was introduced into alfalfa genome by Agrobacterium-mediated transformation. No abnormal phenotype was observed among the transgenic plants when compared with wild type (wt) plants. Significant enhancement of resistance to salt-shock treatment was noted on the rstB transgenic (T0) plants. Transgenic second-generation (T1) seeds showed improved germination rate and seedling growth under salt-stress condition. Hindered Na(+) accumulation, but enhanced Ca(2+) accumulation was observed on the rstB T1 plants when subjected to salt-stresses. Enhanced calcium accumulation in transgenic plants was also verified by cytohistochemical localization of calcium. Under salt-stress of 50mM NaCl, about 15% of the transgenic plants finished their life-cycle but the wt plants had no flower formation. The results demonstrated that the expression of rstB gene improved salt tolerance in transgenic alfalfa.

  20. Transient protein-protein interactions perturb E. coli metabolome and cause gene dosage toxicity

    PubMed Central

    Bhattacharyya, Sanchari; Bershtein, Shimon; Yan, Jin; Argun, Tijda; Gilson, Amy I; Trauger, Sunia A; Shakhnovich, Eugene I

    2016-01-01

    Gene dosage toxicity (GDT) is an important factor that determines optimal levels of protein abundances, yet its molecular underpinnings remain unknown. Here, we demonstrate that overexpression of DHFR in E. coli causes a toxic metabolic imbalance triggered by interactions with several functionally related enzymes. Though deleterious in the overexpression regime, surprisingly, these interactions are beneficial at physiological concentrations, implying their functional significance in vivo. Moreover, we found that overexpression of orthologous DHFR proteins had minimal effect on all levels of cellular organization – molecular, systems, and phenotypic, in sharp contrast to E. coli DHFR. Dramatic difference of GDT between ‘E. coli’s self’ and ‘foreign’ proteins suggests the crucial role of evolutionary selection in shaping protein-protein interaction (PPI) networks at the whole proteome level. This study shows how protein overexpression perturbs a dynamic metabolon of weak yet potentially functional PPI, with consequences for the metabolic state of cells and their fitness. DOI: http://dx.doi.org/10.7554/eLife.20309.001 PMID:27938662

  1. Immobilisation of barley aleurone layers enables parallelisation of assays and analysis of transient gene expression in single cells.

    PubMed

    Zór, Kinga; Mark, Christina; Heiskanen, Arto; Madsen, Claus Krogh; Dufva, Martin; Emnéus, Jenny; Brinch-Pedersen, Henrik; Finnie, Christine

    2017-09-01

    The barley aleurone layer is an established model system for studying phytohormone signalling, enzyme secretion and programmed cell death during seed germination. Most analyses performed on the aleurone layer are end-point assays based on cell extracts, meaning each sample is only analysed at a single time point. By immobilising barley aleurone layer tissue on polydimethylsiloxane pillars in the lid of a multiwell plate, continuous monitoring of living tissue is enabled using multiple non-destructive assays in parallel. Cell viability and menadione reducing capacity were monitored in the same aleurone layer samples over time, in the presence or absence of plant hormones and other effectors. The system is also amenable to transient gene expression by particle bombardment, with simultaneous monitoring of cell death. In conclusion, the easy to handle and efficient experimental setup developed here enables continuous monitoring of tissue samples, parallelisation of assays and single cell analysis, with potential for time course studies using any plant tissue that can be immobilised, for example leaves or epidermal peels. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Increased accumulation of anthocyanins in Fragaria chiloensis fruits by transient suppression of FcMYB1 gene.

    PubMed

    Salvatierra, Ariel; Pimentel, Paula; Moya-León, María Alejandra; Herrera, Raúl

    2013-06-01

    Anthocyanins and proanthocyanidins (PAs), flavonoid-derived metabolites with different physiological roles, are produced by plants in a coordinated manner during fruit development by the action of transcription factors (TFs). These regulatory proteins have either an activating or repressing effect over structural genes from the biosynthetic pathway under their control. FaMYB1, a TF belonging to the R2R3-MYB family and isolated from commercial strawberry fruit (Fragaria×ananassa), was reported as a transcriptional repressor and its heterologous over-expression in tobacco flowers suppressed flavonoid-derived compound accumulation. FcMYB1, an ortholog of FaMYB1 isolated from the white Chilean strawberry (Fragaria chiloensis ssp. chiloensis f. chiloensis), showed higher transcript levels in white (F. chiloensis) than in red (F.×ananassa cv. Camarosa) fruits. In order to assess its contribution to the discolored phenotype in F. chiloensis, FcMYB1 was transiently down-regulated in planta using an RNAi-based approach. Quantitative real-time PCR on FcMYB1 down-regulated fruits resulted an up-regulation of anthocyanidin synthase (ANS) and a strong repression of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) transcript accumulation. In addition, these fruits showed increased concentrations of anthocyanins and undetectable levels of flavan 3-ols. Altogether, these results indicate a role for FcMYB1 in regulation of the branching-point of the anthocyanin/PA biosynthesis determining the discolored phenotype of the white Chilean strawberry fruit.

  3. Solulin reduces infarct volume and regulates gene-expression in transient middle cerebral artery occlusion in rats.

    PubMed

    Ryang, Yu-Mi; Dang, Jon; Kipp, Markus; Petersen, Karl-Uwe; Fahlenkamp, Astrid V; Gempt, Jens; Wesp, Dominik; Rossaint, Rolf; Beyer, Cordian; Coburn, Mark

    2011-11-14

    Thrombolysis after acute ischemic stroke has only proven to be beneficial in a subset of patients. The soluble recombinant analogue of human thrombomodulin, Solulin, was studied in an in vivo rat model of acute ischemic stroke. Male SD rats were subjected to 2 hrs of transient middle cerebral artery occlusion (tMCAO). Rats treated with Solulin intravenously shortly before reperfusion were compared to rats receiving normal saline i.v. with respect to infarct volumes, neurological deficits and mortality. Gene expression of IL-6, IL-1β, TNF-α, MMP-9, CD11B and GFAP were semiquantitatively analyzed by rtPCR of the penumbra. 24 hrs after reperfusion, rats were neurologically tested, euthanized and infarct volumes determined. Solulin significantly reduced mean total (p=0.001), cortical (p=0.002), and basal ganglia (p=0.036) infarct volumes. Hippocampal infarct volumes (p=0.191) were not significantly affected. Solulin significantly downregulated the expression of IL-1β (79%; p<0.001), TNF-α (59%; p=0.001), IL-6 (47%; p=0.04), and CD11B (49%; p=0.001) in the infarcted cortex compared to controls. Solulin reduced mean total, cortical and basal ganglia infarct volumes and regulated a subset of cytokines and proteases after tMCAO suggesting the potency of this compound for therapeutic interventions.

  4. Solulin reduces infarct volume and regulates gene-expression in transient middle cerebral artery occlusion in rats

    PubMed Central

    2011-01-01

    Background Thrombolysis after acute ischemic stroke has only proven to be beneficial in a subset of patients. The soluble recombinant analogue of human thrombomodulin, Solulin, was studied in an in vivo rat model of acute ischemic stroke. Methods Male SD rats were subjected to 2 hrs of transient middle cerebral artery occlusion (tMCAO). Rats treated with Solulin intravenously shortly before reperfusion were compared to rats receiving normal saline i.v. with respect to infarct volumes, neurological deficits and mortality. Gene expression of IL-6, IL-1β, TNF-α, MMP-9, CD11B and GFAP were semiquantitatively analyzed by rtPCR of the penumbra. Results 24 hrs after reperfusion, rats were neurologically tested, euthanized and infarct volumes determined. Solulin significantly reduced mean total (p = 0.001), cortical (p = 0.002), and basal ganglia (p = 0.036) infarct volumes. Hippocampal infarct volumes (p = 0.191) were not significantly affected. Solulin significantly downregulated the expression of IL-1β (79%; p < 0.001), TNF-α (59%; p = 0.001), IL-6 (47%; p = 0.04), and CD11B (49%; p = 0.001) in the infarcted cortex compared to controls. Conclusions Solulin reduced mean total, cortical and basal ganglia infarct volumes and regulated a subset of cytokines and proteases after tMCAO suggesting the potency of this compound for therapeutic interventions. PMID:22082476

  5. Estrogen-Responsive Transient Expression Assay Using a Brain Aromatase-Based Reporter Gene in Zebrafish (Danio rerio)

    PubMed Central

    Kim, Dong-Jae; Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Na, Yi-Rang; Park, Sung-Hoon; Lee, Hyun-Kyoung; Dutta, Noton Kumar; Kawakami, Koichi; Park, Jae-Hak

    2009-01-01

    Whereas endogenous estrogens play an important role in the development, maintenance, and function of female and male reproductive organs, xenoestrogens present in the environment disrupt normal endocrine function in humans and wildlife. Various in vivo and in vitro assays have been developed to screen these xenoestrogens. However, traditional in vivo assays are laborious and unsuitable for large-scale screening, and in vitro assays do not necessarily replicate in vivo functioning. To overcome these limitations, we developed a transient expression assay in zebrafish, into which a brain aromatase (cyp19a1b)-based estrogen-responsive reporter gene was introduced. In response to 17β-estradiol (10−6 M) and heptachlor (10−6 M), zebrafish embryos carrying the reporter construct expressed enhanced green fluorescent protein in the olfactory bulb, telencephalon, preoptic area, and mediobasal hypothalamus. This system will serve to model the in vivo conversion and breakdown of estrogenic compounds and thus provide a rapid preliminary screening method to estimate their estrogenicity. PMID:19887024

  6. Escin attenuates cognitive deficits and hippocampal injury after transient global cerebral ischemia in mice via regulating certain inflammatory genes.

    PubMed

    Zhang, Leiming; Fu, Fenghua; Zhang, Xiumei; Zhu, Mei; Wang, Tian; Fan, Huaying

    2010-09-01

    Considerable evidence has been accumulated demonstrating an important role for inflammation in ischemic brain injury and its contribution to greater cerebral damage after ischemia. Blocking the inflammatory reaction promotes neuroprotection and shows therapeutic potential for clinical treatment of ischemic brain injury. Escin, a natural mixture of triterpenoid saponin isolated from the seed of the horse chestnut, demonstrates antiedematous and anti-inflammatory effects. Here we assessed neuroprotective effects of escin with a transient global cerebral ischemia model. Global cerebral ischemia was induced by occluding both common carotid arteries and withdrawing 0.3ml of blood from the tail vein in mice. Treatment with escin was initiated 0.5h after ischemia induction and given once a day for three consecutive days. Then animals were assessed using the Morris water-maze test and step-down passive avoidance test. Acetylcholinesterase (AChE) activity, histological pathology, and expression of inflammatory genes in the hippocampus were determined. The results showed escin significantly improved learning and memory recovery and reduced hippocampal damage in the cerebral ischemic mice. However, donepezil merely improved learning and memory recovery but did not ameliorate hippocampal damage in the cerebral ischemic mice. Furthermore, we found escin significantly downregulated certain inflammatory gene expression and upregulated expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), which was recently reported as a neuroprotective protein in the brain. Our results indicate that inhibition of inflammation and protection of hippocampal neurons by escin may be a potentially useful therapy for ischemic brain injury. Copyright 2010 Elsevier Ltd. All rights reserved.

  7. Plasmid-based transient human stromal cell-derived factor-1 gene transfer improves cardiac function in chronic heart failure

    PubMed Central

    Sundararaman, S; Miller, T J; Pastore, J M; Kiedrowski, M; Aras, R; Penn, M S

    2011-01-01

    We previously demonstrated that transient stromal cell-derived factor-1 alpha (SDF-1) improved cardiac function when delivered via cell therapy in ischemic cardiomyopathy at a time remote from acute myocardial infarction (MI) rats. We hypothesized that non-viral gene transfer of naked plasmid DNA-expressing hSDF-1 could similarly improve cardiac function. To optimize plasmid delivery, we tested SDF-1 and luciferase plasmids driven by the cytomegalovirus (CMV) promoter with (pCMVe) or without (pCMV) translational enhancers or α myosin heavy chain (pMHC) promoter in a rodent model of heart failure. In vivo expression of pCMVe was 10-fold greater than pCMV and pMHC expression and continued over 30 days. We directly injected rat hearts with SDF-1 plasmid 1 month after MI and assessed heart function. At 4 weeks after plasmid injection, we observed a 35.97 and 32.65% decline in fractional shortening (FS) in control (saline) animals and pMHC-hSDF1 animals, respectively, which was sustained to 8 weeks. In contrast, we observed a significant 24.97% increase in animals injected with the pCMVe-hSDF1 vector. Immunohistochemistry of cardiac tissue revealed a significant increase in vessel density in the hSDF-1-treated animals compared with control animals. Increasing SDF-1 expression promoted angiogenesis and improved cardiac function in rats with ischemic heart failure along with evidence of scar remodeling with a trend toward decreased myocardial fibrosis. These data demonstrate that stand-alone non-viral hSDF-1 gene transfer is a strategy for improving cardiac function in ischemic cardiomyopathy. PMID:21472007

  8. Transient receptor potential melastatin 8 gene polymorphism is associated with cold-induced airway hyperresponsiveness in bronchial asthma.

    PubMed

    Naumov, Denis E; Perelman, Juliy M; Kolosov, Victor P; Potapova, Tatyana A; Maksimov, Vladimir N; Zhou, Xiangdong

    2015-11-01

    Cold-induced airway hyperresponsiveness (CAH) is common in bronchial asthma (BA) patients and represents a problem for those living in cold climate. Transient receptor potential melastatin 8 (TRPM8) channel is the main cold temperature sensor in humans that could mediate cold response in asthmatics with CAH. No associations between TRPM8 gene polymorphisms and CAH have been reported. The present study involved 123 BA patients. CAH was assessed by 3-min isocapnic (5% CO2 ) cold air (-20°C) hyperventilation challenge. The c.750G > C (rs11562975), c.1256G > A (rs7593557), c.3048C > T (rs11563208) and c.3174C > G (rs11563071) polymorphisms of TRPM8 gene were genotyped by allele-specific polymerase chain reaction (PCR) and PCR with subsequent restriction fragment length polymorphism analysis. GC genotype and C allele carriers of the c.750G > C (rs11562975) polymorphism were more frequently observed to exhibit CAH. The estimated odds ratio for the GC genotype was 3.73 95%CI (1.48; 9.37), P = 0.005. Furthermore, GC heterozygotes had a prominent decrease in forced expiratory volume in 1 s after the challenge as compared to GG homozygotes (-12% (-16; -8.1) vs -6.45% (-11; -2.1), P < 0.001). GC carriers also had a marked reduction in other spirometric parameters. The GC variant of the TRPM8:c.750G > C (rs11562975) polymorphism is associated with CAH in patients with BA, which suggests a potential role of TRPM8 in CAH development. © 2015 Asian Pacific Society of Respirology.

  9. Immersion infection of germ-free zebrafish with Listeria monocytogenes induces transient expression of innate immune response genes

    PubMed Central

    Shan, Ying; Fang, Chun; Cheng, Changyong; Wang, Yong; Peng, Jinrong; Fang, Weihuan

    2015-01-01

    Zebrafish, Denio rerio, can be an alternative to other classic animal models for human infectious diseases to examine the processes of microbial infections and host–pathogen interactions in vivo because of their small body dimension but large clutch size. We established germ-free zebrafish infection models of Listeria monocytogenes through different routes of infection: oral immersion and injection via yolk sac, brain ventricle and blood island. Immersion of zebrafish larva even with 1010 CFU/mL L. monocytogenes EGDe strain in egg water was unable to cause mortality, but GFP-expressing bacteria in the gut lumen can be observed in frozen sections. Several selected maker genes of the innate immune system, including cyp1a, irg1l, il1b, and mmp9, were significantly induced by oral immersion not only with strain EGDe, but also with strain M7 and L. innocua, though to a lesser degree (P < 0.01). Such induction appears to be transient with peak at 48 h post-infection, but returned to basal level at 72 h post-infection. Of the three injection routes, mortality after infection by yolk sac was 80% in early stage of infection. Few eggs can survive and hatch. Injection into zebrafish embryos via brain ventricle or blood island led to progressive lethal infection. L. mocytogenes EGDe showed steady replication in the fish embryos and was far more pathogenic than strain M7, which is consistent with findings in the murine model. We conclude that zebrafish can serve as susceptible and microscopically visible infection models for L. monocytogenes via different routes and can be applied to further studies on the interactions between bacterial virulence factors and host immune responses. PMID:25972853

  10. Marek's disease virus-induced transient paralysis is associated with cytokine gene expression in the nervous system.

    PubMed

    Abdul-Careem, M F; Hunter, B D; Sarson, A J; Mayameei, A; Zhou, H; Sharif, S

    2006-01-01

    Marek's disease (MD)-associated transient paralysis (TP) was experimentally induced in chickens by intraperitoneal inoculation of RB1B strain of Marek's disease virus (MDV). Between 7 and 11 days post-infection (d.p.i.), neck and limb paralysis was observed in 18% of infected chickens, which was associated with various degrees of edema, vacuolation, perivascular cuffing of mononuclear cells, and glial cell infiltration mainly in the cerebrum, cerebellum, and brain stem. The chickens that were infected but did not progress to develop TP until 12 d.p.i. also had similar lesions suggestive of encephalitis in the cerebrum, cerebellum, and brain stem. Chickens infected with MDV had more interleukin (IL)-6, IL-12, and interferon (IFN)-gamma in their brain tissues compared to uninfected chickens. Moreover, IL-18 was significantly increased in brain tissues of birds showing clinical signs of TP compared to uninfected birds. Importantly, the expression of IL-6, IL-18, and IFN- gamma in brain tissues of MDV-infected chickens with signs of TP was significantly increased compared to that in asymptomatic MDV-infected birds. MDV genome load in the brain of chickens showing clinical signs of TP was higher than that in asymptomatic MDV-infected chickens but was not statistically significant. The lesions in the cervical, thoracic, and lumbar spinal cord segments in MDVinfected chickens were characterized mainly by perivascular cuffing of mononuclear cells irrespective of the group. The expression of mRNA for IL-18 and IFN-gamma genes was not significantly different in spinal cord tissues of chickens with TP compared to clinically normal, MDV-infected and noninfected chickens. These results suggest possible underlying immunologic mechanisms for MDV-induced TP.

  11. Long-term stability of marker gene expression in Prunus subhirtella: a model fruit tree species.

    PubMed

    Maghuly, Fatemeh; da Câmara Machado, Artur; Leopold, Stephan; Khan, Mahmood Ali; Katinger, Hermann; Laimer, Margit

    2007-01-01

    Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. Since trees are particularly suited for long-term evaluations of the impact of the technology, Prunus subhirtella autumno rosa (PAR) was chosen as model fruit tree species and transformed with a reporter gene (uidA) under the control of the 35S promoter. Using Southern and GUS fluorometric techniques, we compared transgene copy numbers and observed stability of transgene expression levels in 34 different transgenic plants, grown under in vitro, greenhouse and screenhouse conditions, over a period of 9 years. An influence of grafting on gene expression was not observed. No silenced transgenic plant was detected. Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs, confirming the value of PAR as model species to study season-dependent regulation in mature stone fruit tissues. While the Agrobacterium-derived Prunus transformants contained one to two copies of the transgenes, 91% of the transgenic events also contained various lengths of the bacterial plasmid backbone, indicating that the Agrobacterium-mediated transformation is not as precise as previously perceived. The implications for public acceptance and future applications are discussed.

  12. Regulation of brain-derived neurotrophic factor gene expression after transient middle cerebral artery occlusion with and without brain damage.

    PubMed

    Kokaia, Z; Zhao, Q; Kokaia, M; Elmér, E; Metsis, M; Smith, M L; Siesjö, B K; Lindvall, O

    1995-11-01

    Levels of mRNA for c-fos, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), TrkB, and TrkC were studied using in situ hybridization in the rat brain at different reperfusion times after unilateral middle cerebral artery occlusion (MCAO). Short-term (15 min) MCAO, which does not cause neuronal death, induced elevated BDNF mRNA expression confined to ipsilateral frontal and cingulate cortices outside the ischemic area. With a longer duration of MCAO (2 h), which leads to cortical infarction, the increase was more marked and elevated BDNF mRNA levels were also detected bilaterally in dentate granule cells and CA1 and CA3 pyramidal neurons. Maximum expression was found after 2 h of reperfusion. At 24 h BDNF mRNA expression had returned to control values. In the ischemic core of the parietal cortex only scattered neurons were expressing high levels of BDNF mRNA after 15 min and 2 h of MCAO. Analysis of different BDNF transcripts showed that MCAO induced a marked increase of exon III mRNA but only small increases of exon I and II mRNAs in cortex and hippocampus. In contrast to BDNF mRNA, elevated expression of c-fos mRNA was observed in the entire ipsilateral cerebral cortex, including the ischemic core, after both 15 min and 2 h of MCAO. Two hours of MCAO also induced transient, bilateral increases of NGF and TrkB mRNA levels and a decrease of NT-3 mRNA expression, confined to dentate granule cells. The upregulation of BDNF mRNA expression in cortical neurons after MCAO is probably triggered by glutamate through a spreading depression-like mechanism. The lack of response of the BDNF gene in the ischemic core may be due to suppression of signal transduction or transcription factor synthesis caused by the ischemia. The observed pattern of gene expression after MCAO agrees well with a neuroprotective role of BDNF in cortical neurons. However, elevated levels of NGF and BDNF protein could also increase synaptic efficacy in the

  13. CASE-REPORT Dysregulation of gene expression in a patient with depressive disorder after transient ischemic attack confirmed by a neurophysiological neuromarker.

    PubMed

    Trystuła, M; Żychowska, M; Wilk-Frańczuk, M; Kropotov, J D; Pąchalska, M

    2017-02-16

    The aim of this study was to evaluate dysregulation of gene expression associated with the cellular stress response in a patient with a post-"warning stroke" depressive disorder confirmed by the presence of a neurophysiological neuromarker through the use of quantitative EEG and event-related potentials. The patient was tested for seven genes associated with the stress reaction: HSPA1A, HSPB1, IL6, IL10, CRP, and HSF-1 along with NF-κB, compared to gene expression in health controls. A 54-year-old patient with a past history of schizophrenia (at the age of 20), and of transient ischemic attack (at the age of 53) and depressive disorder confirmed by functional, cognitive, emotional, and affectional diagnostics underwent additional testing for expression of the genes associated with stress response. The expression of genes coding for heat shock protein (HSPA1A, HSPB1), interleukins (IL6, IL10), and C-reactive protein was tested along with factors that regulate their expression. The results of the tests conducted on this patient were compared with 42 healthy control subjects. Diagnostic testing revealed upregulation in expression of these genes, presenting as increased expression of the target genes and of the regulatory genes. A post-"warning stroke" depressive disorder appears to be associated with overexpression of the genes coding for HSP and interleukins. Further research on larger groups of people may provide grounds for treatment modification.

  14. Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

    PubMed

    Liu, Lin-Lin; Zhao, Hui; Ma, Teng-Fei; Ge, Fei; Chen, Ce-Shi; Zhang, Ya-Ping

    2015-01-01

    Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct), and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2) expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

  15. Fasting during the suckling-weaning transient period of rats induces inflammatory gene expression in the adipose tissue and peripheral leukocytes.

    PubMed

    Honma, Kazue; Mawatari, Riko; Iikeda, Misa; Mochizuki, Kazuki; Goda, Toshinao

    2016-01-01

    Nutritional deficiency during developmental stages could be associated with subsequent development of inflammation-related metabolic abnormalities. In this study, we examined the effects of a 3-d fast during the suckling-weaning transient period of rats, and subsequent intake of high-fat-high-sucrose (HF) and low-fat-high-starch (LF) diets in adulthood, on the expression of inflammatory genes in adipose tissue and peripheral leukocytes. Male Sprague-Dawley rats were deprived of food for 3 d during the suckling-weaning transient period, and were subsequently fed an HF or LF diet for 14 wk from 17 wk of age. Serum monocyte chemoattractant protein-1 (MCP-1) concentration and mRNA levels of inflammatory genes in mesenteric adipose tissues were assessed at 31 wk of age. The mRNA levels of inflammatory genes at 0 h and 2 h after oral glucose load at 30 wk of age in peripheral leukocytes were measured. Fasting induced circulating MCP-1 protein in rats fed an LF diet but not an HF diet. The HF diet induced high mRNA levels of tumor necrosis factor-α, interleukin-1β, and S100 proteins in peripheral leukocytes at 2 h after glucose load in fasted rats when compared with controls. Expression of CD11c, an activated macrophage marker, was induced in the fasted group given an HF diet during adulthood. Fasting rats during the suckling-weaning transient period and an HF diet intake during adulthood enhance inflammation by promoting the expression of inflammatory genes in adipose tissue and peripheral leukocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Agrobacterium-mediated genetic transformation of Prunus salicina

    USDA-ARS?s Scientific Manuscript database

    We report Agrobacterium tumefaciens-mediated transformation from hypocotyls slices of two Prunus salicina varieties, 'Angeleno' and 'Larry Anne', using a modification of the technique previously described for P. domestica. Regeneration rates on thidiazuron (TDZ) and indole-3-butyric acid (IBA) supp...

  17. Agrobacterium-mediated genetic transformation of Fraxinus americana hypocotyls

    Treesearch

    Kaitlin J. Palla; Paula M. Pijut

    2015-01-01

    An Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for white ash (Fraxinus americana) using hypocotyls as the initial explants. Hypocotyls isolated from mature embryos germinated on Murashige and Skoog (MS) medium supplemented with 22.2 µM 6-benzyladenine (BA) and 0.5 µM...

