A Thermostable β-Glucuronidase Obtained by Directed Evolution as a Reporter Gene in Transgenic Plants

PubMed Central

Xiong, Ai-Sheng; Peng, Ri-He; Zhuang, Jing; Chen, Jian-Min; Zhang, Bin; Zhang, Jian; Yao, Quan-Hong

2011-01-01

A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter. PMID:22096498

Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata.

PubMed

Gherbi, Hassen; Nambiar-Veetil, Mathish; Zhong, Chonglu; Félix, Jessy; Autran, Daphné; Girardin, Raphaël; Vaissayre, Virginie; Auguy, Florence; Bogusz, Didier; Franche, Claudine

2008-05-01

In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the beta-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP(+) hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.

Mechanism of Gene Expression of Arabidopsis Glutathione S-Transferase, AtGST1, and AtGST11 in Response to Aluminum Stress1

PubMed Central

Ezaki, Bunichi; Suzuki, Masakatsu; Motoda, Hirotoshi; Kawamura, Masako; Nakashima, Susumu; Matsumoto, Hideaki

2004-01-01

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5′-upstream region of each gene was fused to a β-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress. PMID:15047894

Alteration of gene expression by restriction enzymes electroporated into plant cells.

PubMed

Ashraf, M; Altschuler, M; Galasinski, S; Griffiths, T D

1993-06-01

The alteration in the expression of a beta-glucuronidase (GUS) reporter gene was used to monitor the effect of restriction endonucleases electroporated into the tobacco (Nicotiana tabacum L.) protoplasts. Restriction enzyme (RE) Hind III which does not have a recognition site within the gene cassette, had little effect on enzyme activity. In contrast restriction endonucleases Hae III and Sau3A1 which possess 8 and 16 recognition sites in the GUS cassette, were found to reduce the enzyme activity by 89% and 94% respectively when compared to control electroporations. Restriction-site mutation analysis (RSM) and Southern blot analysis indicated the enzymatic degradation of GUS coding sequence by the REs Hae III and Sau3A1. Results of this study suggest that on electroporation, REs can enter into plant cells and alter the expression of the GUS gene. The alteration of gene expression is thus correlated with the digestion of GUS template DNA. Future applications of this technique could include addressing fundamental questions with regard to DNA repair, site-specific recombination, identifying mutations, insertional mutagenesis, enhancement of stable transformation and gene tagging in plants.

Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum

PubMed Central

2013-01-01

Background Hairpin RNA (hpRNA) transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms. However, in fungi, expression of hairpin RNA transcripts can induce post-transcriptional gene silencing, but in some species can also lead to transcriptional gene silencing, suggesting a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a powerful technique to understand gene function, particularly when gene knockouts are difficult to obtain. We investigated whether the plant pathogenic fungus Fusarium oxysporum possesses a functional gene silencing machinery and whether hairpin RNA transcripts can be employed to effectively induce gene silencing. Results Here we show that, in the phytopathogenic fungus F. oxysporum, hpRNA transgenes targeting either a β-glucuronidase (Gus) reporter transgene (hpGus) or the endogenous gene Frp1 (hpFrp) did not induce significant silencing of the target genes. Expression analysis suggested that the hpRNA transgenes are prone to transcriptional inactivation, resulting in low levels of hpRNA and siRNA production. However, the hpGus RNA can be efficiently transcribed by promoters acquired either by recombination with a pre-existing, actively transcribed Gus transgene or by fortuitous integration near an endogenous gene promoter allowing siRNA production. These siRNAs effectively induced silencing of a target Gus transgene, which in turn appeared to also induce secondary siRNA production. Furthermore, our results suggested that hpRNA transcripts without poly(A) tails are efficiently processed into siRNAs to induce gene silencing. A convergent promoter transgene, designed to express poly(A)-minus sense and antisense Gus RNAs, without an inverted-repeat DNA structure, induced consistent Gus silencing in F. oxysporum. Conclusions These results indicate that F. oxysporum possesses

Demonstration of vaginal colonization with GusA-expressing Lactobacillus jensenii following oral delivery in rhesus macaques

PubMed Central

Lagenaur, Laurel A; Lee, Peter P; Hamer, Dean H; Sanders-Beer, Brigitte E

2012-01-01

The vaginal microbiome, which harbors beneficial Lactobacillus strains, is believed to be a major host defense mechanism for preventing infections of the urogenital tract. It has been suggested that the gastrointestinal tract serves as a reservoir for lactobacilli that colonize the vagina. Using rhesus macaques, we examined whether oral delivery of human vaginal Lactobacillus jensenii-1153-1646, a GusA-producing strain, would result in colonization of the rectum and the vagina. Lactobacilli were identified from the vagina tracts of three macaques on the basis of β-glucuronidase enzyme production, 16S rRNA gene sequence and DNA homology using a repetitive sequence-based polymerase chain reaction. PMID:21907793

The Triticum aestivum non-specific lipid transfer protein (TaLtp) gene family: comparative promoter activity of six TaLtp genes in transgenic rice.