  18. Expression of genes involved in the salicylic acid pathway in type h1 thioredoxin transiently silenced pepper plants during a begomovirus compatible interaction.

    PubMed

    Luna-Rivero, Marianne S; Hernández-Zepeda, Cecilia; Villanueva-Alonzo, Hernán; Minero-García, Yereni; Castell-González, Salvador E; Moreno-Valenzuela, Oscar A

    2016-04-01

    The type-h thioredoxins (TRXs) play a fundamental role in oxidative stress tolerance and defense responses against pathogens. In pepper plants, type-h TRXs participate in the defense mechanism against Cucumber mosaic virus. The goal of this study was to analyze the role of the CaTRXh1-cicy gene in pepper plants during compatible interaction with a DNA virus, the Euphorbia mosaic virus-Yucatan Peninsula (EuMV-YP). The effects of a transient silencing of the CaTRXh1-cicy gene in pepper plants wëre evaluated by observing the accumulation of viral DNA and the visible symptoms of pepper plants under different treatments. The accumulation of salicylic acid (SA) and the relative expression of the defense genes NPR1 and PR10 were also evaluated. Results showed that viral DNA accumulation was higher in transiently CaTRXh1-cicy silenced plants that were also infected with EuMV-YP. Symptoms in these plants were more severe compared to the non-silenced plants infected with EuMV-YP. The SA levels in the EuMV-YP-infected plants were rapidly induced at 1 h post infection (hpi) in comparison to the non-silenced plants inoculated with EuMV-YP. Additionally, in pepper plants infected with EuMV-YP, the expression of NPR1 decreased by up to 41 and 58 % at 28 days post infection (dpi) compared to the non-silenced pepper plants infected with only EuMV-YP and healthy non-inoculated pepper plants, respectively. PR10 gene expression decreased by up to 70 % at 28 dpi. Overall, the results indicate that the CaTRXh1-cicy gene participates in defense mechanisms during the compatible interaction of pepper plants with the EuMV-YP DNA virus.

  19. Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene.

    PubMed

    Narendran, M; Deole, Satish G; Harkude, Satish; Shirale, Dattatray; Nanote, Asaram; Bihani, Pankaj; Parimi, Srinivas; Char, Bharat R; Zehr, Usha B

    2013-08-01

    Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ). Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T1 generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T0 lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.

  20. Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene*

    PubMed Central

    ZHAO, Qi-chao; LIU, Ming-hong; ZHANG, Xian-wen; LIN, Chao-yang; ZHANG, Qing; SHEN, Zhi-cheng

    2015-01-01

    Insect resistance and glyphosate tolerance have been two of the most important traits in the genetic improvement of various crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, Cry1Ac and Cry1Ig, and a modified glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (G10) were combined into a single transferred DNA (T-DNA) fragment and introduced into rice by Agrobacterium-mediated transformation. A transgenic line with single-copy T-DNA insertion named GAI-14 was found to be highly resistant to striped stem borer and rice leaf roller, and tolerant to glyphosate. Analysis of T-DNA border sequence suggested that the transgenes were inserted at the chromosome 3 and appeared to have not interrupted any known or putative genes. A field trial observed no significant difference in the basic agronomic traits between GAI-14 and the recipient rice. PMID:26465130

  1. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

    PubMed Central

    2009-01-01

    Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant

  2. Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice.

    PubMed

    Peng, Baowei; Ye, Peiqing; Blazar, Bruce R; Freeman, Gordon J; Rawlings, David J; Ochs, Hans D; Miao, Carol H

    2008-09-01

    Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid-treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS(+)CD4(+) T cells and activation of CD25(+)Foxp3(+) Tregs in the peripheral blood, spleen, and lymph nodes. CD4(+) T cells from anti-ICOS-treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-beta. Moreover, CD4(+)CD25(+) Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS-treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Phix 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

  3. Transient transcription of the somatostatin gene at the time of estrogen-dependent organization of the sexually dimorphic nucleus of the rat preoptic area.

    PubMed

    Orikasa, Chitose; Kondo, Yasuhiko; Sakuma, Yasuo

    2007-03-01

    In situ hybridization detected a transient, sex-specific transcription of somatostatin gene in the central part of the rat medial preoptic nucleus, which coincides with the sexually dimorphic nucleus of the preoptic area (SDN-POA), during, but not after, the establishment of sex difference. On postnatal d 1 (day of birth), somatostatin mRNA was detected in the SDN-POA of both sexes. On d 8 through 35, the area of somatostatin mRNA-positive cells was significantly larger in males than in females. In males the area attained its maximum size on d 15 and diminished gradually thereafter. In females the area did not change in size during this period. On d 60 expression of somatostatin mRNA was low and not different between sexes. Throughout the observed period, Nissl staining and calbindin immunohistochemistry enabled visualization of the typical SDN-POA in the same region. As with Nissl staining and calbindin immunohistochemistry, somatostatin mRNA hybridization on d 15 revealed a reversal of the sexual dimorphism in the size of the SDN-POA in males that had been neonatally orchidectomized or females given estrogen as pups, showing that sex steroid milieu during the organizational period determines the area occupied by somatostatin mRNA-positive cells. Sex-specific, transient transcription of the somatostatin gene may causally relate to the estrogen-dependent organization of the SDN-POA.

  4. A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature-sensitive processes in plant-pathogen interactions.

    PubMed

    Del Toro, Francisco; Tenllado, Francisco; Chung, Bong-Nam; Canto, Tomas

    2014-10-01

    Localized expression of genes in plants from T-DNAs delivered into plant cells by Agrobacterium tumefaciens is an important tool in plant research. The technique, known as agroinfiltration, provides fast, efficient ways to transiently express or silence a desired gene without resorting to the time-consuming, challenging stable transformation of the host, the use of less efficient means of delivery, such as bombardment, or the use of viral vectors, which multiply and spread within the host causing physiological alterations themselves. A drawback of the agroinfiltration technique is its temperature dependence: early studies have shown that temperatures above 29 °C are nonpermissive to tumour induction by the bacterium as a result of failure in pilus formation. However, research in plant sciences is interested in studying processes at these temperatures, above the 25 °C experimental standard, common to many host-environment and host-pathogen interactions in nature, and agroinfiltration is an excellent tool for this purpose. Here, we measured the efficiency of agroinfiltration for the expression of reporter genes in plants from T-DNAs at the nonpermissive temperature of 30 °C, either transiently or as part of viral amplicons, and envisaged procedures that allow and optimize its use for gene expression at this temperature. We applied this technical advance to assess the performance at 30 °C of two viral suppressors of silencing in agropatch assays [Potato virus Y helper component proteinase (HCPro) and Cucumber mosaic virus 2b protein] and, within the context of infection by a Potato virus X (PVX) vector, also assessed indirectly their effect on the overall response of the host Nicotiana benthamiana to the virus.

  5. A survey of FLS2 genes from multiple citrus species identifies candidates for enhancing disease resistance to Xanthomonas citri ssp. citri.

    PubMed Central

    Shi, Qingchun; Febres, Vicente J; Jones, Jeffrey B; Moore, Gloria A

    2016-01-01

    Pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) is an important component of plant innate immunity. In a previous study, we showed that the PAMP flg22 from Xanthomonas citri ssp. citri (Xflg22), the causal agent of citrus canker, induced PTI in citrus, which correlated with the observed levels of canker resistance. Here, we identified and sequenced two bacterial flagellin/flg22 receptors (FLS2-1 and FLS2-2) from ‘Duncan’ grapefruit (Citrus paradisi, CpFLS2-1 and CpFLS2-2) and ‘Sun Chu Sha’ mandarin (C. reticulata, CrFLS2-1 and CrFLS2-2). We were able to isolate only one FLS2 from ‘Nagami’ kumquat (Fortunella margarita, FmFLS2-1) and gene flanking sequences suggest a rearrangement event that resulted in the deletion of FLS2-2 from the genome. Phylogenetic analysis, gene structure and presence of critical amino acid domains all indicate we identified the true FLS2 genes in citrus. FLS2-2 was more transcriptionally responsive to Xflg22 than FLS2-1, with induced expression levels higher in canker-resistant citrus than in susceptible ones. Interestingly, ‘Nagami’ kumquat showed the highest FLS2-1 steady-state expression levels, although it was not induced by Xflg22. We selected FmFLS2-1, CrFLS2-2 and CpFLS2-2 to further evaluate their capacity to enhance bacterial resistance using Agrobacterium-mediated transient expression assays. Both FmFLS2-1 and CrFLS2-2, the two proteins from canker-resistant species, conferred stronger Xflg22 responses and reduced canker symptoms in leaves of the susceptible grapefruit genotype. These two citrus genes will be useful resources to enhance PTI and achieve resistance against canker and possibly other bacterial pathogens in susceptible citrus types. PMID:27222722

  6. Systems Analysis of Early Host Gene Expression Provides Clues for Transient Mycobacterium avium ssp avium vs. Persistent Mycobacterium avium ssp paratuberculosis Intestinal Infections

    PubMed Central

    Khare, Sangeeta; Drake, Kenneth L.; Lawhon, Sara D.; Nunes, Jairo E. S.; Figueiredo, Josely F.; Rossetti, Carlos A.; Gull, Tamara; Everts, Robin E.; Lewin, Harris. A.; Adams, Leslie Garry

    2016-01-01

    It has long been a quest in ruminants to understand how two very similar mycobacterial species, Mycobacterium avium ssp. paratuberculosis (MAP) and Mycobacterium avium ssp. avium (MAA) lead to either a chronic persistent infection or a rapid-transient infection, respectively. Here, we hypothesized that when the host immune response is activated by MAP or MAA, the outcome of the infection depends on the early activation of signaling molecules and host temporal gene expression. To test our hypothesis, ligated jejuno-ileal loops including Peyer’s patches in neonatal calves were inoculated with PBS, MAP, or MAA. A temporal analysis of the host transcriptome profile was conducted at several times post-infection (0.5, 1, 2, 4, 8 and 12 hours). When comparing the transcriptional responses of calves infected with the MAA versus MAP, discordant patterns of mucosal expression were clearly evident, and the numbers of unique transcripts altered were moderately less for MAA-infected tissue than were mucosal tissues infected with the MAP. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis. Bayesian network modeling identified mechanistic genes, gene-to-gene relationships, pathways and Gene Ontologies (GO) biological processes that are involved in specific cell activation during infection. MAP and MAA had significant different pathway perturbation at 0.5 and 12 hours post inoculation. Inverse processes were observed between MAP and MAA response for epithelial cell proliferation, negative regulation of chemotaxis, cell-cell adhesion mediated by integrin and regulation of cytokine-mediated signaling. MAP inoculated tissue had significantly lower expression of phagocytosis receptors such as mannose receptor and complement receptors. This study reveals that perturbation of genes and cellular pathways during MAP infection resulted in host evasion by mucosal membrane barrier weakening to access entry in the ileum

  7. Systems Analysis of Early Host Gene Expression Provides Clues for Transient Mycobacterium avium ssp avium vs. Persistent Mycobacterium avium ssp paratuberculosis Intestinal Infections.

    PubMed

    Khare, Sangeeta; Drake, Kenneth L; Lawhon, Sara D; Nunes, Jairo E S; Figueiredo, Josely F; Rossetti, Carlos A; Gull, Tamara; Everts, Robin E; Lewin, Harris A; Adams, Leslie Garry

    It has long been a quest in ruminants to understand how two very similar mycobacterial species, Mycobacterium avium ssp. paratuberculosis (MAP) and Mycobacterium avium ssp. avium (MAA) lead to either a chronic persistent infection or a rapid-transient infection, respectively. Here, we hypothesized that when the host immune response is activated by MAP or MAA, the outcome of the infection depends on the early activation of signaling molecules and host temporal gene expression. To test our hypothesis, ligated jejuno-ileal loops including Peyer's patches in neonatal calves were inoculated with PBS, MAP, or MAA. A temporal analysis of the host transcriptome profile was conducted at several times post-infection (0.5, 1, 2, 4, 8 and 12 hours). When comparing the transcriptional responses of calves infected with the MAA versus MAP, discordant patterns of mucosal expression were clearly evident, and the numbers of unique transcripts altered were moderately less for MAA-infected tissue than were mucosal tissues infected with the MAP. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis. Bayesian network modeling identified mechanistic genes, gene-to-gene relationships, pathways and Gene Ontologies (GO) biological processes that are involved in specific cell activation during infection. MAP and MAA had significant different pathway perturbation at 0.5 and 12 hours post inoculation. Inverse processes were observed between MAP and MAA response for epithelial cell proliferation, negative regulation of chemotaxis, cell-cell adhesion mediated by integrin and regulation of cytokine-mediated signaling. MAP inoculated tissue had significantly lower expression of phagocytosis receptors such as mannose receptor and complement receptors. This study reveals that perturbation of genes and cellular pathways during MAP infection resulted in host evasion by mucosal membrane barrier weakening to access entry in the ileum

  8. Morphological and physiological characteristics of transgenic cherry tomato mutant with HBsAg gene.

    PubMed

    Guan, Z J; Guo, B; Huo, Y L; Hao, H Y; Wei, Y H

    2011-08-01

    HBsAg gene was previously introduced into cherry tomato (Lycopersicum esculentum var. cerasiforme) by Agrobacterium-mediated transformation. To investigate the side effect of HBsAg gene in cherry tomato, we analyzed morphological and physiological characteristics of the transgenic mutant N244. The process was performed under field conditions. The results suggested that the mutant N244 exhibited morphological, cytological and physiological variation. First of all, compared with the wild plants NK, N244 had fleshy and dark green leaves, the fewer notches of leaf edge, more adventitious roots and barren seeds. Moreover, the chromosome of N244 were found to be triploid (n = 36) by flow cytometric analysis. Furthermore, N244 has obvious physiological alterations, as compared to NK. It was speculated that transformation of the genes probably led to ploidy variation, and further caused phenotype and physiological changes of plants. Our study will reveal side effects of the mutants, and promote cultivation of transgenic plants in the field.

  9. Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice.

    PubMed

    Nishizawa-Yokoi, Ayako; Nonaka, Satoko; Saika, Hiroaki; Kwon, Yong-Ik; Osakabe, Keishi; Toki, Seiichi

    2012-12-01

    Evidence for the involvement of the nonhomologous end joining (NHEJ) pathway in Agrobacterium-mediated transferred DNA (T-DNA) integration into the genome of the model plant Arabidopsis remains inconclusive. Having established a rapid and highly efficient Agrobacterium-mediated transformation system in rice (Oryza sativa) using scutellum-derived calli, we examined here the involvement of the NHEJ pathway in Agrobacterium-mediated stable transformation in rice. Rice calli from OsKu70, OsKu80 and OsLig4 knockdown (KD) plants were infected with Agrobacterium harboring a sensitive emerald luciferase (LUC) reporter construct to evaluate stable expression and a green fluorescent protein (GFP) construct to monitor transient expression of T-DNA. Transient expression was not suppressed, but stable expression was reduced significantly, in KD plants. Furthermore, KD-Ku70 and KD-Lig4 calli exhibited an increase in the frequency of homologous recombination (HR) compared with control calli. In addition, suppression of OsKu70, OsKu80 and OsLig4 induced the expression of HR-related genes on treatment with DNA-damaging agents. Our findings suggest strongly that NHEJ is involved in Agrobacterium-mediated stable transformation in rice, and that there is a competitive and complementary relationship between the NHEJ and HR pathways for DNA double-strand break repair in rice.

  10. Transient Global Cerebral Ischemia Induces RNF213, a Moyamoya Disease Susceptibility Gene, in Vulnerable Neurons of the Rat Hippocampus CA1 Subregion and Ischemic Cortex.

    PubMed

    Sato-Maeda, Mika; Fujimura, Miki; Rashad, Sherif; Morita-Fujimura, Yuiko; Niizuma, Kuniyasu; Sakata, Hiroyuki; Ikawa, Shuntaro; Tominaga, Teiji

    2017-09-01

    The RING finger protein 213 (RNF213) is an important susceptibility gene for moyamoya disease (MMD) and is also implicated in other types of intracranial major artery stenosis/occlusion (ICAS); however, the role of RNF213 in the development of ICAS including MMD is unclear. The constitutive expression of the RNF213 gene is relatively weak in brain tissue, while information regarding the expression patterns of the RNF213 gene under cerebral ischemia, which is one of characteristic pathologies associated with ICAS, is currently limited. Our objective was to address this critical issue, and we investigated Rnf213 mRNA expression in rat brains after 5 minutes of transient global cerebral ischemia (tGCI) by occluding the common carotid arteries coupled with severe hypotension. Rnf213 gene expression patterns were investigated with in situ RNA hybridization and a real-time polymerase chain reaction (PCR) from 1 to 72 hours after tGCI. In situ RNA hybridization revealed a significant increase in Rnf213 mRNA levels in the hippocampus CA1 sub-region 48 hours after tGCI. The significant induction of the Rnf213 gene was also evident in the ischemic cortex. Double staining of Rnf213 mRNA with NeuN immunohistochemistry revealed Rnf213 hybridization signal expression exclusively in neurons. The real-time PCR analysis confirmed the induction of the Rnf213 gene after tGCI. The up-regulation of the Rnf213 gene in vulnerable neurons in the hippocampus CA1 after tGCI suggests its involvement in forebrain ischemia, which is an underlying pathology of MMD. Further investigations are needed to elucidate its exact role in the pathophysiology of ICAS including MMD. Copyright © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  11. Genetic transformation of Begonia tuberhybrida by Ri rol genes.

    PubMed

    Kiyokawa, S; Kikuchi, Y; Kamada, H; Harada, H

    1996-04-01

    We have developed an Agrobacterium -mediated transformation system for commercial Begonia species. The leaf explants of Begonia semperflorens, Begonia x hiemalis and B. tuberhybrida were inoculated with Agrobacterium tumefaciens LBA4404 harboring a binary vector pBI121 which contains rolA, B and C genes of an agropine type Ri plasmid (pRiA4b). Kanamycin resistant shoots of B. tuberhybrida were obtained on MS agar medium supplemented with 0.1 mg/l NAA, 0.5 mg/l BA, 500 mg/l claforan and 100 mg/l kanamycin. These shoots exhibited GUS activity and Southern analysis showed a single copy insertion into the genome. When the transgenic plants were transferred to soil, they displayed the phenotype specific to the transgenic plants by A. rhizogenes such as dwarfness, delay of flowering, and wrinkled leaves and petals.

  12. Expression of the synthetic neutralizing epitope gene of porcine epidemic diarrhea virus in tobacco plants without nicotine.

    PubMed

    Kang, Tae-Jin; Kim, Young-Sook; Jang, Yong-Suk; Yang, Moon-Sik

    2005-03-18

    Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in pigs, leading to death with a high mortality. In this study, the synthetic neutralizing epitope gene of PEDV, which was optimized based on the coding sequence of tobacco plant genes being modified, was expressed in the no-nicotine tobacco plants. The synthetic gene was cloned into the plant expression vector under the control of CaMV 35S promoter and transformed by an Agrobacterium-mediated transformation. The amount of synthetic epitope protein of PEDV detected in the transgenic tobacco plants was approximately 2.1% of the total soluble plant protein, which was approximately five-fold higher than that expressed with the native gene.

  13. A simple and reliable multi-gene transformation method for switchgrass.

    PubMed

    Ogawa, Yoichi; Shirakawa, Makoto; Koumoto, Yasuko; Honda, Masaho; Asami, Yuki; Kondo, Yasuhiro; Hara-Nishimura, Ikuko

    2014-07-01

    A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape. Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the 'herbicide dipping method'. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes.

  14. Molecular Transfer of Nematode Resistance Genes

    PubMed Central

    Williamson, V. M.; Ho, J.-Y.; Ma, H. M.

    1992-01-01

    Recombinant DNA techniques have been used to introduce agronomically valuable traits, including resistance to viruses, herbicides, and insects, into crop plants. Introduction of these genes into plants frequently involves Agrobacterium-mediated gene transfer. The potential exists for applying this technology to nematode control by introducing genes conferring resistance to nematodes. Transferred genes could include those encoding products detrimental to nematode development or reproduction as well as cloned host resistance genes. Host genes that confer resistance to cyst or root-knot nematode species have been identified in many plants. The best characterized is Mi, a gene that confers resistance to root-knot nematodes in tomato. A map-based cloning approach is being used to isolate the gene. For development of a detailed map of the region of the genome surrounding Mi, DNA markers genetically linked to Mi have been identified and analyzed in tomato lines that have undergone a recombination event near Mi. The molecular map will be used to identify DNA corresponding to Mi. We estimate that a clone of Mi will be obtained in 2-5 years. An exciting prospect is that introduction of this gene will confer resistance in plant species without currently available sources of resistance. PMID:19282989

  15. Computational identification of conserved transcription factor binding sites upstream of genes induced in rat brain by transient focal ischemic stroke.

    PubMed

    Pulliam, John V K; Xu, Zhenfeng; Ford, Gregory D; Liu, Cuimei; Li, Yonggang; Stovall, Kyndra C; Cannon, Virginetta S; Tewolde, Teclemichael; Moreno, Carlos S; Ford, Byron D

    2013-02-07

    Microarray analysis has been used to understand how gene regulation plays a critical role in neuronal injury, survival and repair following ischemic stroke. To identify the transcriptional regulatory elements responsible for ischemia-induced gene expression, we examined gene expression profiles of rat brains following focal ischemia and performed computational analysis of consensus transcription factor binding sites (TFBS) in the genes of the dataset. In this study, rats were sacrificed 24 h after middle cerebral artery occlusion (MCAO) stroke and gene transcription in brain tissues following ischemia/reperfusion was examined using Affymetrix GeneChip technology. The CONserved transcription FACtor binding site (CONFAC) software package was used to identify over-represented TFBS in the upstream promoter regions of ischemia-induced genes compared to control datasets. CONFAC identified 12 TFBS that were statistically over-represented from our dataset of ischemia-induced genes, including three members of the Ets-1 family of transcription factors (TFs). Microarray results showed that mRNA for Ets-1 was increased following tMCAO but not pMCAO. Immunohistochemical analysis of Ets-1 protein in rat brains following MCAO showed that Ets-1 was highly expressed in neurons in the brain of sham control animals. Ets-1 protein expression was virtually abolished in injured neurons of the ischemic brain but was unchanged in peri-infarct brain areas. These data indicate that TFs, including Ets-1, may influence neuronal injury following ischemia. These findings could provide important insights into the mechanisms that lead to brain injury and could provide avenues for the development of novel therapies. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Transient Expression of Fez Family Zinc Finger 2 Protein Regulates the Brn3b Gene in Developing Retinal Ganglion Cells.

    PubMed

    Qu, Chunsheng; Bian, Dandan; Li, Xue; Xiao, Jian; Wu, Chunping; Li, Yue; Jiang, Tian; Zhou, Xiangtian; Qu, Jia; Chen, Jie-Guang

    2016-04-01

    Retinal ganglion cells (RGCs) are projection neurons in the neural retina that relay visual information from the environment to the central nervous system. The early expression of MATH5 endows the post-mitotic precursors with RGC competence and leads to the activation ofBrn3bthat marks committed RGCs. Nevertheless, this fate commitment process and, specifically, regulation ofBrn3bremain elusive. To explore the molecular mechanisms underlying RGC generation in the mouse retina, we analyzed the expression and function of Fez family zinc finger 2 (FEZF2), a transcription factor critical for the development of projection neurons in the cerebral cortex.Fezf2mRNA and protein were transiently expressed at embryonic day 16.5 in the inner neuroblast layer and the prospective ganglion cell layer of the retina, respectively. Knockout ofFezf2in the developing retina reduced BRN3B+ cells and increased apoptotic cell markers.Fezf2knockdown by retinalin uteroelectroporation diminished BRN3B but not the coexpressed ISLET1 and BRN3A, indicating that the BRN3B decrease was the cause, not the result, of the overall reduction of BRN3B+ RGCs in theFezf2knockout retina. Moreover, the mRNA and promoter activity ofBrn3bwere increasedin vitroby FEZF2, which bound to a 5' regulatory fragment in theBrn3bgenomic locus. These results indicate that transient expression ofFezf2in the retina modulates the transcription ofBrn3band the survival of RGCs. This study improves our understanding of the transcriptional cascade required for the specification of RGCs and provides novel insights into the molecular basis of retinal development.

  17. Transient osteoporosis.

    PubMed

    Korompilias, Anastasios V; Karantanas, Apostolos H; Lykissas, Marios G; Beris, Alexandros E

    2008-08-01

    Transient osteoporosis is characterized primarily by bone marrow edema. The disease most commonly affects the hip, knee, and ankle in middle-aged men. Its cause remains unknown. The hallmark that separates transient osteoporosis from other conditions presenting with a bone marrow edema pattern is its self-limited nature. Laboratory tests usually do not contribute to the diagnosis. Plain radiographs may reveal regional osseous demineralization. Magnetic resonance imaging is used primarily for early diagnosis and monitoring disease progression. Early differentiation from more aggressive conditions with long-term sequelae is essential to avoid unnecessary treatment. Clinical entities such as transient osteoporosis of the hip and regional migratory osteoporosis are spontaneously resolving conditions. However, early differential diagnosis and surgical treatment are crucial for the patient with osteonecrosis of the hip or knee.

  18. Regulation of KChIP2 potassium channel beta subunit gene expression underlies the gradient of transient outward current in canine and human ventricle.

    PubMed

    Rosati, B; Pan, Z; Lypen, S; Wang, H S; Cohen, I; Dixon, J E; McKinnon, D

    2001-05-15

    Expression of four members of the KChIP family of potassium channel beta subunits was examined in canine heart. Only one member of the gene family, KChIP2, was expressed in heart. There was a steep gradient of KChIP2 mRNA expression across the canine ventricular free wall. KChIP2 mRNA was 25-fold more abundant in the epicardium than in the endocardium, and this gradient paralleled the gradient in transient outward current (Ito) expression. In contrast, Kv4.3 potassium channel alpha subunit mRNA was expressed at equal levels across the ventricular wall. There was no difference in the pharmacological sensitivity of epicardial and endocardial Ito channels to flecainide, suggesting that the current is produced by the same channel in the two tissues. A similar gradient of KChIP2 expression was found across the ventricular wall of human heart, but not rat heart. It is concluded that transcriptional regulation of the KChIP2 beta subunit gene, rather than the Kv4.3 [alpha] subunit gene, is the primary determinant regulating the transmural gradient of Ito expression in the ventricular free wall of canine and human heart.

  19. Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L.

    PubMed

    Ishizaki, Kimitsune; Johzuka-Hisatomi, Yasuyo; Ishida, Sakiko; Iida, Shigeru; Kohchi, Takayuki

    2013-01-01

    The liverwort Marchantia polymorpha is an emerging model organism on account of its ideal characteristics for molecular genetics in addition to occupying a crucial position in the evolution of land plants. Here we describe a method for gene targeting by applying a positive/negative selection system for reduction of non-homologous random integration to an efficient Agrobacterium-mediated transformation system using M. polymorpha sporelings. The targeting efficiency was evaluated by knocking out the NOP1 gene, which impaired air-chamber formation. Homologous recombination was observed in about 2% of the thalli that passed the positive/negative selection. With the advantage of utilizing the haploid gametophytic generation, this strategy should facilitate further molecular genetic analysis of M. polymorpha, in which many of the mechanisms found in land plants are conserved, yet in a less complex form.

  20. Overexpression of a Chitinase Gene from Trichoderma asperellum Increases Disease Resistance in Transgenic Soybean.

    PubMed

    Zhang, Fuli; Ruan, Xianle; Wang, Xian; Liu, Zhihua; Hu, Lizong; Li, Chengwei

    2016-12-01

    In the present study, a chi gene from Trichoderma asperellum, designated Tachi, was cloned and functionally characterized in soybean. Firstly, the effects of sodium thiosulfate on soybean Agrobacterium-mediated genetic transformation with embryonic tip regeneration system were investigated. The transformation frequency was improved by adding sodium thiosulfate in co-culture medium for three soybean genotypes. Transgenic soybean plants with constitutive expression of Tachi showed increased resistance to Sclerotinia sclerotiorum compared to WT plants. Meanwhile, overexpression of Tachi in soybean exhibited increased reactive oxygen species (ROS) level as well as peroxidase (POD) and catalase (SOD) activities, decreased malondialdehyde (MDA) content, along with diminished electrolytic leakage rate after S. sclerotiorum inoculation. These results suggest that Tachi can improve disease resistance in plants by enhancing ROS accumulation and activities of ROS scavenging enzymes and then diminishing cell death. Therefore, Tachi represents a candidate gene with potential application for increasing disease resistance in plants.

  1. Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L.

    PubMed Central

    Ishizaki, Kimitsune; Johzuka-Hisatomi, Yasuyo; Ishida, Sakiko; Iida, Shigeru; Kohchi, Takayuki

    2013-01-01

    The liverwort Marchantia polymorpha is an emerging model organism on account of its ideal characteristics for molecular genetics in addition to occupying a crucial position in the evolution of land plants. Here we describe a method for gene targeting by applying a positive/negative selection system for reduction of non-homologous random integration to an efficient Agrobacterium-mediated transformation system using M. polymorpha sporelings. The targeting efficiency was evaluated by knocking out the NOP1 gene, which impaired air-chamber formation. Homologous recombination was observed in about 2% of the thalli that passed the positive/negative selection. With the advantage of utilizing the haploid gametophytic generation, this strategy should facilitate further molecular genetic analysis of M. polymorpha, in which many of the mechanisms found in land plants are conserved, yet in a less complex form. PMID:23524944

  2. Transfer and expression of the rabbit defensin NP-1 gene in lettuce (Lactuca sativa).

    PubMed

    Song, D; Xiong, X; Tu, W F; Yao, W; Liang, H W; Chen, F J; He, Z Q

    2017-01-23

    Lettuce (Lactuca sativa L.) is an annual plant of the daisy family, Asteraceae, with high food and medicinal value. However, the crop is susceptible to several viruses that are transmitted by aphids and is highly vulnerable to post-harvest diseases, as well as insect and mammal pests and fungal and bacterial diseases. Here, the rabbit defensin gene NP-1 was transferred into lettuce by Agrobacterium-mediated transformation to obtain a broad-spectrum disease-resistant lettuce. Transgenic lettuce plants were selected and regenerated on selective media. The presence of the NP-1 gene in these plants was confirmed by western blot analyses. Resistance tests revealed native defensin NP-1 expression conferred partial resistance to Bacillus subtilis and Pseudomonas aeruginosa, which suggests new possibilities for lettuce disease resistance.

  3. Spaceflight induces both transient and heritable alterations in DNA methylation and gene expression in rice (Oryza sativa L.).

    PubMed

    Ou, Xiufang; Long, Likun; Zhang, Yunhong; Xue, Yiqun; Liu, Jingchun; Lin, Xiuyun; Liu, Bao

    2009-03-09

    Spaceflight represents a complex environmental condition in which several interacting factors such as cosmic radiation, microgravity and space magnetic fields are involved, which may provoke stress responses and jeopardize genome integrity. Given the inherent property of epigenetic modifications to respond to intrinsic as well as external perturbations, it is conceivable that epigenetic markers like DNA methylation may undergo alterations in response to spaceflight. We report here that extensive alteration in both DNA methylation and gene expression occurred in rice plants subjected to a spaceflight, as revealed by a set of characterized sequences including 6 transposable elements (TEs) and 11 cellular genes. We found that several features characterize the alterations: (1) All detected alterations are hypermethylation events; (2) whereas alteration in both CG and CNG methylation occurred in the TEs, only alteration in CNG methylation occurred in the cellular genes; (3) alteration in expression includes both up- and down-regulations, which did not show a general correlation with alteration in methylation; (4) altered methylation patterns in both TEs and cellular genes are heritable to progenies at variable frequencies; however, stochastic reversion to wild-type patterns and further de novo changes in progenies are also apparent; and (5) the altered expression states in both TEs and cellular genes are also heritable to selfed progenies but with markedly lower transmission frequencies than altered DNA methylation states. Furthermore, we found that a set of genes encoding for the various putative DNA methyltransferases, 5-methylcytosine DNA glycosylases, the SWI/SNF chromatin remodeller (DDM1) and siRNA-related proteins are extremely sensitive to perturbation by spaceflight, which might be an underlying cause for the altered methylation patterns in the space-flown plants. We discuss implications of spaceflight-induced epigenetic variations with regard to health safety

  4. Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations.

    PubMed

    Lawrence, Richard J; Pikaard, Craig S

    2003-10-01

    Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss-of-function alleles impractical. We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes. Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A. thaliana and A. arenosa. We demonstrate that A. suecica can be genetically transformed using the floral dip method for Agrobacterium-mediated transformation. Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs. Expressing a double-stranded (ds) RNA corresponding to the A. thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A. thaliana- and A. arenosa-derived centromere repeats. These results indicate that a single RNAi-inducing transgene can dominantly repress multiple orthologs.

  5. Transient generalized glucocorticoid hypersensitivity.

    PubMed

    Nicolaides, Nicolas C; Lamprokostopoulou, Agaristi; Polyzos, Alexandros; Kino, Tomoshige; Katsantoni, Eleni; Triantafyllou, Panagiota; Christophoridis, Athanasios; Katzos, George; Dracopoulou, Maria; Sertedaki, Amalia; Chrousos, George P; Charmandari, Evangelia

    2015-12-01

    Transient generalized glucocorticoid hypersensitivity is a rare disorder characterized by increased tissue sensitivity to glucocorticoids and compensatory hypo-activation of the hypothalamic-pituitary-adrenal axis. The condition itself and the underlying molecular mechanisms have not been elucidated. To present the clinical manifestations, endocrinologic evaluation and transcriptomic profile in a patient with transient generalized glucocorticoid hypersensitivity. A 9-year-old girl presented with an 8-month history of clinical manifestations suggestive of Cushing syndrome. Endocrinologic evaluation revealed undetectable 08:00 h ACTH (<1 pg/mL) and cortisol (0·025 μg/dL) concentrations, which remained decreased throughout the 24-h period and did not respond to stimulation with ovine CRH. The disease gradually resolved spontaneously over the ensuing 3 months. Sequencing of the human glucocorticoid receptor gene revealed no mutations or polymorphisms. Western blot analysis in peripheral blood mononuclear cells revealed equal protein expression of hGRα of the patient in the disease and postresolution phases compared with a control subject. Transcriptomic analysis in peripheral blood mononuclear cells in the disease and postresolution phases identified 903 differentially expressed genes. Of these, 106 genes were up-regulated and 797 were down-regulated in the disease compared with the resolution phase. Bioinformatics analysis on the differentially expressed gene networks revealed Nuclear Factor-κB as the predominant transcription factor influencing the expression of the majority of differentially expressed genes. Our findings indicate that a transient postreceptor defect, or a virus- or bacterium-encoded molecule, may have enhanced glucocorticoid signal transduction, leading to transient generalized glucocorticoid hypersensitivity and hypo-activation of the HPA axis. © 2015 Stichting European Society for Clinical Investigation Journal Foundation.

  6. Activation of gene transcription via CIM0216, a synthetic ligand of transient receptor potential melastatin-3 (TRPM3) channels.

    PubMed

    Rubil, Sandra; Thiel, Gerald

    2017-01-02

    Several compounds have been proposed to stimulate TRPM3 Ca(2+) channels. We recently showed that stimulation of TRPM3 channels with pregnenolone sulfate activated the transcription factor AP-1, while other proposed TRPM3 ligands (nifedipine, D-erythro-sphingosine) exhibited either no or TRPM3-independent effects on gene transcription. Here, we have analyzed the transcriptional activity of CIM0216, a synthetic TRPM3 ligand proposed to have a higher potency and affinity for TRPM3 than pregnenolone sulfate. The results show that CIM0216 treatment of HEK293 cells expressing TRPM3 channels activated AP-1 and stimulated the transcriptional activation potential of c-Jun and c-Fos, 2 basic region leucine zipper transcription factors that constitute AP-1. CIM0216-induced gene transcription was attenuated by knock-down of TRPM3 or treatment with mefenamic acid, a TRPM3 inhibitor. CIM0216 was similarly or less capable in activating TRPM3-mediated gene transcription, suggesting that pregnenolone sulfate is still the ligand of choice for changing the gene expression pattern via TRPM3.

  7. Transient restoration of gene rearrangement at multiple T cell receptor loci in gamma-irradiated scid mice

    PubMed Central

    1996-01-01

    The developmental arrest of thymocytes from scid mice, deficient in variable, (diversity), and joining, or V(D)J recombination, can be overcome by sublethal gamma-irradiation. Since previous studies focused on restoration of rearrangement of the T cell receptor (TCR) beta locus, productive rearrangement of which is selected for, we sought to examine to what extent locus specificity and cellular selection contributed to the observed effects. We report here that irradiation of newborn scid mice induces normal V-D-J rearrangements of the TCR delta locus, which like TCR beta, is also actively rearranged in CD(4-)CD(8-) (double negative) thymocytes. In contrast, no complete V-J alpha rearrangements were detected. Instead, we detected substantial levels of hairpin-terminated coding ends at the 5' end of the J alpha locus, demonstrating that TCR alpha rearrangements manifest the effects of the scid mutation. Irradiation, therefore, transiently compensates for the effects of the scid mutation in a locus-nonspecific manner in thymocytes, resulting in a burst of normal TCR beta and delta rearrangements. Irradiation also allows the development of cells that can initiate but fail to complete V(D)J recombination events at the TCR alpha locus, which is normally inaccessible in scid thymocytes. PMID:8760795

  8. Analysis of clustered point mutations in the human ribosomal RNA gene promoter by transient expression in vivo.

    PubMed Central

    Jones, M H; Learned, R M; Tjian, R

    1988-01-01

    We have mapped the cis regulatory elements required in vivo for initiation at the human rRNA promoter by RNA polymerase I. Transient expression in COS-7 cells was used to evaluate the transcription phenotype of clustered base substitution mutations in the human rRNA promoter. The promoter consists of two major elements: a large upstream region, composed of several domains, that lies between nucleotides -234 and -107 relative to the transcription initiation site and affects transcription up to 100-fold and a core element that lies between nucleotides -45 and +20 and affects transcription up to 1000-fold. The upstream region is able to retain partial function when positioned within 100-160 nucleotides of the transcription initiation site, but it cannot stimulate transcription from distances of greater than or equal to 600 nucleotides. In addition, we demonstrate, using mouse-human hybrid rRNA promoters, that the sequences responsible for human species-specific transcription in vivo appear to reside in both the core and upstream elements, and sequences from the mouse rRNA promoter cannot be substituted for them. Images PMID:3422449

  9. Identification of differentially expressed genes involved in transient regeneration of the neonatal C57BL/6J mouse heart by digital gene expression profiling.

    PubMed

    Liu, Ming; Zhu, Jin-Gai; Yu, Zhang-Bin; Song, Gui-Xian; Shen, Ya-Hui; Liu, Yao-Qiu; Zhu, Chun; Qian, Ling-Mei

    2014-06-01

    Accumulating evidence has revealed that the mammalian heart possesses a measurable capacity for renewal. Neonatal mice retain a regenerative capacity over a short time-frame (≤6 days), but this capacity is lost by 7 days of age. In the present study, differential gene expression profiling of mouse cardiac tissue was performed to further elucidate the mechanisms underlying this process. The global gene expression patterns of the neonatal C57BL/6J mouse heart were examined at three key time-points (1, 6 and 7 days old) using digital gene expression analysis. In the distribution of total clean tags, high-expression tags (>100 copies) were found to be predominant, whereas low expression tags (<5 copies) occupied the majority of distinct tag distributions. In total, 306 differentially expressed genes (DEGs) were detected in cardiac tissue, with the expression levels of 115 genes upregulated and those of 191 genes downregulated in 7-day-old mice compared with expression levels in 1- and 6-day-old mice, respectively. The expression levels of five DEGs were confirmed using quantitative polymerase chain reaction. Gene ontology analysis revealed a large proportion of DEGs distributed throughout the cell, and these DEGs were associated with binding as well as catalytic, hydrolase, transferase and molecular transducer activities. Furthermore, these genes were involved in cellular, metabolic and developmental processes, as well as biological regulation and signaling pathways. Pathway analysis identified the oxidative phosphorylation pathway to be the process most significantly putatively affected by the differential expression of these genes. These data provide the basis for future analysis of the gene expression patterns that regulate the molecular mechanism of cardiac regeneration.

  10. Over-Expression of a Tobacco Nitrate Reductase Gene in Wheat (Triticum aestivum L.) Increases Seed Protein Content and Weight without Augmenting Nitrogen Supplying

    PubMed Central

    Zhao, Xiao-Qiang; Nie, Xuan-Li; Xiao, Xing-Guo

    2013-01-01

    Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, “Nongda146” and “Jimai6358”, by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying. PMID:24040315

  11. A gene encoding a chloroplast-targeted lipoxygenase in tomato leaves is transiently induced by wounding, systemin, and methyl jasmonate.

    PubMed Central

    Heitz, T; Bergey, D R; Ryan, C A

    1997-01-01

    We investigated the relationship between the expression of lipoxygenase (LOX) genes and the systemin-dependent wound response in tomato (Lycopersicon esculentum) leaves. A polymerase chain reaction-based approach was used to isolate two tomato Lox cDNAs, called TomLoxC and TomLoxD. Both TomLOXC and TomLOXD amino acid sequences possess an N-terminal extension of about 60 residues that were shown by in vitro uptake to function as transit peptides, targeting these proteins into the chloroplast. Within 30 to 50 min following wounding or systemin or methyl jasmonate treatments, the TomLoxD mRNA level increased and reached a maximum between 1 and 2 h. TomLoxC mRNA was not detectable in leaves and was not found following wounding, but it was found in ripening fruits, indicating that the two tomato Lox genes are regulated in different tissues by different processes. The results suggest that the TomLoxD gene is up-regulated in leaves in response to wounding and encodes a chloroplast LOX that may play a role as a component of the octadecanoid defense-signaling pathway. PMID:9232884

  12. Promoter analyses and transcriptional profiling of eggplant polyphenol oxidase 1 gene (SmePPO1) reveal differential response to exogenous methyl jasmonate and salicylic acid.

    PubMed

    Shetty, Santoshkumar M; Chandrashekar, Arun; Venkatesh, Yeldur P

    2012-05-01

    The transcriptional regulation of multigenic eggplant (Solanum melongena) polyphenol oxidase genes (SmePPO) is orchestrated by their corresponding promoters which mediate developmentally regulated expression in response to myriad biotic and abiotic factors. However, information on structural features of SmePPO promoters and modulation of their expression by plant defense signals are lacking. In the present study, SmePPOPROMOTERs were cloned by genome walking, and their transcription start sites (TSS) were determined by RLM-RACE. Extensive sequence analyses revealed the presence of evolutionarily conserved and over-represented putative cis-acting elements involved in light-regulated transcription, biosynthetic pathways (phenylpropanoid/flavonoid), hormone signaling (abscisic acid, gibberellic acid, jasmonate and salicylate), elicitor and stress responses (cold/dehydration responses), sugar metabolism and plant defense signaling (W-BOX/WRKY) that are common to SmePPOPROMOTER1 and 2. The TSS for SmePPO genes are located 9-15bp upstream of ATG with variable lengths of 5' untranslated regions. Transcriptional profiling of SmePPOs in eggplant seedlings has indicated differential response to methyl jasmonate (MeJA) or salicylic acid (SA) treatment. In planta, while MeJA elicited expression of all the six SmePPOs, SA was only able to induce the expression of SmePPO4-6. Interestingly, in dual treatment, SA considerably repressed the MeJA-induced expression of SmePPOs. Functional dissection of SmePPOPROMOTER1 by deletion analyses using Agrobacterium-mediated transient expression in tobacco leaves has shown that MeJA enhances the SmePPOPROMOTER1-β-glucuronidase (GUS) expression in vivo, while SA does not. Histochemical and quantitative GUS assays have also indicated the negative effect of SA on MeJA-induced expression of SmePPOPROMOTER1. By combining in silico analyses, transcriptional profiling and expression of SmePPOPROMOTER1-GUS fusions, the role of SA on the modulation

  13. Problems associated with gene transfer and opportunities for microgravity environments

    SciTech Connect

    Tennessen, D.J.

    1997-01-01

    The method of crop improvement by gene transfer is becoming increasingly routine with transgenic foods and ornamental crops now being marketed to consumers. However, biological processes of plants, and the physical barriers of current protocols continue to limit the application of gene transfer in many commercial crops. The goal of this paper is to outline the current limitations of gene transfer and to hypothesize possible opportunities for use of microgravity to overcome such limitations. The limitations detailed in this paper include host-range specificity of {ital Agrobacterium} mediated transformation, probability of gene insertion, position effects of the inserted genes, gene copy number, stability of foreign gene expression in host plants, and regeneration of recalcitrant plant species. Microgravity offers an opportunity for gene transfer where cell growth kinetics, DNA synthesis, and genetic recombination rates can be altered. Such biological conditions may enhance the ability for recombination of reporter genes and other genes of interest to agriculture. Proposed studies would be useful for understanding instability of foreign gene expression and may lead to stable transformed plants. Other aspects of gene transfer in microgravity are discussed. {copyright} {ital 1997 American Institute of Physics.}

  14. Problems associated with gene transfer and opportunities for microgravity environments

    NASA Astrophysics Data System (ADS)

    Tennessen, Daniel J.

    1997-01-01

    The method of crop improvement by gene transfer is becoming increasingly routine with transgenic foods and ornamental crops now being marketed to consumers. However, biological processes of plants, and the physical barriers of current protocols continue to limit the application of gene transfer in many commercial crops. The goal of this paper is to outline the current limitations of gene transfer and to hypothesize possible opportunities for use of microgravity to overcome such limitations. The limitations detailed in this paper include host-range specificity of Agrobacterium mediated transformation, probability of gene insertion, position effects of the inserted genes, gene copy number, stability of foreign gene expression in host plants, and regeneration of recalcitrant plant species. Microgravity offers an opportunity for gene transfer where cell growth kinetics, DNA synthesis, and genetic recombination rates can be altered. Such biological conditions may enhance the ability for recombination of reporter genes and other genes of interest to agriculture. Proposed studies would be useful for understanding instability of foreign gene expression and may lead to stable transformed plants. Other aspects of gene transfer in microgravity are discussed.

  15. Replication of the rRNA and legumin genes in synchronized root cells of pea (Pisum sativum): evidence for transient EcoR I sites in replicating rRNA genes.

    PubMed

    Hof, J V; Hernandez, P; Bjerknes, C A; Kraszewska, E K; Lamm, S S

    1987-03-01

    The temporal pattern of replication of the rRNA and legumin genes differs in synchronized pea root cells. The relative number of rRNA genes replicated hourly during the first five hours of S phase ranges between 5 and 10 percent. In late S phase, during hours six through nine, the number of rRNA genes replicated increases reaching a maximum of about 25 percent at the ninth hour. Unlike the rRNA genes, the legumin genes have a wave-like pattern of replication peaking in early S phase at the third hour and again in late S phase at the eighth hour.Replicating rDNA, isolated by benzoylated naphthoylated DEAE-column chromatography, has EcoR I restriction sites that are absent in non-replicating rDNA sequences. The cleavage of these sites is independent of the time of rDNA replication. The transient nature of the EcoR I sites suggests that they exist in a hemimethylated state in parental DNA.The two Hind III repeat-size classes of rDNA of var. Alaska peas are replicated simultaneously as cells progress through S phase. Thus, even if the 9.0 kb and 8.6 kb repeat classes are located on different chromosomes, their temporal order of replication is the same.

  16. Self-entanglement of long linear DNA vectors using transient non-B-DNA attachment points: a new concept for improvement of non-viral therapeutic gene delivery.

    PubMed

    Tolmachov, Oleg E

    2012-05-01

    The cell-specific and long-term expression of therapeutic transgenes often requires a full array of native gene control elements including distal enhancers, regulatory introns and chromatin organisation sequences. The delivery of such extended gene expression modules to human cells can be accomplished with non-viral high-molecular-weight DNA vectors, in particular with several classes of linear DNA vectors. All high-molecular-weight DNA vectors are susceptible to damage by shear stress, and while for some of the vectors the harmful impact of shear stress can be minimised through the transformation of the vectors to compact topological configurations by supercoiling and/or knotting, linear DNA vectors with terminal loops or covalently attached terminal proteins cannot be self-compacted in this way. In this case, the only available self-compacting option is self-entangling, which can be defined as the folding of single DNA molecules into a configuration with mutual restriction of molecular motion by the individual segments of bent DNA. A negatively charged phosphate backbone makes DNA self-repulsive, so it is reasonable to assume that a certain number of 'sticky points' dispersed within DNA could facilitate the entangling by bringing DNA segments into proximity and by interfering with the DNA slipping away from the entanglement. I propose that the spontaneous entanglement of vector DNA can be enhanced by the interlacing of the DNA with sites capable of mutual transient attachment through the formation of non-B-DNA forms, such as interacting cruciform structures, inter-segment triplexes, slipped-strand DNA, left-handed duplexes (Z-forms) or G-quadruplexes. It is expected that the non-B-DNA based entanglement of the linear DNA vectors would consist of the initial transient and co-operative non-B-DNA mediated binding events followed by tight self-ensnarement of the vector DNA. Once in the nucleoplasm of the target human cells, the DNA can be disentangled by type II

  17. Construction of a stepwise gene integration system by transient expression of actinophage R4 integrase in cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Takahashi, Hideo; Shirai, Makoto

    2014-08-01

    The integrase of actinophage R4, which belongs to the large serine-recombinase family, catalyzes site-specific recombination between two distinct attachment site sequences of the phage (attP) and actinomycete Streptomyces parvulus 2297 chromosome (attB). We previously reported that R4 integrase (Sre) catalyzed site-specific recombination both in vivo and in vitro. In the present study, a Sre-based system was developed for the stepwise site-specific integration of multiple genes into the chromosome of cyanobacterium Synechocystis sp. PCC 6803 (hereafter PCC 6803). A transgene-integrated plasmid with two attP sites and a non-replicative sre-containing plasmid were co-introduced into attB-inserted PCC 6803 cells. The transiently expressed Sre catalyzed highly efficient site-specific integration between one of the two attP sites on the integration plasmid and the attB site on the chromosome of PCC 6803. A second transgene-integrated plasmid with an attB site was integrated into the residual attP site on the chromosome by repeating site-specific recombination. The transformation frequencies (%) of the first and second integrations were approximately 5.1 × 10(-5) and 8.2 × 10(-5), respectively. Furthermore, the expression of two transgenes was detected. This study is the first to apply the multiple gene site-specific integration system based on R4 integrase to cyanobacteria.

  18. Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2.