PubMed

Boutrot, Freddy; Meynard, Donaldo; Guiderdoni, Emmanuel; Joudrier, Philippe; Gautier, Marie-Françoise

2007-03-01

Plant non-specific lipid transfer proteins (nsLTPs) are encoded by a multigene family and support physiological functions, which remain unclear. We adapted an efficient ligation-mediated polymerase chain reaction (LM-PCR) procedure that enabled isolation of 22 novel Triticum aestivum nsLtp (TaLtp) genes encoding types 1 and 2 nsLTPs. A phylogenetic tree clustered the wheat nsLTPs into ten subfamilies comprising 1-7 members. We also studied the activity of four type 1 and two type 2 TaLtp gene promoters in transgenic rice using the 1-Glucuronidase reporter gene. The activities of the six promoters displayed both overlapping and distinct features in rice. In vegetative organs, these promoters were active in leaves and root vascular tissues while no beta-Glucuronidase (GUS) activity was detected in stems. In flowers, the GUS activity driven by the TaLtp7.2a, TaLtp9.1a, TaLtp9.2d, and TaLtp9.3e gene promoters was associated with vascular tissues in glumes and in the extremities of anther filaments whereas only the TaLtp9.4a gene promoter was active in anther epidermal cells. In developing grains, GUS activity and GUS immunolocalization data evidenced complex patterns of activity of the TaLtp7.1a, TaLtp9.2d, and TaLtp9.4a gene promoters in embryo scutellum and in the grain epicarp cell layer. In contrast, GUS activity driven by TaLtp7.2a, TaLtp9.1a, and TaLtp9.3e promoters was restricted to the vascular bundle of the embryo scutellum. This diversity of TaLtp gene promoter activity supports the hypothesis that the encoded TaLTPs possess distinct functions in planta.

Isolation and characterization of an Arabidopsis biotin carboxylase gene and its promoter.

PubMed

Bao, X; Shorrosh, B S; Ohlrogge, J B

1997-11-01

In the plastids of most plants, acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a multisubunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protien (BCCP), and carboxytransferase (alpha-CT, beta-CT) subunits. To better understand the regulation of this enzyme, we have isolated and sequenced a BC genomic clone from Arabidopsis and partially characterized its promoter. Fifteen introns were identified. The deduced amino acid sequence of the mature BC protein is highly conserved between Arabidopsis and tobacco (92.6% identity). BC expression was evaluated using northern blots and BC/GUS fusion constructs in transgenic Arabidopsis. GUS activity in the BC/GUS transgenics as well as transcript level of the native gene were both found to be higher in silique and flower than in root and leaf. Analysis of tobacco suspension cells transformed with truncated BC promoter/GUS gene fusions indicated the region from -140 to +147 contained necessary promoter elements which supported basal gene expression. A positive regulatory region was found to be located between -2100 and -140, whereas a negative element was possibly located in the first intron. In addition, several conserved regulatory elements were identified in the BC promoter. Surprisingly, although BC is a low-abundance protein, the expression of BC/GUS fusion constructs was similar to 35S/GUS constructs.

Effects of sulfate concentrations on the expression of a soybean seed storage protein gene and its reversibility in transgenic Arabidopsis thaliana.

PubMed

Hirai, M Y; Fujiwara, T; Chino, M; Naito, S

1995-10-01

Transgenic expression of genes encoding the alpha' and beta subunits of beta-conglycinin, one of the major seed storage proteins of soybean (Glycine max [L.] Merr.), was analyzed in Arabidopsis thaliana (L.) Heynh. under conditions of sulfate deficiency. Temporal patterns of expression of both the intact beta subunit gene and the beta subunit gene promoter fused to the beta-glucuronidase (GUS) gene are similar in soil-less cultures using rockwool, suggesting that the response to sulfate deficiency is regulated mainly at the level of transcription. In hydroponic cultures with various concentrations of sulfate, expression of both the intact beta subunit gene and the beta subunit gene promoter-GUS fusion gene were negatively correlated to increased sulfate concentrations in the culture medium. Transfer of transgenic A. thaliana plants carrying the beta subunit gene promoter-GUS fusion from sulfate-deficient to sulfate-sufficient control medium caused GUS activity in developing siliques to be repressed within two days. A reverse shift, where the plants were transferred from the control to sulfate-deficient medium, caused GUS activity to become higher than that in seeds of the control plants within two days. These results indicate that the expression of the beta subunit gene promoter responds rapidly to changes of sulfate availability.

Endophytic Herbaspirillum seropedicae expresses nif genes in gramineous plants.