    PubMed Central

    Wilkinson, J Q; Crawford, N M

    1991-01-01

    Chlorate, the chlorine analog of nitrate, is a herbicide that has been used to select mutants impaired in the process of nitrate assimilation. In Arabidopsis thaliana, mutations at any one of eight distinct loci confer resistance to chlorate. The molecular identities of the genes at these loci are not known; however, one of these loci--chl3--maps very near the nitrate reductase structural gene NIA2. Through the isolation, characterization, and genetic analysis of new chlorate-resistant mutants generated by gamma irradiation, we have been able to demonstrate that the CHL3 gene and the NIA2 gene are identical. Three new chlorate-resistant mutants were identified that had deletions of the entire NIA2 gene. These nia2 null mutants were viable and still retained 10% of wild-type nitrate reductase activity in the leaves of the plants. All three deletion mutations were found to be new alleles of chl3. Introduction of the NIA2 gene back into these chl3 mutants by Agrobacterium-mediated transformation partially complemented their mutant phenotype. From these data, we conclude that Arabidopsis has at least two functional nitrate reductase genes and that the NIA2 gene product accounts for the majority of the leaf nitrate reductase activity and chlorate sensitivity of Arabidopsis plants. PMID:1840922

  19. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

    PubMed

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2016-04-01

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

  20. Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

    PubMed Central

    Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Forthal, Donald; Mao, Lingjun; -Abanto, Segundo Hernandez; Urban, Lori; Landucci, Gary; Fischer, Rainer; Jiang, Xiaoming

    2013-01-01

    Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production. PMID:23533588

  1. Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana.

    PubMed

    Khakimov, Bekzod; Kuzina, Vera; Erthmann, Pernille Ø; Fukushima, Ery Odette; Augustin, Jörg M; Olsen, Carl Erik; Scholtalbers, Jelle; Volpin, Hanne; Andersen, Sven Bode; Hauser, Thure P; Muranaka, Toshiya; Bak, Søren

    2015-11-01

    The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs). © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons

  2. [Transgenic Belarussian-bred potato plants expressing genes for antimicrobial peptides of the cecropin-melittin type].

    PubMed

    Vutto, N L; Gapeeva, T A; Pundik, A N; Tret'iakova, T G; Volotovskiĭ, I D

    2010-12-01

    Binary vectors for Agrobacterium-mediated transformation were constructed to express the genes for antimicrobial peptides (APs) of the cectropin-melittin type under the control of the cauliflower mosaic virus 35S RNA promoter in plants. It was shown with Escherichia coli and Agrobacterium tumefaciens cells that the cassettes could be cloned in pB1121-based vectors with deletion of the 3-D-glycuronidase gene only in the orientation opposite to that of the original vector. Transgenic potato plants were obtained using the Belarussian varieties Odyssey, Vetraz, and Scarb. Their cells expressed the MsrA1 or CEMA peptides of the cecropin-melittin type. The expression was shown to confer higher resistance to bacterial (Erwinia carotovora) infection and extremely high resistance to fungal (Phytophtora infestans and Alternarla solani) infections.

  3. Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker.

    PubMed

    Fang, Weiguo; Zhang, Yongjun; Yang, Xingyong; Zheng, Xuelian; Duan, Hui; Li, Yi; Pei, Yan

    2004-01-01

    Beauveria bassiana has been investigated for use in the biological control of several insects in agricultural practice. To understand the molecular basis of virulence and host specificity and to improve the entomopathogenicity of B. bassiana, we have developed a simple, highly efficient and reliable Agrobacterium-mediated transformation method for B. bassiana using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. Most transformants contained single copies of T-DNA and the T-DNA inserts were stably inherited after five generations. With this highly efficient transformation method for B. bassiana, we also obtained two putative T-DNA-tagged mutants that may have altered growth habits or virulence. Thus, the described protocol could provide a useful tool to manipulate the genetic make-up and to tag genes that may be important for virulence or growth and development of B. bassiana.

  4. [Construction of the plant expression vector with hepatitis a capsid protein fusion gene and genetic transformation of Citrus. Sinensis Osbeck].

    PubMed

    Hu, Rong; Wei, Hong; Chen, Shan-Chun; He, Yong-Rui

    2004-07-01

    The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems. The construction of the plant expression vector pBI121-A was reported, which contained a fusion gene encoding hepatitis A capsid proteins. The gene was located between the left and right Ti border sequences under the control of CaMV35S promoter. The vector was identified via PCR and restriction enzyme analysis and was introduced into Agrobacterium tumerifacience LBA4404. The transgenic Citrus plants were produced by Agrobacterium-mediated transformation of epicotyl segments.13 putatively transformed plants through the kanamycin selection were micrografted onto the seedlings. The presence and integration of the transgene had been verified by PCR analysis. The result showed that five transformants were integrated and the transformation efficiency was 4.1%.

  5. Efficient transformation and artificial miRNA gene silencing in Lemna minor

    PubMed Central

    Cantó-Pastor, Alex; Mollá-Morales, Almudena; Ernst, Evan; Dahl, William; Zhai, Jixian; Yan, Yiheng; Meyers, Blake; Shanklin, John; Martienssen, Robert

    2015-01-01

    Lack of genetic tools in the Lemnaceae (duckweed) has impeded full implementation of this organism as model for biological research, despite its rapid doubling time, simple architecture and unusual metabolic characteristics. Here we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a Magnesium Chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic Lemna minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae. PMID:24989135

  6. Production of transgenic kiwifruit plants harboring the SbtCry1Ac gene.

    PubMed

    Zhang, H Y; Liu, H M; Liu, X Z

    2015-07-28

    The kiwifruit (Actinidia chinensis Planch.) is an economically and nutritionally important fruit crop that has a remarkably high vitamin C content and is popular throughout the world. However, kiwifruit plants are vulnerable to attack from pests, and effective pest control is urgently required. Transgenic kiwifruit plants containing the synthetic chimeric gene SbtCry1Ac that encodes the insecticidal protein btCrylAc were obtained through an Agrobacterium-mediated transformation of kiwifruit leaf discs. The kanamycin resistance of the transgenic plants was then analyzed. Results from polymerase chain reactions and genomic DNA Southern blot analyses indicated that SbtCrylAc had been integrated into the genomes of these plants. The results of insect bioassays revealed that the average Oraesia excavate inhibition rate of plants tested at 10 days post-infestation was 75.2%. To our knowledge, this is the first study that has developed insect-resistant transgenic kiwifruit plants.

  7. Transient transfection of human CDNF gene reduces the 6-hydroxydopamine-induced neuroinflammation in the rat substantia nigra.

    PubMed

    Nadella, Rasajna; Voutilainen, Merja H; Saarma, Mart; Gonzalez-Barrios, Juan A; Leon-Chavez, Bertha A; Jiménez, Judith M Dueñas; Jiménez, Sergio H Dueñas; Escobedo, Lourdes; Martinez-Fong, Daniel

    2014-12-16

    The anti-inflammatory effect of the cerebral dopamine neurotrophic factor (CDNF) was shown recently in primary glial cell cultures, yet such effect remains unknown both in vivo and in 6-hydroxydopamine (6-OHDA) models of Parkinson's disease (PD). We addressed this issue by performing an intranigral transfection of the human CDNF (hCDNF) gene in the critical period of inflammation after a single intrastriatal 6-OHDA injection in the rat. At day 15 after lesion, the plasmids p3xNBRE-hCDNF or p3xNBRE-EGFP, coding for enhanced green florescent protein (EGFP), were transfected into the rat substantia nigra (SN) using neurotensin (NTS)-polyplex. At day 15 post-transfection, we measured nitrite and lipoperoxide levels in the SN. We used ELISA to quantify the levels of TNF-α, IL-1β, IL-6, endogenous rat CDNF (rCDNF) and hCDNF. We also used qRT-PCR to measure rCDNF and hCDNF transcripts, and immunofluorescence assays to evaluate iNOS, CDNF and glial cells (microglia, astrocytes and Neuron/Glial type 2 (NG2) cells). Intact SNs were additional controls. In the SN, 6-OHDA triggered nitrosative stress, increased inflammatory cytokines levels, and activated the multipotent progenitor NG2 cells, which convert into astrocytes to produce rCDNF. In comparison with the hemiparkinsonian rats that were transfected with the EGFP gene or without transfection, 6-OHDA treatment and p3xNBRE-hCDNF transfection increased the conversion of NG2 cells into astrocytes resulting in 4-fold increase in the rCDNF protein levels. The overexpressed CDNF reduced nitrosative stress, glial markers and IL-6 levels in the SN, but not TNF-α and IL-1β levels. Our results show the anti-inflammatory effect of CDNF in a 6-OHDA rat of Parkinson's disease. Our results also suggest the possible participation of TNF-α, IL-1β and IL-6 in rCDNF production by astrocytes, supporting their anti-inflammatory role.

  8. Fibronectin affects transient MMP2 gene expression through DNA demethylation changes in non-invasive breast cancer cell lines.

    PubMed

    Pereira, Isabela T; Ramos, Edneia A S; Costa, Erico T; Camargo, Anamaria A; Manica, Graciele C M; Klassen, Liliane M B; Chequin, Andressa; Braun-Prado, Karin; Pedrosa, Fábio de O; Souza, Emanuel M; Costa, Fabricio F; Klassen, Giseli

    2014-01-01

    Metastasis accounts for more than 90% of cancer deaths. Cells from primary solid tumors may invade adjacent tissues and migrate to distant sites where they establish new colonies. The tumor microenvironment is now recognized as an important participant in the signaling that induces cancer cell migration. An essential process for metastasis is extracellular matrix (ECM) degradation by metalloproteases (MMPs), which allows tumor cells to invade local tissues and to reach blood vessels. The members of this protein family include gelatinase A, or MMP-2, which is responsible for the degradation of type IV collagen, the most abundant component of the basal membrane, that separates epithelial cells in the stroma. It is known that fibronectin is capable of promoting the expression of MMP-2 in MCF7 breast cancer cells in culture. In addition, it was already shown that the MMP2 gene expression is regulated by epigenetic mechanisms. In this work, we showed that fibronectin was able to induce MMP2 expression by 30% decrease in its promoter methylation. In addition, a histone marker for an open chromatin conformation was significantly increased. These results indicate a new role for fibronectin in the communication between cancer cells and the ECM, promoting epigenetic modifications.

  9. Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

    PubMed Central

    Mignaqui, Ana Clara; Ruiz, Vanesa; Perret, Sylvie; St-Laurent, Gilles; Singh Chahal, Parminder; Transfiguracion, Julia; Sammarruco, Ayelén; Gnazzo, Victoria; Durocher, Yves; Wigdorovitz, Andrés

    2013-01-01

    Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV. PMID:23977353

  10. The potentiating effect of calcitonin gene-related peptide on transient receptor potential vanilloid-1 activity and the electrophysiological responses of rat trigeminal neurons to nociceptive stimuli.

    PubMed

    Chatchaisak, Duangthip; Connor, Mark; Srikiatkhachorn, Anan; Chetsawang, Banthit

    2017-02-15

    Growing evidence suggests that calcitonin gene-related peptide (CGRP) participates in trigeminal nociceptive responses. However, the role of CGRP in sensitization or desensitization of nociceptive transduction remains poorly understood. In this study, we sought to further investigate the CGRP-induced up-regulation of transient receptor potential vanilloid-1 (TRPV1) and the responses of trigeminal neurons to nociceptive stimuli. Rat trigeminal ganglion (TG) organ cultures and isolated trigeminal neurons were incubated with CGRP. An increase in TRPV1 levels was observed in CGRP-incubated TG organ cultures. CGRP potentiated capsaicin-induced increase in phosphorylated CaMKII levels in the TG organ cultures. The incubation of the trigeminal neurons with CGRP significantly increased the inward currents in response to capsaicin challenge, and this effect was inhibited by co-incubation with the CGRP receptor antagonist, BIBN4068BS or the inhibitor of protein kinase A, H-89. These findings reveal that CGRP acting on trigeminal neurons may play a significant role in facilitating cellular events that contribute to the peripheral sensitization of the TG in nociceptive transmission.

  11. Production process reproducibility and product quality consistency of transient gene expression in HEK293 cells with anti-PD1 antibody as the model protein.

    PubMed

    Ding, Kai; Han, Lei; Zong, Huifang; Chen, Junsheng; Zhang, Baohong; Zhu, Jianwei

    2017-03-01

    Demonstration of reproducibility and consistency of process and product quality is one of the most crucial issues in using transient gene expression (TGE) technology for biopharmaceutical development. In this study, we challenged the production consistency of TGE by expressing nine batches of recombinant IgG antibody in human embryonic kidney 293 cells to evaluate reproducibility including viable cell density, viability, apoptotic status, and antibody yield in cell culture supernatant. Product quality including isoelectric point, binding affinity, secondary structure, and thermal stability was assessed as well. In addition, major glycan forms of antibody from different batches of production were compared to demonstrate glycosylation consistency. Glycan compositions of the antibody harvested at different time periods were also measured to illustrate N-glycan distribution over the culture time. From the results, it has been demonstrated that different TGE batches are reproducible from lot to lot in overall cell growth, product yield, and product qualities including isoelectric point, binding affinity, secondary structure, and thermal stability. Furthermore, major N-glycan compositions are consistent among different TGE batches and conserved during cell culture time.

  12. CHO-S antibody titers >1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines.

    PubMed

    Steger, Krista; Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-04-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities.

  13. Transient Transcriptional Regulation of the CYS-C1 Gene and Cyanide Accumulation upon Pathogen Infection in the Plant Immune Response1[C][W

    PubMed Central

    García, Irene; Rosas, Tábata; Bejarano, Eduardo R.; Gotor, Cecilia; Romero, Luis C.

    2013-01-01

    Cyanide is produced concomitantly with ethylene biosynthesis. Arabidopsis (Arabidopsis thaliana) detoxifies cyanide primarily through the enzyme β-cyanoalanine synthase, mainly by the mitochondrial CYS-C1. CYS-C1 loss of function is not toxic for the plant and leads to an increased level of cyanide in cys-c1 mutants as well as a root hairless phenotype. The classification of genes differentially expressed in cys-c1 and wild-type plants reveals that the high endogenous cyanide content of the cys-c1 mutant is correlated with the biotic stress response. Cyanide accumulation and CYS-C1 gene expression are negatively correlated during compatible and incompatible plant-bacteria interactions. In addition, cys-c1 plants present an increased susceptibility to the necrotrophic fungus Botrytis cinerea and an increased tolerance to the biotrophic Pseudomonas syringae pv tomato DC3000 bacterium and Beet curly top virus. The cys-c1 mutation produces a reduction in respiration rate in leaves, an accumulation of reactive oxygen species, and an induction of the alternative oxidase AOX1a and pathogenesis-related PR1 expression. We hypothesize that cyanide, which is transiently accumulated during avirulent bacterial infection and constitutively accumulated in the cys-c1 mutant, uncouples the respiratory electron chain dependent on the cytochrome c oxidase, and this uncoupling induces the alternative oxidase activity and the accumulation of reactive oxygen species, which act by stimulating the salicylic acid-dependent signaling pathway of the plant immune system. PMID:23784464

  14. Cryptococcus neoformans Virulence Gene Discovery through Insertional Mutagenesis

    PubMed Central

    Idnurm, Alexander; Reedy, Jennifer L.; Nussbaum, Jesse C.; Heitman, Joseph

    2004-01-01

    Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37°C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37°C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu+-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their

  15. Transformation of the limonene synthase gene into peppermint (Mentha piperita L.) and preliminary studies on the essential oil profiles of single transgenic plants.

    PubMed

    Krasnyanski, S; May, R A; Loskutov, A; Ball, T M; Sink, K C

    1999-08-01

    Agrobacterium-mediated and direct gene transfer into protoplasts using PEG were both successfully used to produce stable, transformed peppermint plants (Mentha×piperita L. cultivar Black Mitcham) with the limonene synthase gene. Stem internode explants found to possess a high level of organogenesis through adventitious shoot formation were subjected to Agrobacterium tumefaciens disarmed strain GV3101 (pMP90). Following the development of an efficient protoplast-to-plant cycle from stem-isolated protoplasts, they were used in direct gene transformations. In both cases the binary vector pGA643 carrying the nptII/GUS genes, both driven by the CaMV35S promoter, was used in preliminary plant-transformation studies. Later, GUS was replaced with the limonene synthase gene. Kanamycin was used as a selective agent in all transformation experiments to obtain both transformed protoplast-derived calli as well as putative transgenic shoots regenerated from internode explants. Both types of transformation resulted in transgenic plants which were detected using PCR and confirmed by Southern-blot hybridizations. Southern analysis revealed that the method of Agrobacterium-mediated transformation is superior to the direct DNA uptake into protoplasts with regard to the stability of the insert during the transformation event. Single transgenic plants were grown to 10% flowering in a greenhouse and the plants derived both by the Agrobacterium and the protoplast-derived methods were generally observed to have essential oil profiles characterized by a high-menthone, low-menthol, high-menthofuran and -pulegone content in comparison to a typical mid-west peppermint. Limonene varied only slightly, around 1.2%, in transgenic plants produced by both methods.

  16. Characterization of the 2,3-Oxidosqualene Cyclase Gene from Antrodia cinnamomea and Enhancement of Cytotoxic Triterpenoid Compound Production.

    PubMed

    Lin, Yan-Liang; Lee, Yi-Ru; Tsao, Nai-Wen; Wang, Sheng-Yang; Shaw, Jei-Fu; Chu, Fang-Hua

    2015-07-24

    Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.

  17. Cloning and sequence analysis of the Korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutralizing epitope in plants.

    PubMed

    Kang, Tae-Jin; Seo, Jo-Eun; Kim, Dae-Hyuk; Kim, Tae-Geum; Jang, Yong-Suk; Yang, Moon-Sik

    2005-06-01

    Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in pigs and leads to death with a high mortality rate, which has been reported notably in Korea. The spike (S) gene of the PEDV isolated in Korea was cloned and sequenced. The nucleotide sequence encoding the entire S gene open reading frame of Korean strain was 4161 bases long encoding 1387 amino acids. The neutralizing epitope of Korean PEDV (K-COE) was expressed in tobacco plants using Agrobacterium-mediated protein transformation. The recombinant K-COE constituted up to 0.1% of the total soluble protein in the leaves of transgenic tobacco plants. The result of this study opens the way for the development of an edible vaccine against PEDV infection in Korea.

  18. Repression of Global Protein Synthesis by Eif1a-Like Genes That Are Expressed Specifically in the Two-Cell Embryos and the Transient Zscan4-Positive State of Embryonic Stem Cells

    PubMed Central

    Hung, Sandy S. C.; Wong, Raymond C. B.; Sharov, Alexei A.; Nakatake, Yuhki; Yu, Hong; Ko, Minoru S. H.

    2013-01-01

    Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1–Eif1al10) and 9 pseudogenes (Eif1al-ps1–Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state. PMID:23649898

  19. Transient transformation of plants.

    PubMed

    Jones, Huw D; Doherty, Angela; Sparks, Caroline A

    2009-01-01

    Transient expression in plants is a valuable tool for many aspects of functional genomics and promoter testing. It can be used both to over-express and to silence candidate genes. It is also scaleable and provides a viable alternative to microbial fermentation and animal cell culture for the production of recombinant proteins. It does not depend on chromosomal integration of heterologous DNA so is a relatively facile procedure and can lead to high levels of transgene expression. Recombinant DNA can be introduced into plant cells via physical methods, via Agrobacterium or via viral vectors.

  20. Retraction: "An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus".

    PubMed

    2015-11-01

    Retracted: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus Volume 33, Issue 5, 949–956, Article first published online: 28 February 2003. The above article, first published online on 28 February 2003 in Wiley Online Library (wileyonlinelibrary.com), and in volume 33, pp. 949–956, has been retracted by agreement between the authors, the journal Editor in Chief, Christoph Benning, and John Wiley & Sons Ltd.This notice updates and replaces a recent correction notice, published on 8 June 2015.In the above article, it has recently been noted that the original Figure 3b in this paper was assembled incorrectly and included image duplications. As the original data are no longer available for assembly of a corrected figure, the experiment was repeated, in agreement with the editors, by co-author S. Rivas. The data from the repeated experiment, presented below together with the original figure legend, lead to the same interpretation and conclusions as in the original paper.Since publication of the above notice the corresponding author has become aware of additional image duplications involving the loading control lanes of Figures 2g, 3a, 4e and 4f. The authors accept that integrity of the scientific literature is compromised by the data manipulation and, for that reason, they wish to retract the article. However, researchers wishing to use the method described in this paper can still obtain the necessary clones from the corresponding author (dcb40@cam.ac.uk). The authors apologise for having allowed this flawed article to be published.

  1. The Arabidopsis bZIP Gene AtbZIP63 Is a Sensitive Integrator of Transient Abscisic Acid and Glucose Signals1[W][OA

    PubMed Central

    Matiolli, Cleverson Carlos; Tomaz, Juarez Pires; Duarte, Gustavo Turqueto; Prado, Fernanda Manso; Del Bem, Luiz Eduardo Vieira; Silveira, Amanda Bortolini; Gauer, Luciane; Corrêa, Luiz Gustavo Guedes; Drumond, Rodrigo Duarte; Viana, Américo José Carvalho; Di Mascio, Paolo; Meyer, Christian; Vincentz, Michel

    2011-01-01

    Glucose modulates plant metabolism, growth, and development. In Arabidopsis (Arabidopsis thaliana), Hexokinase1 (HXK1) is a glucose sensor that may trigger abscisic acid (ABA) synthesis and sensitivity to mediate glucose-induced inhibition of seedling development. Here, we show that the intensity of short-term responses to glucose can vary with ABA activity. We report that the transient (2 h/4 h) repression by 2% glucose of AtbZIP63, a gene encoding a basic-leucine zipper (bZIP) transcription factor partially involved in the Snf1-related kinase KIN10-induced responses to energy limitation, is independent of HXK1 and is not mediated by changes in ABA levels. However, high-concentration (6%) glucose-mediated repression appears to be modulated by ABA, since full repression of AtbZIP63 requires a functional ABA biosynthetic pathway. Furthermore, the combination of glucose and ABA was able to trigger a synergistic repression of AtbZIP63 and its homologue AtbZIP3, revealing a shared regulatory feature consisting of the modulation of glucose sensitivity by ABA. The synergistic regulation of AtbZIP63 was not reproduced by an AtbZIP63 promoter-5′-untranslated region::β-glucuronidase fusion, thus suggesting possible posttranscriptional control. A transcriptional inhibition assay with cordycepin provided further evidence for the regulation of mRNA decay in response to glucose plus ABA. Overall, these results indicate that AtbZIP63 is an important node of the glucose-ABA interaction network. The mechanisms by which AtbZIP63 may participate in the fine-tuning of ABA-mediated abiotic stress responses according to sugar availability (i.e., energy status) are discussed. PMID:21844310

  2. Potential role of melastatin-related transient receptor potential cation channel subfamily M gene expression in the pathogenesis of urinary bladder cancer

    PubMed Central

    Ceylan, Gülay Güleç; Önalan, Ebru Etem; Kuloğlu, Tuncay; Aydoğ, Gülten; Keleş, İbrahim; Tonyali, Şenol; Ceylan, Cavit

    2016-01-01

    Urinary bladder cancer is one of the most common malignancies of the urinary tract. Ion channels and calcium homeostasis are involved in almost all basic cellular mechanisms. The transient receptor potential cation channel subfamily M (TRPM) takes its name from the melastatin protein, which is classified as potential tumor suppressor. To the best of our knowledge, there have been no previous studies in the literature investigating the role of these ion channels in bladder cancer. The present study aimed to determine whether bladder cancer is associated with mRNA expression levels of TRPM ion channel genes, and whether there is the potential to conduct further studies to establish novel treatment modalities. The present study included a total of 47 subjects, of whom 40 were bladder cancer patients and 7 were controls. Following the histopathological evaluation for bladder carcinoma, the mRNA and protein expression of TRPM were examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry in tumor and normal tissues, in order to determine whether there is a difference in the expression of these channels in tumor and normal tissues. Immunoreactivity for TRPM2, TRPM4, TRPM7 and TRPM8 was observed in epithelial bladder cells in the two groups. RT-qPCR revealed a significant increase in TRPM7 expression in bladder cancer tissue compared to the controls (healthy bladder tissue), whereas no differences in TRPM2 or TRPM4 expression levels were observed. There were significant reductions in the expression levels of TRPM5 and TRPM8 in bladder cancer tissues. In the present study, the effects of TRP ion channels on the formation of bladder cancer was investigated. This study is instructive for TRPM2, TRPM4, TRPM5, TRPM7 and TRPM8 and their therapeutic role in bladder cancer. The results support the fact that these gens can be novel targets and can also be tested for during the treatment of bladder cancer. PMID:28101241

  3. Expression of the apyrase-like APY1 genes in roots of Medicago truncatula is induced rapidly and transiently by stress and not by Sinorhizobium meliloti or Nod factors.

    PubMed

    Navarro-Gochicoa, Maria-Teresa; Camut, Sylvie; Niebel, Andreas; Cullimore, Julie V

    2003-03-01

    The model legume Medicago truncatula contains at least six apyrase-like genes, five of which (MtAPY1;1, MtAPY1;2, MtAPY1;3, MtAPY1;4, and MtAPY1;5) are members of a legume-specific family, whereas a single gene (MtAPY2) has closer homologs in Arabidopsis. Phylogenetic analysis has revealed that the proteins encoded by these two plant gene families are more similar to yeast (Saccharomyces cerevisiae) GDA1 and to two proteins encoded by newly described mammalian genes (ENP5 and 6) than they are to mammalian CD39- and CD39-like proteins. Northern analyses and analyses of the frequencies of expressed sequence tags (ESTs) in different cDNA libraries suggest that in roots, leaves, and flowers, the more highly expressed genes are MtAPY1;3/MtAPY2, MtAPY1;3/MtAPY1;5 and MtAPY1;2/MtAPY1;3 respectively. In roots, at least four of the MtAPY1 genes are induced transiently within 3 to 6 h by a stress response that seems to be ethylene independent because it occurs after treatment with an ethylene synthesis inhibitor and also in the skl ethylene-insensitive mutant. This response also occurs in roots of the following symbiotic mutants: dmi1, dmi2, dmi3, nsp, hcl, pdl, lin, and skl. No evidence was obtained for a rapid, transient, and specific induction of the MtAPY genes in roots in response to rhizobia or rhizobial lipochitooligosaccharidic Nod factors. Thus, our data suggest that the apyrase-like genes, which in several legumes have been implicated to play a role in the legume-rhizobia symbiosis (with some members being described as early nodulin genes), are not regulated symbiotically by rhizobia in M. truncatula.

  4. Expression of the Apyrase-Like APY1 Genes in Roots of Medicago truncatula Is Induced Rapidly and Transiently by Stress and Not by Sinorhizobium meliloti or Nod Factors1

    PubMed Central

    Navarro-Gochicoa, Maria-Teresa; Camut, Sylvie; Niebel, Andreas; Cullimore, Julie V.

    2003-01-01

    The model legume Medicago truncatula contains at least six apyrase-like genes, five of which (MtAPY1;1, MtAPY1;2, MtAPY1;3, MtAPY1;4, and MtAPY1;5) are members of a legume-specific family, whereas a single gene (MtAPY2) has closer homologs in Arabidopsis. Phylogenetic analysis has revealed that the proteins encoded by these two plant gene families are more similar to yeast (Saccharomyces cerevisiae) GDA1 and to two proteins encoded by newly described mammalian genes (ENP5 and 6) than they are to mammalian CD39- and CD39-like proteins. Northern analyses and analyses of the frequencies of expressed sequence tags (ESTs) in different cDNA libraries suggest that in roots, leaves, and flowers, the more highly expressed genes are MtAPY1;3/MtAPY2, MtAPY1;3/MtAPY1;5 and MtAPY1;2/MtAPY1;3 respectively. In roots, at least four of the MtAPY1 genes are induced transiently within 3 to 6 h by a stress response that seems to be ethylene independent because it occurs after treatment with an ethylene synthesis inhibitor and also in the skl ethylene-insensitive mutant. This response also occurs in roots of the following symbiotic mutants: dmi1, dmi2, dmi3, nsp, hcl, pdl, lin, and skl. No evidence was obtained for a rapid, transient, and specific induction of the MtAPY genes in roots in response to rhizobia or rhizobial lipochitooligosaccharidic Nod factors. Thus, our data suggest that the apyrase-like genes, which in several legumes have been implicated to play a role in the legume-rhizobia symbiosis (with some members being described as early nodulin genes), are not regulated symbiotically by rhizobia in M. truncatula. PMID:12644663

  5. Overexpression of a Chimeric Gene, OsDST-SRDX, Improved Salt Tolerance of Perennial Ryegrass

    PubMed Central

    Cen, Huifang; Ye, Wenxing; Liu, Yanrong; Li, Dayong; Wang, Kexin; Zhang, Wanjun

    2016-01-01

    The Drought and Salt Tolerance gene (DST) encodes a C2H2 zinc finger transcription factor, which negatively regulates salt tolerance in rice (Oryza sativa). Phylogenetic analysis of six homologues of DST genes in different plant species revealed that DST genes were conserved evolutionarily. Here, the rice DST gene was linked to an SRDX domain for gene expression repression based on the Chimeric REpressor gene-Silencing Technology (CRES-T) to make a chimeric gene (OsDST-SRDX) construct and introduced into perennial ryegrass by Agrobacterium-mediated transformation. Integration and expression of the OsDST-SRDX in transgenic plants were tested by PCR and RT-PCR, respectively. Transgenic lines overexpressing the OsDST-SRDX fusion gene showed obvious phenotypic differences and clear resistance to salt-shock and to continuous salt stresses compared to non-transgenic plants. Physiological analyses including relative leaf water content, electrolyte leakage, proline content, malondialdehyde (MDA) content, H2O2 content and sodium and potassium accumulation indicated that the OsDST-SRDX fusion gene enhanced salt tolerance in transgenic perennial ryegrass by altering a wide range of physiological responses. To our best knowledge this study is the first report of utilizing Chimeric Repressor gene-Silencing Technology (CRES-T) in turfgrass and forage species for salt-tolerance improvement. PMID:27251327

  6. In vivo evolution of the gp90 gene and consistently low plasma viral load during transient immune suppression demonstrate the safety of an attenuated equine infectious anemia virus (EIAV) vaccine.

    PubMed

    Ma, Jian; Jiang, Chenggang; Lin, Yuezhi; Wang, Xuefeng; Zhao, Liping; Xiang, Wenhua; Shao, Yiming; Shen, Rongxian; Kong, Xiangang; Zhou, Jianhua

    2009-01-01

    To study the in vivo evolution of the attenuated Chinese equine infectious anemia virus (EIAV) vaccine, viral gp90 gene variation and virus replication in immunosuppressed hosts were investigated. The results showed that after vaccination, the gp90 gene followed an evolutionary trend of declining diversity. The trend coincided with the maturation of immunity to EIAV, and eventually, the gp90 gene became highly homologous. The sequences of these predominant quasispecies were consistently detected up to 18 months after vaccination. Furthermore, after transient immune suppression with dexamethasone, the plasma viral RNA copy number of the vaccine strain in three vaccinated ponies remained consistently below the "pathogenic threshold" level, while the viral load increased by 25,000-fold in the positive control of an inapparent carrier of the parental virulent strain. This study is the first to provide evidence for the safety of an attenuated lentiviral vaccine with decreased genomic diversity and consistently low viral replication under suppressed immunity.

  7. Discrepancy in Expression of β-Secretase and Amyloid-β Protein Precursor in Alzheimer-Related Genes in the Rat Medial Temporal Lobe Cortex Following Transient Global Brain Ischemia.

    PubMed

    Pluta, Ryszard; Kocki, Janusz; Ułamek-Kozioł, Marzena; Petniak, Alicja; Gil-Kulik, Paulina; Januszewski, Sławomir; Bogucki, Jacek; Jabłoński, Mirosław; Brzozowska, Judyta; Furmaga-Jabłońska, Wanda; Bogucka-Kocka, Anna; Czuczwar, Stanisław J

    2016-01-01

    Brain ischemia may be causally related with Alzheimer's disease. Presumably, β-secretase and amyloid-β protein precursor gene expression changes may be associated with Alzheimer's disease neuropathology. Consequently, we have examined quantitative changes in both β-secretase and amyloid-β protein precursor genes in the medial temporal lobe cortex with the use of quantitative rtPCR analysis following 10-min global brain ischemia in rats with survival of 2, 7, and 30 days. The greatest significant overexpression of β-secretase gene was noted on the 2nd day, while on days 7-30 the expression of this gene was only modestly downregulated. Amyloid-β protein precursor gene was downregulated on the 2nd day, but on days 7-30 postischemia, there was a significant reverse tendency. Thus, the demonstrated alterations indicate that the considerable changes of expression of β-secretase and amyloid-β protein precursor genes may be connected with a response of neurons in medial temporal lobe cortex to transient global brain ischemia. Finally, the ischemia-induced gene changes may play a key role in a late and slow onset of Alzheimer-type pathology.

  8. A simple Gateway-assisted construction system of TALEN genes for plant genome editing.

    PubMed

    Kusano, Hiroaki; Onodera, Hitomi; Kihira, Miho; Aoki, Hiromi; Matsuzaki, Hikaru; Shimada, Hiroaki

    2016-07-25

    TALEN is an artificial nuclease being applied for sequence-specific genome editing. For the plant genome editing, a pair of TALEN genes is expressed in the cells, and a binary plasmid for Agrobacterium-mediated transformation should be assembled. We developed a novel procedure using the Gateway-assisted plasmids, named Emerald-Gateway TALEN system. We constructed entry vectors, pPlat plasmids, for construction of a desired TALEN gene using Platinum Gate TALEN kit. We also created destination plasmid, pDual35SGw1301, which allowed two TALEN genes to both DNA strands to recruit using Gateway technology. Resultant TALEN genes were evaluated by the single-strand annealing (SSA) assay in E. coli cells. By this assay, the TALENs recognized the corresponding targets in the divided luciferase gene, and induced a specific recombination to generate an active luciferase gene. Using the TALEN genes constructed, we created a transformant potato cells in which a site-specific mutation occurred at the target site of the GBSS gene. This suggested that our system worked effectively and was applicable as a convenient tool for the plant genome editing.

  9. A simple Gateway-assisted construction system of TALEN genes for plant genome editing

    PubMed Central

    Kusano, Hiroaki; Onodera, Hitomi; Kihira, Miho; Aoki, Hiromi; Matsuzaki, Hikaru; Shimada, Hiroaki

    2016-01-01

    TALEN is an artificial nuclease being applied for sequence-specific genome editing. For the plant genome editing, a pair of TALEN genes is expressed in the cells, and a binary plasmid for Agrobacterium-mediated transformation should be assembled. We developed a novel procedure using the Gateway-assisted plasmids, named Emerald–Gateway TALEN system. We constructed entry vectors, pPlat plasmids, for construction of a desired TALEN gene using Platinum Gate TALEN kit. We also created destination plasmid, pDual35SGw1301, which allowed two TALEN genes to both DNA strands to recruit using Gateway technology. Resultant TALEN genes were evaluated by the single-strand annealing (SSA) assay in E. coli cells. By this assay, the TALENs recognized the corresponding targets in the divided luciferase gene, and induced a specific recombination to generate an active luciferase gene. Using the TALEN genes constructed, we created a transformant potato cells in which a site-specific mutation occurred at the target site of the GBSS gene. This suggested that our system worked effectively and was applicable as a convenient tool for the plant genome editing. PMID:27452606

  10. Cloning of galactinol synthase gene from Ammopiptanthus mongolicus and its expression in transgenic Photinia serrulata plants.

    PubMed

    Song, Jian; Liu, Jing; Weng, Manli; Huang, Yanyan; Luo, Lei; Cao, Pengxiu; Sun, Haiwei; Liu, Jie; Zhao, Jinhong; Feng, Dianqi; Wang, Bin

    2013-01-15

    A cold induced galactinol synthase gene (AmGS) and its promoter sequence were identified and cloned from the cold-tolerant tree Ammopiptanthus mongolicus by using cDNA-AFLP, RACE-PCR and TAIL-PCR strategies combined with its expression pattern analysis after cold inducing treatment. Accession number of the AmGS gene in GenBank is DQ519361. The open reading frame (ORF) region of the AmGS gene is 987 nucleotides encoding for 328 amino acid residues and a stop codon. The genomic DNA sequence of AmGS gene contains 3 exons and 2 introns. Moreover, a variety of temporal gene expression patterns of AmGS was detected, which revealed the up-regulation of AmGS gene in stresses of cold, ABA and others. Then the AmGS gene was transformed into Photinia serrulata tree by Agrobacterium-mediated transformation, and the transgenic plants exhibited higher cold-tolerance comparing with non-transformed plants. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Gene transfection of HEK cells on supermacroporous polyacrylamide monoliths: a comparison of transient and stable recombinant protein expression in perfusion culture.

    PubMed

    Cheeks, Matthew C; Edwards, Alexander D; Arnot, Christopher J; Slater, Nigel K H

    2009-12-31

    Transient and continuous recombinant protein expression by HEK cells was evaluated in a perfused monolithic bioreactor. Highly porous synthetic cryogel scaffolds (10 ml bed volume) were characterised by scanning electron microscopy and tested as cell substrates. Efficient seeding was achieved (94% inoculum retained, with 91-95% viability). Metabolite monitoring indicated continuous cell growth, and endpoint cell density was estimated by genomic DNA quantification to be 5.2 x 10(8), 1.1 x 10(9) and 3.5 x 10(10) at day 10, 14 and 18. Culture of stably transfected cells allowed continuous production of the Drosophila cytokine Spätzle by the bioreactor at the same rate as in monolayer culture (total 1.2mg at day 18) and this protein was active. In transient transfection experiments more protein was produced per cell compared with monolayer culture. Confocal microscopy confirmed homogenous GFP expression after transient transfection within the bioreactor. Monolithic bioreactors are thus a flexible and powerful tool for manufacturing recombinant proteins.

  12. Transient drainage summary report

    SciTech Connect

    1996-09-01

    This report summarizes the history of transient drainage issues on the Uranium Mill Tailings Remedial Action (UMTRA) Project. It defines and describes the UMTRA Project disposal cell transient drainage process and chronicles UMTRA Project treatment of the transient drainage phenomenon. Section 4.0 includes a conceptual cross section of each UMTRA Project disposal site and summarizes design and construction information, the ground water protection strategy, and the potential for transient drainage.

  13. Transient, recurrent, white matter lesions in x-linked Charcot-Marie-tooth disease with novel mutation of gap junction protein beta 1 gene in China: a case report.

    PubMed

    Zhao, Yuan; Xie, Yanchen; Zhu, Xiaoquan; Wang, Huigang; Li, Yao; Li, Jimei

    2014-08-03

    Transient white matter lesions have been rarely reported in X-linked Charcot-Marie-Tooth disease type 1. We describe a 15-year-old boy who presented transient and recurrent weakness of the limbs for 5 days. His mother, his mother's mother and his mother's sister presented pes cavus. MRI and electrophysiology were performed in the proband. Gap junction protein beta l gene was analyzed by PCR-sequencing in the proband and his parents. The electrophysiological studies showed a mixed demyelinating and axonal sensorimotor neuropathy. MRI showed white matter lesions in the internal capsule, corpus callosum and periventricular areas, which showed almost complete resolution after two months. T278G mutation in Gap junction protein beta l gene was detected in the proband and his mother. This case report highlights that the novel T278G mutation of Gap junction protein beta l maybe could result in X-linked Charcot-Marie-Tooth disease type 1 with predominant leucoencephalopathy. The white matter changes in MRI of X-linked Charcot-Marie-Tooth disease type 1 patient are reversible.

  14. Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs

    PubMed Central

    2011-01-01

    Background Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses. Results All genes encoded by four distinct begomoviruses (African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a Potato virus X (PVX) vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct. Conclusions Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case. PMID

  15. Random T-DNA mutagenesis identifies a Cu-Zn-superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum

    USDA-ARS?s Scientific Manuscript database

    Agrobacterium-mediated transformation (AMT) was used to identify potential virulence factors in Sclerotinia sclerotiorum. Screening AMT transformants identified two mutants showing significantly reduced virulence. The mutants showed similar growth rate, colony morphology, and sclerotial and oxalate ...

  16. Maternal riboflavin deficiency, resulting in transient neonatal-onset glutaric aciduria Type 2, is caused by a microdeletion in the riboflavin transporter gene GPR172B.

    PubMed

    Ho, Gladys; Yonezawa, Atsushi; Masuda, Satohiro; Inui, Ken-ichi; Sim, Keow G; Carpenter, Kevin; Olsen, Rikke K J; Mitchell, John J; Rhead, William J; Peters, Gregory; Christodoulou, John

    2011-01-01

    Riboflavin, or vitamin B2, is a precursor to flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) molecules, required in biological oxidation-reduction reactions. We previously reported a case of a newborn female who had clinical and biochemical features of multiple acyl-CoA dehydrogenation deficiency (MADD), which was corrected by riboflavin supplementation. The mother was then found to be persistently riboflavin deficient, suggesting that a possible genetic defect in riboflavin transport in the mother was the cause of the transient MADD seen in the infant. Two recently-identified riboflavin transporters G protein-coupled receptor 172B (GPR172B or RFT1) and riboflavin transporter 2 (C20orf54 or RFT2) were screened for mutations. Two missense sequence variations, c.209A>G [p.Q70R] and c.886G>A [p.V296M] were found in GPR172B. In vitro functional studies of both missense variations showed that riboflavin transport was unaffected by these variations. Quantitative real-time PCR revealed a de novo deletion in GPR172B spanning exons 2 and 3 in one allele from the mother. We postulate that haploinsufficiency of this riboflavin transporter causes mild riboflavin deficiency, and when coupled with nutritional riboflavin deficiency in pregnancy, resulted in the transient riboflavin-responsive disease seen in her newborn infant. This is the first report of a genetic defect in riboflavin transport in humans. © 2010 Wiley-Liss, Inc.

  17. Oligodeoxynucleotides Can Transiently Up- and Downregulate CHS Gene Expression in Flax by Changing DNA Methylation in a Sequence-Specific Manner

    PubMed Central

    Dzialo, Magdalena; Szopa, Jan; Czuj, Tadeusz; Zuk, Magdalena

    2017-01-01

    Chalcone synthase (CHS) has been recognized as an essential enzyme in the phenylpropanoid biosynthesis pathway. Apart from the leading role in the production of phenolic compounds with many valuable biological activities beneficial to biomedicine, CHS is well appreciated in science. Genetic engineering greatly facilitates expanding knowledge on the function and genetics of CHS in plants. The CHS gene is one of the most intensively studied genes in flax. In our study, we investigated engineering of the CHS gene through genetic and epigenetic approaches. Considering the numerous restrictions concerning the application of genetically modified (GM) crops, the main purpose of this research was optimization of the plant's modulation via epigenetics. In our study, plants modified through two methods were compared: a widely popular agrotransformation and a relatively recent oligodeoxynucleotide (ODN) strategy. It was recently highlighted that the ODN technique can be a rapid and time-serving antecedent in quick analysis of gene function before taking vector-mediated transformation. In order to understand the molecular background of epigenetic variation in more detail and evaluate the use of ODNs as a tool for predictable and stable gene engineering, we concentrated on the integration of gene expression and gene-body methylation. The treatment of flax with a series of short oligonucleotides homologous to a different part of CHS gene isoforms revealed that those directed to regulatory gene regions (5′- and 3′-UTR) activated gene expression, directed to non-coding region (introns) caused gen activity reduction, while those homologous to a coding region may have a variable influence on its activity. Gene expression changes were accompanied by changes in its methylation status. However, only certain (CCGG) motifs along the gene sequence were affected. The analyzed DNA motifs of the CHS flax gene are more accessible for methylation when located within a CpG island. The

  18. Species-dependent expression of the hyoscyamine 6 beta-hydroxylase gene in the pericycle.

    PubMed

    Kanegae, T; Kajiya, H; Amano, Y; Hashimoto, T; Yamada, Y

    1994-06-01

    The tropane alkaloid scopolamine is synthesized in the pericycle of branch roots in certain species of the Solanaceae. The enzyme responsible for the synthesis of scopolamine from hyoscyamine is hyoscyamine 6 beta-hydroxylase (H6H). The gene for H6H was isolated from Hyoscyamus niger. It has an exon/intron organization very similar to those for ethylene-forming enzymes, suggesting a common evolutionary origin. The 827-bp 5' flanking region of the H6H gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to three solanaceous species by Agrobacterium-mediated transformation systems: H. niger and belladonna (Atropa belladonna), which have high and low levels, respectively, of H6H mRNA in the root, and tobacco (Nicotiana tabacum), which has no endogenous H6H gene. Histochemical analysis showed that GUS expression occurred in the pericycle and at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of hairy roots and plants of transgenic tobacco. In transgenic hairy roots and regenerated plants of belladonna, the root meristem was stained with GUS activity, except for a few transformants in which the vascular cylinder was also stained. These studies indicate that the cell-specific expression of the H6H gene is controlled by some genetic regulation specific to scopolamine-producing plants.

  19. Investigation of horizontal gene transfer in poplar/Amanita muscaria ectomycorrhizas.

    PubMed

    Zhang, Chi; Hampp, Rüdiger; Nehls, Uwe

    2005-01-01

    Fine roots of forest trees form together with certain soil fungi symbiotic structures (ectomycorrhizas), where fungal hyphae are in intimate contact with plant cells. Due to root cell degeneration, plant DNA is released and could be taken up by the fungus. The possibility that horizontal gene transfer might result in a risk for the environment should be evaluated before a massive release of genetically engineered trees into nature occurs, even though only a few convincing examples of horizontal gene transfer are known. Transgenic poplars containing a construct of the Streptomyces hygroscopicus bar gene under the control of the Cochliobolus heterostrophus GPD (glyceraldehyde-3-phosphate dehydrogenase) promoter were generated by Agrobacterium-mediated transformation. The functionality of this construct in the ectomycorrhizal model fungus Amanita muscaria was previously verified by protoplast-based fungal transformation. 35,000 ectomycorrhizas, formed between transgenic poplars and non-transgenic A. muscaria hyphae, were isolated and transferred to selective agar plates. Putative herbicide-resistant fungal colonies were obtained after the first round of selection. However, none of these colonies survived a transfer onto fresh selection medium, nor did they contain the bar gene, indicating that no horizontal gene transfer from poplar to A. muscaria occurred during symbiosis under axenic conditions. However, since ectomycorrhizas are associated under natural conditions with viruses, bacteria and other fungi, these additional associations should be evaluated in future.

  20. Molecular cloning and characterization of a gene regulating flowering time from Alfalfa (Medicago sativa L.).

    PubMed

    Zhang, Tiejun; Chao, Yuehui; Kang, Junmei; Ding, Wang; Yang, Qingchuan

    2013-07-01

    Genes that regulate flowering time play crucial roles in plant development and biomass formation. Based on the cDNA sequence of Medicago truncatula (accession no. AY690425), the LFY gene of alfalfa was cloned. Sequence similarity analysis revealed high homology with FLO/LFY family genes of other plants. When fused to the green fluorescent protein, MsLFY protein was localized in the nucleus of onion (Allium cepa L.) epidermal cells. The RT-qPCR analysis of MsLFY expression patterns showed that the expression of MsLFY gene was at a low level in roots, stems, leaves and pods, and the expression level in floral buds was the highest. The expression of MsLFY was induced by GA3 and long photoperiod. Plant expression vector was constructed and transformed into Arabidopsis by the agrobacterium-mediated methods. PCR amplification with the transgenic Arabidopsis genome DNA indicated that MsLFY gene had integrated in Arabidopsis genome. Overexpression of MsLFY specifically caused early flowering under long day conditions compared with non-transgenic plants. These results indicated MsLFY played roles in promoting flowering time.

  1. Transient Expression of Secretory IgA In Planta is Optimal Using a Multi-Gene Vector and may be Further Enhanced by Improving Joining Chain Incorporation

    PubMed Central

    Westerhof, Lotte B.; Wilbers, Ruud H. P.; van Raaij, Debbie R.; van Wijk, Christina Z.; Goverse, Aska; Bakker, Jaap; Schots, Arjen

    2016-01-01

    Secretory IgA (sIgA) is a crucial antibody in host defense at mucosal surfaces. It is a promising antibody isotype in a variety of therapeutic settings such as passive vaccination and treatment of inflammatory disorders. However, heterologous production of this heteromultimeric protein complex is still suboptimal. The challenge is the coordinate expression of the four required polypeptides; the alpha heavy chain, the light chain, the joining chain, and part of the polymeric-Ig-receptor called the secretory component, in a 4:4:1:1 ratio. We evaluated the transient expression of three sIgAκ variants, harboring the heavy chain isotype α1, α2m1, or α2m2, of the clinical antibody Ustekinumab in planta. Ustekinumab is directed against the p40 subunit that is shared by the pro-inflammatory cytokines interleukin (IL)-12 and IL-23. A sIgA variant of this antibody may enable localized treatment of inflammatory bowel disease. Of the three different sIgA variants we obtained the highest yield with sIgA1κ reaching up to 373 μg sIgA/mg total soluble protein. The use of a multi-cassette vector containing all four expression cassettes was most efficient. However, not the expression strategy, but the incorporation of the joining chain turned out to be the limiting step for sIgA production. Our data demonstrate that transient expression in planta is suitable for the economic production of heteromultimeric protein complexes such as sIgA. PMID:26793201

  2. Transient Expression of Secretory IgA In Planta is Optimal Using a Multi-Gene Vector and may be Further Enhanced by Improving Joining Chain Incorporation.

    PubMed

    Westerhof, Lotte B; Wilbers, Ruud H P; van Raaij, Debbie R; van Wijk, Christina Z; Goverse, Aska; Bakker, Jaap; Schots, Arjen

    2015-01-01

    Secretory IgA (sIgA) is a crucial antibody in host defense at mucosal surfaces. It is a promising antibody isotype in a variety of therapeutic settings such as passive vaccination and treatment of inflammatory disorders. However, heterologous production of this heteromultimeric protein complex is still suboptimal. The challenge is the coordinate expression of the four required polypeptides; the alpha heavy chain, the light chain, the joining chain, and part of the polymeric-Ig-receptor called the secretory component, in a 4:4:1:1 ratio. We evaluated the transient expression of three sIgAκ variants, harboring the heavy chain isotype α1, α2m1, or α2m2, of the clinical antibody Ustekinumab in planta. Ustekinumab is directed against the p40 subunit that is shared by the pro-inflammatory cytokines interleukin (IL)-12 and IL-23. A sIgA variant of this antibody may enable localized treatment of inflammatory bowel disease. Of the three different sIgA variants we obtained the highest yield with sIgA1κ reaching up to 373 μg sIgA/mg total soluble protein. The use of a multi-cassette vector containing all four expression cassettes was most efficient. However, not the expression strategy, but the incorporation of the joining chain turned out to be the limiting step for sIgA production. Our data demonstrate that transient expression in planta is suitable for the economic production of heteromultimeric protein complexes such as sIgA.

  3. Cloning of TPS gene from eelgrass species Zostera marina and its functional identification by genetic transformation in rice.

    PubMed

    Zhao, Feng; Li, Qiuying; Weng, Manli; Wang, Xiuliang; Guo, Baotai; Wang, Li; Wang, Wei; Duan, Delin; Wang, Bin

    2013-12-01

    The full-length cDNA sequence (2613 bp) of the trehalose-6-phosphate synthase (TPS) gene of eelgrass Zostera marina (ZmTPS) was identified and cloned. Z. marina is a kind of seed-plant growing in sea water during its whole life history. The open reading frame (ORF) region of ZmTPS gene encodes a protein of 870 amino acid residues and a stop codon. The corresponding genomic DNA sequence is 3770 bp in length, which contains 3 exons and 2 introns. The ZmTPS gene was transformed into rice variety ZH11 via Agrobacterium-mediated transformation method. After antibiotic screening, molecular characterization, salt-tolerance and trehalose content determinations, two transgenic lines resistant to 150 mM NaCL solutions were screened. Our study results indicated that the ZmTPS gene was integrated into the genomic DNA of the two transgenic rice lines and could be expressed well. Moreover, the detection of the transformed ZmTPS gene in the progenies of the two transgenic lines was performed from T1 to T4 generations; and results suggested that the transformed ZmTPS gene can be transmitted from parent to the progeny in transgenic rice. © 2013.

  4. Transgenic tobacco plants overexpressing the Nicta; CycD3; 4 gene demonstrate accelerated growth rates.

    PubMed

    Guo, Jia; Wang, Myeong Hyeon

    2008-07-31

    D-type cyclins control the onset of cell division and the response to extracellular signals during the G1 phase. In this study, we transformed a D-type cyclin gene, Nicta;CycD3;4, from Nicotiana tabacum using an Agrobacterium-mediated method. A predicted 1.1 kb cyclin gene was present in all of the transgenic plants, but not in wild-type. Northern analyses showed that the expression level of the Nicta;CycD3;4 gene in all of the transgenic plants was strong when compared to the wild-type plants, suggesting that Nicta;CycD3;4 gene driven by the CaMV 35S promoter was being overexpressed. Our results revealed that transgenic plants overexpressing Nicta;CycD3;4 had an accelerated growth rate when compared to wild-type plants, and that the transgenic plants exhibited a smaller cell size and a decreased cell population in young leaves when compared to wild-type plants.

  5. Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

    PubMed

    Yao, Lin; Tan, Chong; Song, Jinzhu; Yang, Qian; Yu, Lijie; Li, Xinling

    2016-01-01

    Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669bp) and pksT-2 (7901bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase-acyltransferase domains through Agrobacterium-mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

  6. Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana.

    PubMed Central

    McElver, J; Tzafrir, I; Aux, G; Rogers, R; Ashby, C; Smith, K; Thomas, C; Schetter, A; Zhou, Q; Cushman, M A; Tossberg, J; Nickle, T; Levin, J Z; Law, M; Meinke, D; Patton, D

    2001-01-01

    The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications. PMID:11779812

  7. Perception of acoustic transients

    NASA Astrophysics Data System (ADS)

    Howard, J. H., Jr.

    1984-01-01

    The research investigates the role of knowledge based or top-down processing in the perception of nonlinguistic, transient signals. The experiments address issues in transient pattern classification, target observation, attentional focusing, auditory induction, and computer based performance aids. The theoretical significance and naval relevance of the research is considered.

  8. Gene environment interaction in periphery and brain converge to modulate behavioral outcomes: Insights from the SP1 transient early in life interference rat model

    PubMed Central

    Asor, Eyal; Ben-Shachar, Dorit

    2016-01-01

    It is generally assumed that behavior results from an interaction between susceptible genes and environmental stimuli during critical life stages. The present article reviews the main theoretical and practical concepts in the research of gene environment interaction, emphasizing the need for models simulating real life complexity. We review a novel approach to study gene environment interaction in which a brief post-natal interference with the expression of multiple genes, by hindering the activity of the ubiquitous transcription factor specificity protein 1 (Sp1) is followed by later-in-life exposure of rats to stress. Finally, this review discusses the role of peripheral processes in behavioral responses, with the Sp1 model as one example demonstrating how specific behavioral patterns are linked to modulations in both peripheral and central physiological processes. We suggest that models, which take into account the tripartite reciprocal interaction between the central nervous system, peripheral systems and environmental stimuli will advance our understanding of the complexity of behavior. PMID:27679768

  9. Single nucleotide polymorphisms and genotypes of transient receptor potential ion channel and acetylcholine receptor genes from isolated B lymphocytes in myalgic encephalomyelitis/chronic fatigue syndrome patients.

    PubMed

    Marshall-Gradisnik, Sonya; Johnston, Samantha; Chacko, Anu; Nguyen, Thao; Smith, Peter; Staines, Donald

    2016-12-01

    Objective The pathomechanism of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is unknown; however, a small subgroup of patients has shown muscarinic antibody positivity and reduced symptom presentation following anti-CD20 intervention. Given the important roles of calcium (Ca(2+)) and acetylcholine (ACh) signalling in B cell activation and potential antibody development, we aimed to identify relevant single nucleotide polymorphisms (SNPs) and genotypes in isolated B cells from CFS/ME patients. Methods A total of 11 CFS/ME patients (aged 31.82 ± 5.50 years) and 11 non-fatigued controls (aged 33.91 ± 5.06 years) were included. Flow cytometric protocols were used to determine B cell purity, followed by SNP and genotype analysis for 21 mammalian TRP ion channel genes and nine mammalian ACh receptor genes. SNP association and genotyping analysis were performed using ANOVA and PLINK analysis software. Results Seventy-eight SNPs were identified in nicotinic and muscarinic acetylcholine receptor genes in the CFS/ME group, of which 35 were in mAChM3. The remaining SNPs were identified in nAChR delta (n = 12), nAChR alpha 9 (n = 5), TRPV2 (n = 7), TRPM3 (n = 4), TRPM4 (n = 1) mAChRM3 2 (n = 2), and mAChRM5 (n = 3) genes. Nine genotypes were identified from SNPs in TRPM3 (n = 1), TRPC6 (n = 1), mAChRM3 (n = 2), nAChR alpha 4 (n = 1), and nAChR beta 1 (n = 4) genes, and were located in introns and 3' untranslated regions. Odds ratios for these specific genotypes ranged between 7.11 and 26.67 for CFS/ME compared with the non-fatigued control group. Conclusion This preliminary investigation identified a number of SNPs and genotypes in genes encoding TRP ion channels and AChRs from B cells in patients with CFS/ME. These may be involved in B cell functional changes, and suggest a role for Ca(2+) dysregulation in AChR and TRP ion channel signalling in the pathomechanism of CFS/ME.

  10. Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.).

    PubMed

    Goel, D; Singh, A K; Yadav, V; Babbar, S B; Bansal, K C

    2010-09-01

    Abiotic stresses, especially salinity and drought, are major limiting factors for plant growth and crop productivity. In an attempt to develop salt and drought tolerant tomato, a DNA cassette containing tobacco osmotin gene driven by a cauliflower mosaic virus 35S promoter was transferred to tomato (Solanum lycopersicum) via Agrobacterium-mediated transformation. Putative T0 transgenic plants were screened by PCR analysis. The selected transformants were evaluated for salt and drought stress tolerance by physiological analysis at T1 and T2 generations. Integration of the osmotin gene in transgenic T1 plants was verified by Southern blot hybridization. Transgenic expression of the osmotin gene was verified by RT-PCR and northern blotting in T1 plants. T1 progenies from both transformed and untransformed plants were tested for salt and drought tolerance by subjecting them to different levels of NaCl stress and by withholding water supply, respectively. Results from different physiological tests demonstrated enhanced tolerance to salt and drought stresses in transgenic plants harboring the osmotin gene as compared to the wild-type plants. The transgenic lines showed significantly higher relative water content, chlorophyll content, proline content, and leaf expansion than the wild-type plants under stress conditions. The present investigation clearly shows that overexpression of osmotin gene enhances salt and drought stress tolerance in transgenic tomato plants.

  11. [Inducible expression of a promoter of the gene encoding barley beta-1, 3-glucanase isoenzyme GIII in transgenic rice].

    PubMed

    Li, Yun-Feng; Zhu, Rui; Xie, Long-Xu; Xu, Pei-Lin

    2005-04-01

    A promoter of the gene encoding beta-1, 3-glucanase isoenzyme GIII was amplified from barley genomic DNA using PCR. The GIII gene promoter, designated P(GIII), was ligated upstream of the gus report gene and pGIII-gus fusion fragment was then cloned into a binary vector pCAMBIA1300 for Agrobacterium-mediated transformation of rice (Oryza sativa L. cv. Taipei 309). PCR analyses indicated that the fusion gene was present in all T0 transgenic plants. The integration of the gene into rice genomic DNA was further confirmed by Southern blot. Histochemical staining and spectrofluorophotometric analyses of transgenic rice leaves showed that the GUS activity was increased after treatment with SA and fungal elicitor. Northern blot analysis also showed expression of such induction. Histochemical staining of T(1) rice seeds displayed marked GUS activity after induction with SA and elicitor, while no GUS activity was detected in untreated T(1) rice seeds. The results presented here strongly suggested that P(GIII) could be pathogen-inducible promoter.

  12. Expression of Talaromyces thermophilus lipase gene in Trichoderma reesei by homologous recombination at the cbh1 locus.

    PubMed

    Zhang, Xu; Xia, Liming

    2017-03-01

    CBH1 (cellobiohydrolase) comprises the majority of secreted proteins by Trichoderma reesei. For expression of Talaromyces thermophilus lipase gene in T. reesei, a self-designed CBH1 promoter was applied to drive the lipase gene expression cassette which was bracketed by flanking sequences of cbh1 gene for homologous recombination. Protoplast and Agrobacterium-mediated plasmid transformations were performed and compared, resultantly, transformation mediated by Agrobacterium was overall proved to be more efficient. Stable integration of lipase gene into chromosomal DNA of T. reesei transformants was verified by PCR. After shaking flask fermentation, lipase activity of transformant reached 375 IU mL(-1), whereas no cellobiohydrolase activity was detected. SDS-PAGE analysis further showed an obvious protein band about 39 kDa and no CBH1 band in fermentation broth, implying lipase gene was successfully extracellularly expressed in T. reesei via homologous recombination at cbh1 locus. This study herein would benefit genetic engineering of filamentous fungi and industrial application of thermo-alkaline lipase like in paper making and detergents addition.

  13. Transgenics in groundnut (Arachis hypogaea L.) expressing cry1AcF gene for resistance to Spodoptera litura (F.).

    PubMed

    Keshavareddy, G; Rohini, S; Ramu, S V; Sundaresha, S; Kumar, A R V; Kumar, P Ananda; Udayakumar, M

    2013-07-01

    Large number of primary transgenic events were generated in groundnut by an Agrobacterium mediated, in planta transformation method to assess the efficacy of cry1AcF against the Spodoptera litura. The amplification of required size fragment of 750 bp with npt II primers and 901 bp with cry1AcF gene primers confirmed the integration of the gene. The expression of the cry gene was ascertained by ELISA in T2 generation, and the maximum concentration of cry protein in transgenic plants reached approximately 0.82 μg/g FW. Further, Southern blot analysis of ten T2 transgenic plants proved that transgene had been integrated in the genome of all the plants and Northern analysis of the same plants demonstrated the active expression of cry1AcF gene. The highest mean % larval mortalities 80.0 and 85.0 with an average mean % larval mortalities 16.25 (n = 369) and 26.0 (n = 80) were recorded in T1 and T2 generations, respectively. Segregation analysis of the selected lines in the T3 generation demonstrated homozygous nature. This clearly proved that though there is considerable improvement in average mean % larval mortality in T2 generation, the cry1AcF gene was effective against S. litura only to some extent.

  14. Assessment of inheritance pattern and agronomic performance of transgenic rapeseed having harpin Xooc-encoding hrf2 gene.

    PubMed

    Huo, Rong; Wang, Yu; Ma, Ling-Li; Qiao, Jun-Qing; Shao, Min; Gao, Xue-Wen

    2010-10-01

    hrf2 gene is a member of the harpin-encoding gene family of rice-pathogenic bacterium Xanthomonas oryzae pv. oryzicola. In our previous studies, we observed that harpin(Xooc) could elicit hypersensitive cell death in non-host plants, induce disease and insect resistance in plants, and enhance plant growth. In this study, the rapeseed cultivar, Yangyou 4, was genetically engineered via Agrobacterium-mediated transformation to express the hrf2 gene. Polymerase chain reaction (PCR) and southern blot analyses of T(1) generation of transgenic rapeseed revealed stable integration and expression of the inserted gene hrf2. In addition, the resistance to Sclerotinia sclerotiorum was greatly enhanced. A comparison between agronomic characters of transgenic and control lines displayed significant differences in terms of plant height, stem width, number of pods per plant, number of seeds per pod, 1,000-seed weight, and seed yield per plant. Among lines with resistance to S. sclerotiorum, T(1)1 had improved agronomic traits compared with controls with a 22.7% seed yield increase. These results suggest that the introduction of the hrf2 gene into rapeseed can be an effective strategy for enhancing resistance to S. sclerotiorum.

  15. Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae.

    PubMed

    Zhang, Baolong; Yang, Yuwen; Chen, Tianzi; Yu, Wengui; Liu, Tingli; Li, Hongjuan; Fan, Xiaohui; Ren, Yongzhe; Shen, Danyu; Liu, Li; Dou, Daolong; Chang, Youhong

    2012-01-01

    Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.

  16. Transient Receptor Potential Vanilloid 1 Gene Deficiency Ameliorates Hepatic Injury in a Mouse Model of Chronic Binge Alcohol-Induced Alcoholic Liver Disease

    PubMed Central

    Liu, Huilin; Beier, Juliane I.; Arteel, Gavin E.; Ramsden, Christopher E.; Feldstein, Ariel E.; McClain, Craig J.; Kirpich, Irina A.

    2016-01-01

    Experimental alcohol-induced liver injury is exacerbated by a high polyunsaturated fat diet rich in linoleic acid. We postulated that bioactive oxidized linoleic acid metabolites (OXLAMs) play a critical role in the development/progression of alcohol-mediated hepatic inflammation and injury. OXLAMs are endogenous ligands for transient receptor potential vanilloid 1 (TRPV1). Herein, we evaluated the role of signaling through TRPV1 in an experimental animal model of alcoholic liver disease (ALD). Chronic binge alcohol administration increased plasma OXLAM levels, specifically 9- and 13-hydroxy-octadecadienoic acids. This effect was associated with up-regulation of hepatic TRPV1. Exposure of hepatocytes to these OXLAMs in vitro resulted in activation of TRPV1 signal transduction with increased intracellular Ca2+ levels. Genetic depletion of TRPV1 did not blunt hepatic steatosis caused by ethanol, but prevented hepatic injury. TRPV1 deficiency protected from hepatocyte death and prevented the increase in proinflammatory cytokine and chemokine expression, including tumor necrosis factor-α, IL-6, macrophage inflammatory protein-2, and monocyte chemotactic protein 1. TRPV1 depletion markedly blunted ethanol-mediated induction of plasminogen activator inhibitor-1, an important alcohol-induced hepatic inflammation mediator, via fibrin accumulation. This study indicates, for the first time, that TRPV1 receptor pathway may be involved in hepatic inflammatory response in an experimental animal model of ALD. TRPV1-OXLAM interactions appear to play a significant role in hepatic inflammation/injury, further supporting an important role for dietary lipids in ALD. PMID:25447051

  17. Virus induced gene silencing in Lolium temulentum

    USDA-ARS?s Scientific Manuscript database

    Lolium temulentum L. is valuable as a model species for studying abiotic stress in closely related forage and turf grasses, many of which are polyploid outcrossing species. As with most monocot species, Agrobacterium-mediated transformation of L. temulentum is still challenging, time consuming and n...

  18. Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants

    NASA Astrophysics Data System (ADS)

    Liew, O. W.; Chong, Jenny P. C.; Asundi, Anand K.

    2005-04-01

    This work focuses on developing a portable fibre optic fluorescence analyser for rapid identification of genetically modified plants tagged with a fluorescent marker gene. Independent transgenic tobacco plant lines expressing the enhanced green fluorescence protein (EGFP) gene were regenerated following Agrobacterium-mediated gene transfer. Molecular characterisation of these plant lines was carried out at the DNA level by PCR screening to confirm their transgenic status. Conventional transgene expression analysis was then carried out at the RNA level by RT-PCR and at the protein level by Western blotting using anti-GFP rabbit antiserum. The amount of plant-expressed EGFP on a Western blot was quantified against known amounts of purified EGFP by scanning densitometry. The expression level of EGFP in transformed plants was found to range from 0.1 - 0.6% of total extractable protein. A comparison between conventional western analysis of transformants and direct spectroscopic quantification using the fibre optic fluorescence analyser was made. The results showed that spectroscopic measurements of fluorescence emission from strong EGFP expressors correlated positively with Western blot data. However, the fluorescence analyser was also able to identify weakly expressing plant transformants below the detection limit of colorimetric Western blotting.

  19. Transgenic Sugarcane Resistant to Sorghum mosaic virus Based on Coat Protein Gene Silencing by RNA Interference

    PubMed Central

    Guo, Jinlong; Gao, Shiwu; Lin, Qinliang; Wang, Hengbo; Que, Youxiong; Xu, Liping

    2015-01-01

    As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused by Potyvirus sugarcane mosaic virus (SCMV) and/or Sorghum mosaic virus (SrMV), with additional differences in viral strains. RNA interference (RNAi) is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP) genes was selected as the target gene and the interference sequence with size of 423 bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms. PMID:25685813

  20. Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene

    PubMed Central

    Zhang, Junjie; Liu, Fan; Yao, Lei; Luo, Chen; Yin, Yue; Wang, Guixiang; Huang, Yubi

    2012-01-01

    Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line. PMID:23136521

  1. Overexpression of Arabidopsis FLOWERING LOCUS T (FT) gene improves floral development in cassava (Manihot esculenta, Crantz).

    PubMed

    Adeyemo, O Sarah; Chavarriaga, Paul; Tohme, Joe; Fregene, Martin; Davis, Seth J; Setter, Tim L

    2017-01-01

    Cassava is a tropical storage-root crop that serves as a worldwide source of staple food for over 800 million people. Flowering is one of the most important breeding challenges in cassava because in most lines flowering is late and non-synchronized, and flower production is sparse. The FLOWERING LOCUS T (FT) gene is pivotal for floral induction in all examined angiosperms. The objective of the current work was to determine the potential roles of the FT signaling system in cassava. The Arabidopsis thaliana FT gene (atFT) was transformed into the cassava cultivar 60444 through Agrobacterium-mediated transformation and was found to be overexpressed constitutively. FT overexpression hastened flower initiation and associated fork-type branching, indicating that cassava has the necessary signaling factors to interact with and respond to the atFT gene product. In addition, overexpression stimulated lateral branching, increased the prolificacy of flower production and extended the longevity of flower development. While FT homologs in some plant species stimulate development of vegetative storage organs, atFT inhibited storage-root development and decreased root harvest index in cassava. These findings collectively contribute to our understanding of flower development in cassava and have the potential for applications in breeding.

  2. VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression

    PubMed Central

    Pitzschke, Andrea; Djamei, Armin; Teige, Markus; Hirt, Heribert

    2009-01-01

    The plant pathogen Agrobacterium tumefaciens transforms plant cells by delivering its T-DNA into the plant cell nucleus where it integrates into the plant genome and causes tumor formation. A key role of VirE2-interacting protein 1 (VIP1) in the nuclear import of T-DNA during Agrobacterium-mediated plant transformation has been unravelled and VIP1 was shown to undergo nuclear localization upon phosphorylation by the mitogen-activated protein kinase MPK3. Here, we provide evidence that VIP1 encodes a functional bZIP transcription factor that stimulates stress-dependent gene expression by binding to VIP1 response elements (VREs), a DNA hexamer motif. VREs are overrepresented in promoters responding to activation of the MPK3 pathway such as Trxh8 and MYB44. Accordingly, plants overexpressing VIP1 accumulate high levels of Trxh8 and MYB44 transcripts, whereas stress-induced expression of these genes is impaired in mpk3 mutants. Trxh8 and MYB44 promoters are activated by VIP1 in a VRE-dependent manner. VIP1 strongly enhances expression from a synthetic promoter harboring multiple VRE copies and directly interacts with VREs in vitro and in vivo. Chromatin immunoprecipitation assays of the MYB44 promoter confirm that VIP1 binding to VREs is enhanced under conditions of MPK3 pathway stimulation. These results provide molecular insight into the cellular mechanism of target gene regulation by the MPK3 pathway. PMID:19820165

  3. Overexpression of Arabidopsis FLOWERING LOCUS T (FT) gene improves floral development in cassava (Manihot esculenta, Crantz)

    PubMed Central

    Adeyemo, O. Sarah; Chavarriaga, Paul; Tohme, Joe; Fregene, Martin; Davis, Seth J.

    2017-01-01

    Cassava is a tropical storage-root crop that serves as a worldwide source of staple food for over 800 million people. Flowering is one of the most important breeding challenges in cassava because in most lines flowering is late and non-synchronized, and flower production is sparse. The FLOWERING LOCUS T (FT) gene is pivotal for floral induction in all examined angiosperms. The objective of the current work was to determine the potential roles of the FT signaling system in cassava. The Arabidopsis thaliana FT gene (atFT) was transformed into the cassava cultivar 60444 through Agrobacterium-mediated transformation and was found to be overexpressed constitutively. FT overexpression hastened flower initiation and associated fork-type branching, indicating that cassava has the necessary signaling factors to interact with and respond to the atFT gene product. In addition, overexpression stimulated lateral branching, increased the prolificacy of flower production and extended the longevity of flower development. While FT homologs in some plant species stimulate development of vegetative storage organs, atFT inhibited storage-root development and decreased root harvest index in cassava. These findings collectively contribute to our understanding of flower development in cassava and have the potential for applications in breeding. PMID:28753668

  4. Stimulatory and inhibitory mechanisms of slow muscle-specific myosin heavy chain gene expression in fish: transient and transgenic analysis of torafugu MYH(M86-2) promoter in zebrafish embryos.

    PubMed

    Asaduzzaman, Md; Kinoshita, Shigeharu; Bhuiyan, Sharmin Siddique; Asakawa, Shuichi; Watabe, Shugo

    2013-04-01

    The myosin heavy chain gene, MYHM86-2, exhibited restricted expression in slow muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional mechanisms involved in its expression are still ambiguous. The present study is the first extensive analysis of slow muscle-specific MYHM86-2 promoter in fish for identifying the cis-elements that are crucial for its expression. Combining both transient transfection and transgenic approaches, we demonstrated that the 2614bp 5'-flanking sequences of MYHM86-2 contain a sufficient promoter activity to drive gene expression specific to superficial slow muscle fibers. By cyclopamine treatment, we also demonstrated that the differentiation of such superficial slow muscle fibers depends on hedgehog signaling activity. The deletion analyses defined an upstream fragment necessary for repressing ectopic MYHM86-2 expression in the fast muscle fibers. The transcriptional mechanism that prevents MYHM86-2 expression in the fast muscle fibers is mediated through Sox6 binding elements. We also demonstrated that Sox6 may function as a transcriptional repressor of MYHM86-2 expression. We further discovered that nuclear factor of activated T cells (NFAT) binding elements plays a key role and myocyte enhancer factor-2 (MEF2) binding elements participate in the transcriptional regulation of MYHM86-2 expression.

  5. Luminous Extragalactic Transients

    NASA Astrophysics Data System (ADS)

    O'Brien, Paul; Jonker, Peter; Athena Explosive Transients Working Group

    2015-09-01

    The ESA Athena X-ray observatory mission will combine exceptionally high throughout with high spectral-energy resolution and will revolutionise many aspects of high-energy astrophysics. Many of the most powerful transient sources, including gamma-ray bursts and tidal disruptions events, are bright X-ray sources. Athena will be designed to have a fast-response capability permitting efficient observations of many transients. We will summarise the proposed capability of the mission and illustrate science programs to study transients ranging from the most distant GRBs to nuclear activity in nearby galaxies.

  6. Transient nucleation in glasses

    NASA Technical Reports Server (NTRS)

    Kelton, K. F.

    1991-01-01

    Nucleation rates in condensed systems are frequently not at their steady state values. Such time dependent (or transient) nucleation is most clearly observed in devitrification studies of metallic and silicate glasses. The origin of transient nucleation and its role in the formation and stability of desired phases and microstructures are discussed. Numerical models of nucleation in isothermal and nonisothermal situations, based on the coupled differential equations describing cluster evolution within the classical theory, are presented. The importance of transient nucleation in glass formation and crystallization is discussed.

  7. [Transient epileptic amnesia].

    PubMed

    Muramatsu, Kazuhiro; Yoshizaki, Takahito

    2016-03-01

    Transient amnesia is one of common clinical phenomenon of epilepsy that are encountered by physicians. The amnestic attacks are often associated with persistent memory disturbances. Epilepsy is common among the elderly, with amnesia as a common symptom and convulsions relatively uncommon. Therefore, amnesia due to epilepsy can easily be misdiagnosed as dementia. The term 'transient epileptic amnesia (TEA)' was introduced in the early 1990s by Kapur, who highlighted that amnestic attacks caused by epilepsy can be similar to those occurring in 'transient global amnesia', but are distinguished by features brevity and recurrence. In 1998, Zeman et al. proposed diagnostic criteria for TEA.

  8. Variance-corrected Michaelis-Menten equation predicts transient rates of single-enzyme reactions and response times in bacterial gene-regulation

    NASA Astrophysics Data System (ADS)

    Pulkkinen, Otto; Metzler, Ralf

    2015-12-01

    Many chemical reactions in biological cells occur at very low concentrations of constituent molecules. Thus, transcriptional gene-regulation is often controlled by poorly expressed transcription-factors, such as E.coli lac repressor with few tens of copies. Here we study the effects of inherent concentration fluctuations of substrate-molecules on the seminal Michaelis-Menten scheme of biochemical reactions. We present a universal correction to the Michaelis-Menten equation for the reaction-rates. The relevance and validity of this correction for enzymatic reactions and intracellular gene-regulation is demonstrated. Our analytical theory and simulation results confirm that the proposed variance-corrected Michaelis-Menten equation predicts the rate of reactions with remarkable accuracy even in the presence of large non-equilibrium concentration fluctuations. The major advantage of our approach is that it involves only the mean and variance of the substrate-molecule concentration. Our theory is therefore accessible to experiments and not specific to the exact source of the concentration fluctuations.

  9. Variance-corrected Michaelis-Menten equation predicts transient rates of single-enzyme reactions and response times in bacterial gene-regulation.

    PubMed

    Pulkkinen, Otto; Metzler, Ralf

    2015-12-04

    Many chemical reactions in biological cells occur at very low concentrations of constituent molecules. Thus, transcriptional gene-regulation is often controlled by poorly expressed transcription-factors, such as E.coli lac repressor with few tens of copies. Here we study the effects of inherent concentration fluctuations of substrate-molecules on the seminal Michaelis-Menten scheme of biochemical reactions. We present a universal correction to the Michaelis-Menten equation for the reaction-rates. The relevance and validity of this correction for enzymatic reactions and intracellular gene-regulation is demonstrated. Our analytical theory and simulation results confirm that the proposed variance-corrected Michaelis-Menten equation predicts the rate of reactions with remarkable accuracy even in the presence of large non-equilibrium concentration fluctuations. The major advantage of our approach is that it involves only the mean and variance of the substrate-molecule concentration. Our theory is therefore accessible to experiments and not specific to the exact source of the concentration fluctuations.

  10. Variance-corrected Michaelis-Menten equation predicts transient rates of single-enzyme reactions and response times in bacterial gene-regulation

    PubMed Central

    Pulkkinen, Otto; Metzler, Ralf

    2015-01-01

    Many chemical reactions in biological cells occur at very low concentrations of constituent molecules. Thus, transcriptional gene-regulation is often controlled by poorly expressed transcription-factors, such as E.coli lac repressor with few tens of copies. Here we study the effects of inherent concentration fluctuations of substrate-molecules on the seminal Michaelis-Menten scheme of biochemical reactions. We present a universal correction to the Michaelis-Menten equation for the reaction-rates. The relevance and validity of this correction for enzymatic reactions and intracellular gene-regulation is demonstrated. Our analytical theory and simulation results confirm that the proposed variance-corrected Michaelis-Menten equation predicts the rate of reactions with remarkable accuracy even in the presence of large non-equilibrium concentration fluctuations. The major advantage of our approach is that it involves only the mean and variance of the substrate-molecule concentration. Our theory is therefore accessible to experiments and not specific to the exact source of the concentration fluctuations. PMID:26635080

  11. Heterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice

    PubMed Central

    Stellari, Fabio Franco; Ruscitti, Francesca; Pompilio, Daniela; Ravanetti, Francesca; Tebaldi, Giulia; Macchi, Francesca; Verna, Andrea Elizabeth; Villetti, Gino; Donofrio, Gaetano

    2017-01-01

    Idiopathic pulmonary fibrosis is a very common interstitial lung disease derived from chronic inflammatory insults, characterized by massive scar tissue deposition that causes the progressive loss of lung function and subsequent death for respiratory failure. Bleomycin is used as the standard agent to induce experimental pulmonary fibrosis in animal models for the study of its pathogenesis. However, to visualize the establishment of lung fibrosis after treatment, the animal sacrifice is necessary. Thus, the aim of this study was to avoid this limitation by using an innovative approach based on a double bleomycin treatment protocol, along with the in vivo images analysis of bleomycin-treated mice. A reporter gene construct, containing the luciferase open reading frame under the matrix metalloproteinase-1 promoter control region, was tested on double bleomycin-treated mice to investigate, in real time, the correlation between bleomycin treatment, inflammation, tissue remodeling and fibrosis. Bioluminescence emitted by the lungs of bleomycin-treated mice, corroborated by fluorescent molecular tomography, successfully allowed real time monitoring of fibrosis establishment. The reporter gene technology experienced in this work could represent an advanced functional approach for real time non-invasive assessment of disease evolution during therapy, in a reliable and translational living animal model. PMID:28298912

  12. High energy transients

    NASA Technical Reports Server (NTRS)

    Woosley, S. E.

    1984-01-01

    A meeting was convened on the campus of the University of California at Santa Cruz during the two-week interval July 11 through July 22, 1983. Roughly 100 participants were chosen so as to give broad representation to all aspects of high energy transients. Ten morning review sessions were held in which invited speakers discussed the current status of observations and theory of the above subjects. Afternoon workshops were also held, usually more than one per day, to informally review various technical aspects of transients, confront shortcomings in theoretical models, and to propose productive courses for future research. Special attention was also given to the instrumentation used to study high energy transient and the characteristics and goals of a dedicated space mission to study transients in the next decade were determined. A listing of articles written by various members of the workshop is included.

  13. Transient topographical amnesia.

    PubMed Central

    Stracciari, A; Lorusso, S; Pazzaglia, P

    1994-01-01

    Ten healthy middle aged or elderly women experienced isolated episodes of topographical amnesia without an obvious aetiology. It is likely a benign cognitive disorder, similar to transient global amnesia. PMID:7964826

  14. Transient Astrophysics Observatory (TAO)

    NASA Astrophysics Data System (ADS)

    Racusin, J. L.; TAO Team

    2016-10-01

    The Transient Astrophysics Observatory (TAO) is a NASA MidEx mission concept (formerly known as Lobster) designed to provide simultaneous wide-field gamma-ray, X-ray, and near-infrared observations of the sky.

  15. Transient Ischemic Attack

    MedlinePlus

    A transient ischemic attack (TIA) is a stroke lasts only a few minutes. It happens when the blood supply to part of the brain is briefly blocked. Symptoms of a TIA are like other stroke symptoms, but do not ...

  16. Transgenic Amorphophallus konjac expressing synthesized acyl-homoserine lactonase (aiiA) gene exhibit enhanced resistance to soft rot disease.

    PubMed

    Ban, Huifang; Chai, Xinli; Lin, Yongjun; Zhou, Ying; Peng, Donghai; Zhou, Yi; Zou, Yulan; Yu, Ziniu; Sun, Ming

    2009-12-01

    Amorphophallus konjac is an important economic crop widely used in health products and biomaterials. However, this monocotyledonous plant's production is seriously restricted by soft rot disease. Some Bacillus thuringiensis strains generate an endocellular acyl homoserine lactonase (AiiA), which has inhibitory effect on soft rot pathogen through disrupting the signal molecules (N-acylhomoserine lactones, AHL) of their Quorum Sensing system. The aim of our study is to obtain transgenic A. konjac expressing AiiA protein and exhibiting resistance to soft rot. But till now, there is not any report about exogenous gene transformation in A. konjac. In this research, an Agrobacterium-mediated genetic transformation system was constructed. An aiiA gene was synthesized according to the codon usage in A. konjac. Embryogenic callus was infected with the A. tumefaciens strain EHA105 harboring the plant transformation plasmid pU1301 plus synthesized aiiA gene. After antibiotics screening, 34 plants were obtained. PCR analysis showed that positive amplified fragments were present in 21 out of these 34 lines. Southern blot analysis indicated that aiiA gene had integrated into the genome of A. konjac. Western blotting demonstrated that the target protein of interest was reactive with the antibody against AiiA. Further disease resistance detection revealed that all of the tested transgenic A. konjac lines exhibited high resistance to soft rot bacteria Erwinia carotovora subsp. Carotovora (Ecc) SCG1. The protocol is useful for the quality improvement of A. konjac through genetic transformation.

  17. Transgenic rice plants expressing synthetic cry2AX1 gene exhibits resistance to rice leaffolder (Cnaphalocrosis medinalis).

    PubMed

    Manikandan, R; Balakrishnan, N; Sudhakar, D; Udayasuriyan, V

    2016-06-01

    Bacillus thuringiensis is a major source of insecticidal genes imparting insect resistance in transgenic plants. Level of expression of transgenes in transgenic plants is important to achieve desirable level of resistance against target insects. In order to achieve desirable level of expression, rice chloroplast transit peptide sequence was fused with synthetic cry2AX1 gene to target its protein in chloroplasts. Sixteen PCR positive lines of rice were generated by Agrobacterium mediated transformation using immature embryos. Southern blot hybridization analysis of T0 transgenic plants confirmed the integration of cry2AX1 gene in two to five locations of rice genome and ELISA demonstrated its expression. Concentration of Cry2AX1 in transgenic rice events ranged 5.0-120 ng/g of fresh leaf tissue. Insect bioassay of T0 transgenic rice plants against neonate larvae of rice leaffolder showed larval mortality ranging between 20 and 80 % in comparison to control plant. Stable inheritance and expression of cry2AX1 gene was demonstrated in T1 progenies through Southern and ELISA. In T1 progenies, the highest concentration of Cry2AX1 and mortality of rice leaffolder larvae were recorded as 150 ng/g of fresh leaf tissue and 80 %, respectively. The Cry2AX1 expression even at a very low concentration (120-150 ng/g) in transgenic rice plants was found effective against rice leaffolder larvae.

  18. Cloning and functional analysis of the promoter of a maize starch synthase III gene (ZmDULL1).

    PubMed

    Wu, J D; Jiang, C P; Zhu, H S; Jiang, H Y; Cheng, B J; Zhu, S W

    2015-05-22

    The ZmDULL1 gene encodes a starch synthase and is a determinant of the structure of endosperm starch in maize (Zea mays L.). However, little is known regarding the regulatory mechanism of the ZmDULL1 gene. In this study, we isolated and characterized the ZmDULL1 promoter (PDULL1), which is the 5' flanking region of ZmDULL1 in maize. Sequence analysis showed that several cis-acting elements important for endosperm expression (GCN4_motif and AACA-element) were located within the promoter. A series of PDULL1 deletion derivatives, PDULL1-1-PDULL1-4, from the translation start code (-1676, -1216, -740, and -343) were fused to the β-glucuronidase (GUS) reporter gene. Each deletion construct was transformed into rice using the Agrobacterium-mediated method, and then GUS activity was measured in transgenic plants. The results showed that PDULL1 was an endosperm-specific promoter. Further analysis showed that the promoter sequence (-343 to -1 base pairs) was sufficient for mediating GUS gene expression in endosperm. These results indicate that the region from -343 to -1 base pairs of PDULL1 is valuable for transgenic rice breeding and genetic engineering studies.

  19. Transient multivariable sensor evaluation

    DOEpatents

    Vilim, Richard B.; Heifetz, Alexander

    2017-02-21

    A method and system for performing transient multivariable sensor evaluation. The method and system includes a computer system for identifying a model form, providing training measurement data, generating a basis vector, monitoring system data from sensor, loading the system data in a non-transient memory, performing an estimation to provide desired data and comparing the system data to the desired data and outputting an alarm for a defective sensor.

  20. Gamma ray transients

    NASA Technical Reports Server (NTRS)

    Cline, Thomas L.

    1987-01-01

    The discovery of cosmic gamma ray bursts was made with systems designed at Los Alamos Laboratory for the detection of nuclear explosions beyond the atmosphere. HELIOS-2 was the first gamma ray burst instrument launched; its initial results in 1976, seemed to deepen the mystery around gamma ray transients. Interplanetary spacecraft data were reviewed in terms of explaining the behavior and source of the transients.

  1. Short- and long-term dynamic responses of the metabolic network and gene expression in yeast to a transient change in the nutrient environment.

    PubMed

    Dikicioglu, Duygu; Dunn, Warwick B; Kell, Douglas B; Kirdar, Betul; Oliver, Stephen G

    2012-06-01

    Quantitative data on the dynamic changes in the transcriptome and the metabolome of yeast in response to an impulse-like perturbation in nutrient availability was integrated with the metabolic pathway information in order to elucidate the long-term dynamic re-organization of the cells. This study revealed that, in addition to the dynamic re-organization of the de novo biosynthetic pathways, salvage pathways were also re-organized in a time-dependent manner upon catabolite repression. The transcriptional and the metabolic responses observed for nitrogen catabolite repression were not as severe as those observed for carbon catabolite repression. Selective up- or down regulation of a single member of a paralogous gene pair during the response to the relaxation from nutritional limitation was identified indicating a differentiation of functions among paralogs. Our study highlighted the role of inosine accumulation and recycling in energy homeostasis and indicated possible bottlenecks in the process.

  2. Transient overexpression of cyclin D2/CDK4/GLP1 genes induces proliferation and differentiation of adult pancreatic progenitors and mediates islet regeneration.

    PubMed

    Chen, Shuyuan; Shimoda, Masayuki; Chen, Jiaxi; Matsumoto, Shinichi; Grayburn, Paul A

    2012-02-15

    The molecular mechanism of β-cell regeneration remains poorly understood. Cyclin D2/CDK4 expresses in normal β cells and maintains adult β-cell growth. We hypothesized that gene therapy with cyclin D2/CDK4/GLP-1 plasmids targeted to the pancreas of STZ-treated rats by ultrasound-targeted microbubble destruction (UTMD) would force cell cycle re-entry of residual G(0)-phase islet cells into G(1)/S phase to regenerate β cells. A single UTMD treatment induced β-cell regeneration with reversal of diabetes for 6 mo without evidence of toxicity. We observed that this β-cell regeneration was not mediated by self-replication of pre-existing β cells. Instead, cyclin D2/CDK4/GLP-1 initiated robust proliferation of adult pancreatic progenitor cells that exist within islets and terminally differentiate to mature islets with β cells and α cells.

  3. SmgGDS is a transient nucleolar protein that protects cells from nucleolar stress and promotes the cell cycle by regulating DREAM complex gene expression.

    PubMed

    Gonyo, P; Bergom, C; Brandt, A C; Tsaih, S-W; Sun, Y; Bigley, T M; Lorimer, E L; Terhune, S S; Rui, H; Flister, M J; Long, R M; Williams, C L

    2017-08-14

    The chaperone protein and guanine nucleotide exchange factor SmgGDS (RAP1GDS1) is a key promoter of cancer cell proliferation and tumorigenesis. SmgGDS undergoes nucleocytoplasmic shuttling, suggesting that it has both cytoplasmic and nuclear functions that promote cancer. Previous studies indicate that SmgGDS binds cytoplasmic small GTPases and promotes their trafficking to the plasma membrane. In contrast, little is known about the functions of SmgGDS in the nucleus, or how these nuclear functions might benefit cancer cells. Here we show unique nuclear localization and regulation of gene transcription pathways by SmgGDS. Strikingly, SmgGDS depletion significantly reduces expression of over 600 gene products that are targets of the DREAM complex, which is a transcription factor complex that regulates expression of proteins controlling the cell cycle. The cell cycle regulators E2F1, MYC, MYBL2 (B-Myb) and FOXM1 are among the DREAM targets that are diminished by SmgGDS depletion. E2F1 is well known to promote G1 cell cycle progression, and the loss of E2F1 in SmgGDS-depleted cells provides an explanation for previous reports that SmgGDS depletion characteristically causes a G1 cell cycle arrest. We show that SmgGDS localizes in nucleoli, and that RNAi-mediated depletion of SmgGDS in cancer cells disrupts nucleolar morphology, signifying nucleolar stress. We show that nucleolar SmgGDS interacts with the RNA polymerase I transcription factor upstream binding factor (UBF). The RNAi-mediated depletion of UBF diminishes nucleolar localization of SmgGDS and promotes proteasome-mediated degradation of SmgGDS, indicating that nucleolar sequestration of SmgGDS by UBF stabilizes SmgGDS protein. The ability of SmgGDS to interact with UBF and localize in the nucleolus is diminished by expressing DiRas1 or DiRas2, which are small GTPases that bind SmgGDS and act as tumor suppressors. Taken together, our results support a novel nuclear role for SmgGDS in protecting malignant

  4. Characterization of phenotype resistance to plum pox of transgenic plums expressing plum pox virus capsid gene.

    PubMed

    Ravelonandro, M; Scorza, R; Dunez, J

    1998-09-01

    Resistance to plum pox virus (PPV) infection can be obtained in transgenic plants that express the virus capsid gene. An Agrobacterium-mediated transformation was used to introduce the PPV capsid gene into Prunus domestica plants. Over 11 regenerated plants (clones) were observed for the development of the disease symptoms and analysed for the presence of PPV by enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) through 4 dormancy cycles. The level of protection against PPV was determined in the transformed plants, non-transformed plants, and a control transgenic plant "transformed" with the plasmid vector alone. One clone, C-5, appeared fully protected, while PT-6 and C-4 clones accumulated a low concentration of virus and the rest of the clones was entirely susceptible. Little is known about the mechanisms of resistance to virus infection in transgenic woody plants. To investigate this aspect, comparative studies based on the characteristics of resistant and susceptible clones have been started. A question, whether the phenotype resistance of clone C-5 is similar to that observed in transgenic herbaceous plants or not, has been addressed. Recent progress in this investigation is presented.

  5. The metallothionein gene, TaMT3, from Tamarix androssowii confers Cd2+ tolerance in tobacco.

    PubMed

    Zhou, Boru; Yao, Wenjing; Wang, Shengji; Wang, Xinwang; Jiang, Tingbo

    2014-06-10

    Cadmium (Cd) is a nonessential microelement and low concentration Cd2+ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys)-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS) with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum) through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd2+ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD) activity and chlorophyll concentration, but decreases of peroxidase (POD) activity and malondialdehyde (MDA) accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd2+ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd2+ stress.

  6. Overexpression of a glutamine synthetase gene affects growth and development in sorghum.

    PubMed

    Urriola, Jazmina; Rathore, Keerti S

    2015-06-01

    Nitrogen is a primary macronutrient in plants, and nitrogen fertilizers play a critical role in crop production and yield. In this study, we investigated the effects of overexpressing a glutamine synthetase (GS) gene on nitrogen metabolism, and plant growth and development in sorghum (Sorghum bicolor L., Moench). GS catalyzes the ATP dependent reaction between ammonia and glutamate to produce glutamine. A 1,071 bp long coding sequence of a sorghum cytosolic GS gene (Gln1) under the control of the maize ubiquitin (Ubq) promoter was introduced into sorghum immature embryos by Agrobacterium-mediated transformation. Progeny of the transformants exhibited higher accumulation of the Gln1 transcripts and up to 2.2-fold higher GS activity compared to the non-transgenic controls. When grown under optimal nitrogen conditions, these Gln1 transgenic lines showed greater tillering and up to 2.1-fold increase in shoot vegetative biomass. Interestingly, even under greenhouse conditions, we observed a seasonal component to both these parameters and the grain yield. Our results, showing that the growth and development of sorghum Gln1 transformants are also affected by N availability and other environmental factors, suggest complexity of the relationship between GS activity and plant growth and development. A better understanding of other control points and the ability to manipulate these will be needed to utilize the transgenic technology to improve nitrogen use efficiency of crop plants.

  7. Identification and Characterization of Multiple Intermediate Alleles of the Key Genes Regulating Brassinosteroid Biosynthesis Pathways

    PubMed Central

    Du, Junbo; Zhao, Baolin; Sun, Xin; Sun, Mengyuan; Zhang, Dongzhi; Zhang, Shasha; Yang, Wenyu

    2017-01-01

    Most of the early identified brassinosteroid signaling and biosynthetic mutants are null mutants, exhibiting extremely dwarfed phenotypes and male sterility. These null mutants are usually unable to be directly transformed via a routinely used Agrobacterium-mediated gene transformation system and therefore are less useful for genetic characterization of the brassinosteroid (BR)-related pathways. Identification of intermediate signaling mutants such as bri1–5 and bri1–9 has contributed drastically to the elucidation of BR signaling pathway using both genetic and biochemical approaches. However, intermediate mutants of key genes regulating BR biosynthesis have seldom been reported. Here we report identification of several intermediate BR biosynthesis mutants mainly resulted from leaky transcriptions due to the insertions of T-DNAs in the introns. These mutants are semi-dwarfed and fertile and capable to be transformed. These intermediate mutants could be useful tools for future discovery and analyses of novel components regulating BR biosynthesis and catabolism via genetic modifier screen. PMID:28138331

  8. Changes in Gene Expression Patterns of Circadian-Clock, Transient Receptor Potential Vanilloid-1 and Nerve Growth Factor in Inflamed Human Esophagus.

    PubMed

    Yang, Shu-Chuan; Chen, Chien-Lin; Yi, Chih-Hsun; Liu, Tso-Tsai; Shieh, Kun-Ruey

    2015-09-04

    Circadian rhythm is driven by the molecular circadian-clock system and regulates many physiological functions. Diurnal rhythms in the gastrointestinal tract are known to be related to feeding pattern, but whether these rhythms are also related to the gastrointestinal damage or injuries; for example, gastroesophageal reflux disease (GERD), is unclear. This study was conducted to determine whether expression of circadian-clock genes or factors involved in vagal stimulation or sensitization were altered in the esophagus of GERD patients. Diurnal patterns of PER1, PER2, BMAL1, CRY2, TRPV1, and NGF mRNA expression were found in patient controls, and these patterns were altered and significantly correlated to the GERD severity in GERD patients. Although levels of CRY1, TIM, CB1, NHE3, GDNF, and TAC1 mRNA expression did not show diurnal patterns, they were elevated and also correlated with GERD severity in GERD patients. Finally, strong correlations among PER1, TRPV1, NGF and CRY2 mRNA expression, and among PER2, TRPV1 and CRY2 expression were found. Expression levels of CRY1 mRNA highly correlated with levels of TIM, CB1, NHE3, GDNF and TAC1. This study suggests that the circadian rhythm in the esophagus may be important for the mediation of and/or the response to erosive damage in GERD patients.

  9. Changes in Gene Expression Patterns of Circadian-Clock, Transient Receptor Potential Vanilloid-1 and Nerve Growth Factor in Inflamed Human Esophagus

    PubMed Central

    Yang, Shu-Chuan; Chen, Chien-Lin; Yi, Chih-Hsun; Liu, Tso-Tsai; Shieh, Kun-Ruey

    2015-01-01

    Circadian rhythm is driven by the molecular circadian-clock system and regulates many physiological functions. Diurnal rhythms in the gastrointestinal tract are known to be related to feeding pattern, but whether these rhythms are also related to the gastrointestinal damage or injuries; for example, gastroesophageal reflux disease (GERD), is unclear. This study was conducted to determine whether expression of circadian-clock genes or factors involved in vagal stimulation or sensitization were altered in the esophagus of GERD patients. Diurnal patterns of PER1, PER2, BMAL1, CRY2, TRPV1, and NGF mRNA expression were found in patient controls, and these patterns were altered and significantly correlated to the GERD severity in GERD patients. Although levels of CRY1, TIM, CB1, NHE3, GDNF, and TAC1 mRNA expression did not show diurnal patterns, they were elevated and also correlated with GERD severity in GERD patients. Finally, strong correlations among PER1, TRPV1, NGF and CRY2 mRNA expression, and among PER2, TRPV1 and CRY2 expression were found. Expression levels of CRY1 mRNA highly correlated with levels of TIM, CB1, NHE3, GDNF and TAC1. This study suggests that the circadian rhythm in the esophagus may be important for the mediation of and/or the response to erosive damage in GERD patients. PMID:26337663

  10. RNAi-mediated silencing of MAP kinase signalling genes (Fmk1, Hog1, and Pbs2) in Fusarium oxysporum reduces pathogenesis on tomato plants.

    PubMed

    Pareek, Manish; Rajam, Manchikatla Venkat

    2017-09-01

    Fusarium oxysporum is a soil-borne plant fungal pathogen, and causes colossal losses in several crop plants including tomato. Effective control measures include the use of harmful fungicides and resistant cultivars, but these methods have shown limited success. Conventional methods to validate fungal pathogenic genes are labour intensive. Therefore, an alternative strategy is required to efficiently characterize unknown pathogenic genes. RNA interference (RNAi) has emerged as a potential tool to functionally characterize novel fungal pathogenic genes and also to control fungal diseases. Here, we report an efficient method to produce stable RNAi transformants of F. oxysporum using Agrobacterium-mediated transformation (AMT). We have transformed F. oxysporum spores using RNAi constructs of Fmk1, Hog1, and Pbs2 MAP kinase signalling genes. Fmk1 RNAi fungal transformants showed loss of surface hydrophobicity, reduced invasive growth on tomato fruits and hypo-virulence on tomato seedlings. Hog1 and Pbs2 RNAi transformants showed altered conidial size, and reduced invasive growth and pathogenesis. These results showed that AMT using RNAi constructs is an effective approach for dissecting the role of genes involved in pathogenesis in F. oxysporum and this could be extended for other fungal systems. The obtained knowledge can be easily translated for developing fungal resistant crops by RNAi. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  11. Establishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.).

    PubMed

    Ozawa, Kenjirou

    2009-04-01

    Technologies for transformation of rice have been developed to meet the requirements of functional genomics in order to enable the production of transgenic rice plants with useful agricultural characters. However, many rice varieties are not efficiently transformed by Agrobacterium. We have succeeded in establishing a highly efficient transformation system in rice by co-cultivating rice calli with Agrobacterium on three filter papers moistened with enriched N6 or DKN media instead of using solid media. Rice calli immersed in Agrobacterium suspension (EHA101, Agrobacterium concentration of OD600=0.04) were co-cultured on three pieces of filter paper (9cm in diameter) moistened with 5.5mL of N6 or DKN liquid co-cultivation medium supplemented with 2,4-d (2mg/L), proline (10mM), casein hydrolysate (300mg/L), sucrose (30g/L), glucose (5g/L), l-cysteine (100mg/L) and acetosyringone (15mg/L) at 25°C for 3 days in the dark. Compared with the transformation efficiency of calli co-cultivated on solid media, transformation efficiency was increased by about fivefold by using the filter paper method for many varieties of rice, including those that previously yielded much poor transformation rates.

  12. Refined glufosinate selection in Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill].

    PubMed

    Zeng, P; Vadnais, D A; Zhang, Z; Polacco, J C

    2004-02-01

    Modern genetic analysis and manipulation of soybean ( Glycine max) depend heavily on an efficient and dependable transformation process, especially in public genotypes from which expressed sequence tag (EST), bacterial artificial chromosome and microarray data have been derived. Williams 82 is the subject of EST and functional genomics analyses. However, it has not previously been transformed successfully using either somatic embryogenesis-based or cotyledonary-node transformation methods, the two predominant soybean transformation systems. An advance has recently been made in using antioxidants to enhance Agrobacterium infection of soybean. Nonetheless, an undesirable effect of using these antioxidants is the compromised recovery of transgenic soybean when combined with the use of the herbicide glufosinate as a selective agent. Therefore, we optimized both Agrobacterium infection and glufosinate selection in the presence of L-cysteine for Williams 82. We have recovered transgenic lines of this genotype with an enhanced transformation efficiency using this herbicide selection system.

  13. Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood tree species Fraxinus profunda

    Treesearch

    Micah E. Stevens; Paula M. Pijut

    2014-01-01

    Using mature hypocotyls as the initial explants, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for pumpkin ash (Fraxinus profunda). This transformation protocol is an invaluable tool to combat the highly aggressive, non-native emerald ash borer (EAB), which has the potential to...

  14. Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration

    Treesearch

    Ningxia Du; Paula M. Pijut

    2009-01-01

    A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion...

  15. Enhancers of Agrobacterium-mediated Transformation of Tibouchina semidecandra Selected on the Basis of GFP Expression.

    PubMed

    Yong, Wilson Thau Lym; Henry, Erle Stanley; Abdullah, Janna Ong

    2010-12-01

    Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 μM galactose and 100 μM tyrosine supplemented with 600 μM AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 μM galactose and 50 μM tyrosine with 200 μM AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative T. semidecandra transformants was verified by PCR amplification with specific primers.

  16. Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri.

    PubMed

    Rathod, Jayant Pralhad; Prakash, Gunjan; Pandit, Reena; Lali, Arvind M

    2013-11-01

    Parachlorella kessleri is a unicellular alga which grows in fresh as well as marine water and is commercially important as biomass/lipid feedstock and in bioremediation. The present study describes the successful transformation of marine P. kessleri with the help of Agrobacterium tumefaciens. Transformed marine P. kessleri was able to tolerate more than 10 mg l(-1) hygromycin concentration. Co-cultivation conditions were modulated to allow the simultaneous growth of both marine P. kessleri and A. tumefaciens. For co-cultivation, P. kessleri was shifted from Walne's to tris acetate phosphate medium to reduce the antibiotic requirement during selection. In the present study, the transfer of T-DNA was successful without using acetosyringone. Biochemical and genetic analyses were performed for expression of transgenes by GUS assay and PCR in transformants. Establishment of this protocol would be useful in further genetic modification of oil-bearing Parachlorella species.

  17. Cadophora finlandia and Phialocephala fortinii: Agrobacterium-mediated transformation and functional GFP expression.

    PubMed

    Gorfer, Markus; Klaubauf, Sylvia; Bandian, Dragana; Strauss, Joseph

    2007-07-01

    Hygromycin B resistance was transferred to the sterile mycelia of Cadophora finlandia and Phialocephala fortinii by co-cultivation with Agrobacterium tumefaciens. Constitutively expressed green fluorescent protein (GFP) was also introduced using the same vector. Confocal laser scanning microscopy (CLSM) revealed strong fluorescence of transformants. Both traits were mitotically stable during one year of subculturing on non-selective growth medium. Southern blot analysis showed that the majority of the transformants contained single-copy integrations at random sites in the genome.

  18. Parameters influencing Agrobacterium-mediated transformation system in safflower genotypes AKS-207 and PKV Pink.

    PubMed

    Dhumale, Dipti Raghunath; Shingote, Prashant Raghunath; Dudhare, Mahendra Shankarrao; Jadhav, Pravin Vishwanath; Kale, Prashant Bhaskar

    2016-12-01

    Shoot regeneration in safflower (Carthamus tinctorius 'AKS 207' and 'PKV Pink') genetically transformed using Agrobacterium was used for assessing various constraints to the efficiency of transformation including infection period, virulence induction medium, co-cultivation period, bacterial titre, selection regime, and the natural phenolic compound acetosyringone. Transformation frequency was promising with 8-10-day-old cotyledonary leaf explants. Therefore, explants of that age cultured on Agrobacterium minimal medium (AB) containing 100 µM acetosyringone were infected with Agrobacterium (cell titre 0.5 OD600nm) for 15 min followed by 48 h of co-cultivation on kanamycin-enriched medium (50 mg/L). Transformation of the shoots was confirmed using β-glucuronidase (GUS) histochemical assay and polymerase chain reaction (PCR). With the transformation protocol thus optimized, the transformation frequency as determined using GUS assays was 54.0 % for AKS 207 and 47.6 % for PKV Pink. The corresponding figures using PCR were 27.0 and 33.3 %. The transformed shoots required 10-14 weeks of culture initiation but produced very few roots.

  19. Agrobacterium-mediated transformation of black cherry for flowering control and insect resistance

    Treesearch

    Ying Wang; Paula M. Pijut

    2014-01-01

    Black cherry is one of the most valuable hardwood species for cabinetry, furniture, and veneer. The goal of this study was to develop transgenic black cherry plants with reproductive sterility and enhanced insect resistance. Black cherry TERMINAL FLOWER 1 (PsTFL1) was overexpressed under the control of the CaMV 35S promoter in black cherry via

  20. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    SciTech Connect

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

    1984-10-01

    The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

  1. Cloning and characterization of a Verticillium wilt resistance gene from Gossypium barbadense and functional analysis in Arabidopsis thaliana.

    PubMed

    Zhang, Yan; Wang, Xingfen; Yang, Shuo; Chi, Jina; Zhang, Guiyin; Ma, Zhiying

    2011-11-01

    Verticillium wilt causes enormous loss to yield or quality in many crops. In an effort to help controlling this disease through genetic engineering, we first cloned and characterized a Verticillium wilt resistance gene (GbVe) from cotton (Gossypium barbadense) and analyzed its function in Arabidopsis thaliana. Its nucleotide sequence is 3,819 bp long, with an open reading frame of 3,387 bp, and encoding an 1,128-aa protein precursor. Sequence analysis shows that GbVe produces a leucine-rich repeat receptor-like protein. It shares identities of 55.9% and 57.4% with tomato Ve1 and Ve2, respectively. Quantitative real-time PCR indicated that the Ve gene expression pattern was different between the resistant and susceptible cultivars. In the resistant Pima90-53, GbVe was quickly induced and reached to a peak at 2 h after inoculation, two-fold higher than that of control. We localized the GbVe-GFP fusion protein to the cytomembrane in onion epidermal cells. By inserting GbVe into Arabidopsis via Agrobacterium-mediated transformation, T(3) transgenic lines were obtained. Compared with the wild-type control, GbVe-overexpressing plants had greater levels of resistance to V. dahliae. This suggests that GbVe is a useful gene for improving the plant resistance against fungal diseases.

  2. Enhancing stress tolerance by overexpression of a methionine sulfoxide reductase A (MsrA) gene in Pleurotus ostreatus.

    PubMed

    Yin, Chaomin; Zheng, Liesheng; Zhu, Jihong; Chen, Liguo; Ma, Aimin

    2015-04-01

    Proteins are subjected to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological functions. Methionine as a sulfur-containing amino acid is easily oxidized to methionine sulfoxide (MetSO). The modified methionine can be repaired by methionine sulfoxide reductase (Msr), an enzyme that reverses oxidation of methionine in proteins. In this study, a methionine sulfoxide reductase A (PoMsrA) gene from Pleurotus ostreatus was cloned and characterized. Furthermore, the function of PoMsrA gene was analyzed by overexpression in P. ostreatus via Agrobacterium-mediated transformation. Stable integration of the target gene into the genome of P. ostreatus was confirmed by PCR, fluorescence observation, and Southern blot hybridization. qRT-PCR analysis showed that PoMsrA was highly expressed in the stage of mature and young fruiting bodies as well as the osmotic stress condition of 0.3 M NaCl. Additionally, the transgenic strains with PoMsrA overexpression exhibited an enhanced tolerance to high temperature, high osmotic stress, and oxidative stress. This suggests that PoMsrA is an active player in the protection of the cellular proteins from oxidative stress damage.

  3. Enhancing the Stress Tolerance and Virulence of an Entomopathogen by Metabolic Engineering of Dihydroxynaphthalene Melanin Biosynthesis Genes ▿ †

    PubMed Central

    Tseng, Min N.; Chung, Pei C.; Tzean, Shean S.

    2011-01-01

    Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as UV radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN) melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase genes) cloned from Alternaria alternata were transformed into the amelanotic entomopathogenic fungus Metarhizium anisopliae via Agrobacterium-mediated transformation. Melanin expression in the transformant of M. anisopliae was verified by spectrophotometric methods, liquid chromatography/mass spectrometry (LC/MS), and confocal microscopy. The transformant, especially under stresses, showed notably enhanced antistress capacity and virulence, in terms of germination and survival rate, infectivity, and reduced median time to death (LT50) in killing diamondback moth (Plutella xylostella) larvae compared with the wild type. The possible mechanisms in enhancing the stress tolerance and virulence, and the significance and potential for engineering melanin biosynthesis genes in other biocontrol agents and crops to improve antistress fitness are discussed. PMID:21571888

  4. Transgenic plants containing the phosphinothricin-N-acetyltransferase gene metabolize the herbicide L-phosphinothricin (glufosinate) differently from untransformed plants.

    PubMed

    Dröge, W; Broer, I; Pühler, A

    1992-04-01

    L-Phosphinothricin (L-Pt)-resistant plants were constructed by introducing a modified phosphinothricin-N-acetyl-transferase gene (pat) via Agrobacterium-mediated gene transfer into tobacco (Nicotiana tabacum L), and via direct gene transfer into carrot (Daucus carota L). The metabolism of L-Pt was studied in these transgenic, Pt-resistant plants, as well as in the untransformed species. The degradation of L-Pt, (14)C-labeled specifically at different C-atoms, was analysed by measuring the release of (14)CO2 and by separating the labeled degradation products on thin-layer-chromatography plates. In untransformed tobacco and carrot plants, L-Pt was deaminated to form its corresponding oxo acid 4-methylphosphinico-2-oxo-butanoic acid (PPO), which subsequently was decarboxylated to form 3-methylphosphinico-propanoic acid (MPP). This compound was stable in plants. A third metabolite remained unidentified. The L-Pt was rapidly N-acetylated in herbicide-resistant tobacco and carrot plants, indicating that the degradation pathway of L-Pt into PPO and MPP was blocked. The N-acetylated product, L-N-acetyl-Pt remained stable with regard to degradation, but was found to exist in a second modified form. In addition, there was a pH-dependent, reversible change in the mobility of L-N-acetyl-Pt thin-layer during chromatography.

  5. Germin-like protein 2 gene promoter from rice is responsive to fungal pathogens in transgenic potato plants.

    PubMed

    Munir, Faiza; Hayashi, Satomi; Batley, Jacqueline; Naqvi, Syed Muhammad Saqlan; Mahmood, Tariq

    2016-01-01

    Controlled transgene expression via a promoter is particularly triggered in response to pathogen infiltration. This is significant for eliciting disease-resistant features in crops through genetic engineering. The germins and germin-like proteins (GLPs) are known to be associated with plant and developmental stages. The 1107-bp Oryza sativa root GLP2 (OsRGLP2) gene promoter fused to a β-glucuronidase (GUS) reporter gene was transformed into potato plants through an Agrobacterium-mediated transformation. The OsRGLP2 promoter was activated in response to Fusarium solani (Mart.) Sacc. and Alternaria solani Sorauer. Quantitative real-time PCR results revealed 4-5-fold increase in promoter activity every 24 h following infection. There was a 15-fold increase in OsRGLP2 promoter activity after 72 h of F. solani (Mart.) Sacc. treatment and a 12-fold increase observed with A. solani Sorauer. Our results confirmed that the OsRGLP2 promoter activity was enhanced under fungal stress. Furthermore, a hyperaccumulation of H2O2 in transgenic plants is a clear signal for the involvement of OsRGLP2 promoter region in the activation of specific genes in the potato genome involved in H2O2-mediated defense response. The OsRGLP2 promoter evidently harbors copies of GT-I and Dof transcription factors (AAAG) that act in response to elicitors generated in the wake of pathogen infection.

  6. Structure and expression analysis of the OsCam1-1 calmodulin gene from Oryza sativa L.

    PubMed

    Phean-o-pas, Srivilai; Limpaseni, Tipaporn; Buaboocha, Teerapong

    2008-11-30

    Calmodulin (CaM) proteins, members of the EF-hand family of Ca(2+)- binding proteins, represent important relays in plant calcium signals. Here, OsCam1-1 was isolated by PCR amplification from the rice genome. The gene contains an ORF of 450 base pairs with a single intron at the same position found in other plant Cam genes. A promoter region with a TATA box at position-26 was predicted and fused to a gus reporter gene, and this construct was used to produce transgenic rice by Agrobacterium-mediated transformation. GUS activity was observed in all organs examined and throughout tissues in cross-sections, but activity was strongest in the vascular bundles of leaves and the vascular cylinders of roots. To examine the properties of OsCaM1-1, the encoding cDNA was expressed in Escherichia coli. The electrophoretic mobility shift when incubated with Ca(2+) indicates that recombinant OsCaM1-1 is a functional Ca(2+)-binding protein. In addition, OsCaM1-1 bound the CaMKII target peptide confirming its likely functionality as a calmodulin.

  7. [Study on human aFGF fusion gene transformation with soybean 24 kDa oleosin and expression in safflower].

    PubMed

    Gai, Yu-Hong; Wang, Wang; Jin, Xiao; Wang, Jia-Qi; Li, Hong-Bo; Wang, Gang; Yang, Jing; Wei, Jian

    2013-06-01

    To investigate the expression of acidic fibroblast growth factor (aFGF) in transgenic safflower and lay the foundation for the use of the plant bioreactor large-scale production aFGF. The haFGF gene was transformed into plant preference of the aFGF sequence as a basis for design of primers, plant preferences aFGF gene sequences was amplified by PCR. The vegetable body expression vector was constructed by using digested connection method and then transferred to Agrobacterium tumefaciens EHA105 by the freeze-thaw method. It transferred to safflowers by agrobacterium-mediated transformation method, and identified by PCR, southern blot and RT-PCR. The full-length aFGF gene sequences were amplified through PCR and constructed into plant expression vector with soybean oleosin and promoter, and transformed into safflower. Three independently transformed safflower plant units with point insertion were successfully obtained, which showed the same size of aFGF expression at the transcriptional level. The plant oil body expression vectors were successfully constructed, and the optimal condition for genetic transformation was selected. The transgenic safflower plants were obtained.

  8. Palomar Transient Factory

    NASA Astrophysics Data System (ADS)

    Kulkarni, S.; Quimby, R.

    2010-12-01

    The Palomar Transient Factory (PTF) was designed to explicitly to chart the transient sky with a particular focus on events which lie in the nova-supernova gap. With its innovative two-telescope architecture it achieves both high cadence and large areal rate of coverage. PTF was commissioned during the summer of 2009. PTF is now finding an extragalactic transient every 20 minutes and a Galactic (strong) variable every 10 minutes. Spectroscopy undertaken at Keck and Palomar has allowed us: identify an emerging class of ultra-luminous supernovae, discover luminous red novae, undertake UV spectroscopy of Ia supernovae, discover supernovae powered by something other than Nickel-56, clarification of sub-classes of core collapse and thermo-nuclear explosions, map the systematics of core collapse supernovae, a trove of eclipsing binaries and many others.

  9. Transient Uncoupling Induces Synchronization

    NASA Astrophysics Data System (ADS)

    Schröder, Malte; Mannattil, Manu; Dutta, Debabrata; Chakraborty, Sagar; Timme, Marc

    2015-07-01

    Finding conditions that support synchronization is a fertile and active area of research with applications across multiple disciplines. Here we present and analyze a scheme for synchronizing chaotic dynamical systems by transiently uncoupling them. Specifically, systems coupled only in a fraction of their state space may synchronize even if fully coupled they do not. While for many standard systems coupling strengths need to be bounded to ensure synchrony, transient uncoupling removes this bound and thus enables synchronization in an infinite range of effective coupling strengths. The presented coupling scheme therefore opens up the possibility to induce synchrony in (biological or technical) systems whose parameters are fixed and cannot be modified continuously.

  10. [Construction of a bivalent plant expression vector carrying VvSUC11 and VvSUC12 genes and its genetic transformation in sugar beet].

    PubMed

    Yin, Donglin; Zhu, Jianbo; Wang, Aiying; Xiang, Benchun

    2011-08-01

    We have recombined genes VvSUC11, VvSUC12 from Vitis vinifera L., and root-specific promoters of sweet potato storage protein gene from Ipomoea batatas L. Lam., named as SP1 and SP2. We have constructed a vector pCAMBIA2301-SP1- VvSUC11-SP2-VvSUC12 using pCAMBIA2301 as an original vector. VvSUC11 and VvSUC12 were under the control of root-specific promoters of sweet potato storage protein gene. We transformed the vector into KWS-9103 breeding line of Beta vulgaris L. with Agrobacterium-mediated transformation. We have established the optimal genetic transformation protocol of sugar beet as following: the explants pre-cultured for 4 days were immersed in Agrobacterium suspension of OD(600)=0.5, supplemented with 0.005% Silwet L-77, and followed by a 4-day culture on medium containing cefotaxime, then the buds were selected on medium containing kanamycin and cefotaxime. The percentage of kanamycin-resistant buds was as high as 42%. Results of PCR and RT-PCR proved that the target genes had integrated into sugar beet genome and expressed. It will lay a foundation for further studying their function in Beta vulgaris.

  11. Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea).

    PubMed

    Singh, Natwar; Mishra, Avinash; Jha, Bhavanath

    2014-08-15

    Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T0 transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T1 plants were raised. Transgenic plants (T1) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T1) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Enhanced resistance to blister blight in transgenic tea (Camellia sinensis [L.] O. Kuntze) by overexpression of class I chitinase gene from potato (Solanum tuberosum).

    PubMed

    Singh, H Ranjit; Deka, Manab; Das, Sudripta

    2015-07-01

    Tea is the second most consumed beverage in the world. A crop loss of up to 43 % has been reported due to blister blight disease of tea caused by a fungus, Exobasidium vexans. Thus, it directly affects the tea industry qualitatively and quantitatively. Solanum tuberosum class I chitinase gene (AF153195) is a plant pathogenesis-related gene. It was introduced into tea genome via Agrobacterium-mediated transformation with hygromycin phosphotransferase (hpt) gene conferring hygromycin resistance as plant selectable marker. A total of 41 hygromycin resistant plantlets were obtained, and PCR analysis established 12 plantlets confirming about the stable integration of transgene in the plant genome. Real-time PCR detected transgene expression in four transgenic plantlets (T28, C57, C9, and T31). Resistance to biotrophic fungal pathogen, E. vexans, was tested by detached leaf infection assay of greenhouse acclimated plantlets. An inhibitory activity against the fungal pathogen was evident from the detached leaves from the transformants compared with the control. Fungal lesion formed on control plantlet whereas the transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area. This result suggests that constitutive expression of the potato class I chitinase gene can be exploited to improve resistance to fungal pathogen, E. vexans, in economical perennial plantation crop like tea.

  13. PaCDPK1, a gene encoding calcium-dependent protein kinase from orchid, Phalaenopsis amabilis, is induced by cold, wounding, and pathogen challenge.

    PubMed

    Tsai, Tsung-Mu; Chen, Ying-Ru; Kao, Tien-Wen; Tsay, Wen-Su; Wu, Chiou-Ping; Huang, Ding-Ding; Chen, Wen-Huei; Chang, Ching-Chun; Huang, Hao-Jen

    2007-10-01

    Signaling pathways, specifically calcium and calcium-dependent protein kinase (CDPK), have been implicated in the regulation of stress and developmental signals in plants. Here, we reported the isolation and characterization of an orchid, Phalaenopsis amabilis, CDPK gene, PaCDPK1, by using the rapid amplification of cDNA ends (RACE)-PCR technique. The full length cDNA of 2,310 bp contained an open reading frame for PaCDPK1 consisting of 593 amino acid residues. Sequence alignment indicated that PaCDPK1 shared similarities with other plant CDPKs. PaCDPK1 transcripts were expressed strongly in labellum but not in leaves and roots. In addition, the PaCDPK1 gene was transcriptionally activated in response to low temperature, wounding, and pathogen infection. To identify the regulatory role of the PaCDPK1 promoter, a construct containing the PaCDPK1 promoter fused to a beta-glucuronidase (GUS) gene was transferred into Arabidopsis by Agrobacterium-mediated transformation. GUS staining revealed that PaCDPK1/GUS expression was induced by cold, wounding, and pathogen challenge in leaves and stems of transgenic Arabidopsis. These results suggested that this PaCDPK1 gene promoter could be used as an endogenous promoter for biotechnological purposes in orchids.

  14. Different functions of the histone acetyltransferase HAC1 gene traced in the model species Medicago truncatula, Lotus japonicus and Arabidopsis thaliana.

    PubMed

    Boycheva, Irina; Vassileva, Valya; Revalska, Miglena; Zehirov, Grigor; Iantcheva, Anelia

    2017-03-01

    In eukaryotes, histone acetyltransferases regulate the acetylation of histones and transcription factors, affecting chromatin structural organization, transcriptional regulation, and gene activation. To assess the role of HAC1, a gene encoding for a histone acetyltransferase in Medicago truncatula, stable transgenic lines with modified HAC1 expression in the model plants M. truncatula, Lotus japonicus, and Arabidopsis thaliana were generated by Agrobacterium-mediated transformation and used for functional analyses. Histochemical, transcriptional, flow cytometric, and morphological analyses demonstrated the involvement of HAC1 in plant growth and development, responses to internal stimuli, and cell cycle progression. Expression patterns of a reporter gene encoding beta-glucuronidase (GUS) fused to the HAC1 promoter sequence were associated with young tissues comprised of actively dividing cells in different plant organs. The green fluorescent protein (GFP) signal, driven by the HAC1 promoter, was detected in the nuclei and cytoplasm of root cells. Transgenic lines with HAC1 overexpression and knockdown showed a wide range of phenotypic deviations and developmental abnormalities, which provided lines of evidence for the role of HAC1 in plant development. Synchronization of A. thaliana root tips in a line with HAC1 knockdown showed the involvement of this gene in the acetylation of two core histones during S phase of the plant cell cycle.

  15. Four RNA families with functional transient structures.

    PubMed

    Zhu, Jing Yun A; Meyer, Irmtraud M

    2015-01-01

    Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5' flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5' UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM. All

  16. Four RNA families with functional transient structures

    PubMed Central

    Zhu, Jing Yun A; Meyer, Irmtraud M

    2015-01-01

    Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5′ flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5′ UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM

  17. Lightning-Transient Recorder

    NASA Technical Reports Server (NTRS)

    Grumm, R. L.

    1984-01-01

    Battery-powered system operates for more than one year. Recorder digitizes and records up to 146 current samples at selected intervals during lightning stroke. System continues to store time tags of lightning strokes even if transient current memory is full.

  18. Transient tachypnea - newborn

    MedlinePlus

    ... or reabsorbing it. The first few breaths a baby takes after delivery fill the lungs with air and help to ... goes away within 24 to 48 hours after delivery. In most cases, babies who have had transient tachypnea have no further ...

  19. Transient lingual papillitis.

    PubMed

    Kornerup, Ida M; Senye, Mireya; Peters, Edmund

    2016-01-01

    A case of recurrent, clinically innocuous, but painful papules involving the tongue dorsum of a 25-year-old man is presented. The lesions were interpreted to represent a transient lingual papillitis. This a poorly understood, but benign and self-limited condition involving the tongue fungiform papillae, which does not appear to be widely recognized.

  20. Transient familial hyperbilirubinemia

    MedlinePlus

    ... please enable JavaScript. Transient familial hyperbilirubinemia is a metabolic disorder that is passed down through families. Babies with ... M. Editorial team. Related MedlinePlus Health Topics Jaundice Metabolic Disorders Browse the Encyclopedia A.D.A.M., Inc. ...