PubMed

Roncato-Maccari, Lauren D B; Ramos, Humberto J O; Pedrosa, Fabio O; Alquini, Yedo; Chubatsu, Leda S; Yates, Marshall G; Rigo, Liu U; Steffens, Maria Berenice R; Souza, Emanuel M

2003-07-01

Abstract The interactions between maize, sorghum, wheat and rice plants and Herbaspirillum seropedicae were examined microscopically following inoculation with the H. seropedicae LR15 strain, a Nif(+) (Pnif::gusA) mutant obtained by the insertion of a gusA-kanamycin cassette into the nifH gene of the H. seropedicae wild-type strain. The expression of the Pnif::gusA fusion was followed during the association of the diazotroph with the gramineous species. Histochemical analysis of seedlings of maize, sorghum, wheat and rice grown in vermiculite showed that strain LR15 colonized root surfaces and inner tissues. In early steps of the endophytic association, H. seropedicae colonized root exudation sites, such as axils of secondary roots and intercellular spaces of the root cortex; it then occupied the vascular tissue and there expressed nif genes. The expression of nif genes occurred in roots, stems and leaves as detected by the GUS reporter system. The expression of nif genes was also observed in bacterial colonies located in the external mucilaginous root material, 8 days after inoculation. Moreover, the colonization of plant tissue by H. seropedicae did not depend on the nitrogen-fixing ability, since similar numbers of cells were isolated from roots or shoots of the plants inoculated with Nif(+) or Nif(-) strains.

Strong Magnetic Field Induced Changes of Gene Expression in Arabidopsis

NASA Astrophysics Data System (ADS)

Paul, A.-L.; Ferl, R. J.; Klingenberg, B.; Brooks, J. S.; Morgan, A. N.; Yowtak, J.; Meisel, M. W.

2005-07-01

We review our studies of the biological impact of magnetic field strengths of up to 30 T on transgenic arabidopsis plants engineered with a stress response gene consisting of the alcohol dehydrogenase (Adh) gene promoter driving the β-glucuronidase (GUS) gene reporter. Field strengths in excess of 15 T induce expression of the Adh/GUS transgene in the roots and leaves. Microarray analyses indicate that such field strengths have a far reaching effect on the genome. Wide spread induction of stress-related genes and transcription factors, and a depression of genes associated with cell wall metabolism are prominent examples.

Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco.

PubMed

Chaumont, F; Silva Filho, M de C; Thomas, D; Leterme, S; Boutry, M

1994-02-01

The mitochondrial F1-ATPase beta subunit (ATPase-beta) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH2-terminal extension. By sequencing the mature N. tabacum ATPase-beta, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3' deletions in the sequence coding for the 90 NH2-terminal residues of ATPase-beta. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and beta-glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase-beta remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.

Transcriptional and posttranscriptional regulation of the glycolate oxidase gene in tobacco seedlings.

PubMed

Barak, S; Nejidat, A; Heimer, Y; Volokita, M

2001-03-01

The roles of light and of the putative plastid signal in glycolate oxidase (GLO) gene expression were investigated in tobacco (Nicotiana tabacum cv. Samsun NN) seedlings during their shift from skotomorphogenic to photomorphogenic development. GLO transcript and enzyme activities were detected in etiolated seedlings. Their respective levels increased three- and six-fold during 96 h of exposure to light. The GLO transcript was almost undetectable in seedlings in which chloroplast development was impaired by photooxidation with the herbicide norflurazon. In transgenic tobacco seedlings, photooxidation inhibited the light-dependent increase in GUS activity when it was placed under the regulation of the GLO promoter P(GLO). However, even under these photooxidative conditions, a continuous increase in GUS activity was observed as compared to etiolated seedlings. When GUS expression was driven by the CaMV 35S promoter (P35S), no apparent difference was observed between etiolated, deetiolated and photooxidized seedlings. These observations indicate that the effects of the putative plastid development signal and light on GUS expression can be separated. Translational yield analysis indicated that the translation of the GUS transcript in P(GLO)::GUS seedlings was enhanced 30-fold over that of the GUS transcript in P35S::GUS seedlings. The overall picture emerging from these results is that in etiolated seedlings GLO transcript, though present at a substantial level, is translated at a low rate. Increased GLO transcription is enhanced, however, in response to signals originating from the developing plastids. GLO gene expression is further enhanced at the translational level by a yet undefined light-dependent mechanism.

Encounters with Insignificance in Teaching and Learning: Gus Van Sant's "Elephant"

ERIC Educational Resources Information Center

Sandlos, Karyn

2009-01-01

This article explores how a curriculum of film becomes organized by the teacher's worries about what film may open up in class. The author focuses on her own worries about showing Gus Van Sant's (2003) film, "Elephant," an elliptical and dreamlike study of the murders in 1999 of twelve students and a teacher at Columbine High School, to a class of…

Expression of the patatin-related phospholipase A gene AtPLA IIA in Arabidopsis thaliana is up-regulated by salicylic acid, wounding, ethylene, and iron and phosphate deficiency.

PubMed

Rietz, Steffen; Holk, André; Scherer, Günther F E

2004-09-01

In Arabidopsis thaliana (L.) Heynh., the cytosolic, patatin-related phospholipase A enzymes comprise a family of ten genes designated AtPLAs thought to be involved in auxin and pathogen signalling [A. Holk et al. (2002) Plant Physiol 130:90-101]. One of these, AtPLA IIA, is investigated here by studying its transcriptional regulation through transgenic Arabidopsis plants containing the AtPLA IIA promoter (PIIA) fused to the beta-glucuronidase (GUS) gene. GUS activity appeared in leaves at 10-12 days and became increasingly stronger with age in all leaves. From the same age on, strong GUS activity was visible in the basal stipules of the rosette leaves. PIIA-dependent GUS activity was found in the older parts of the primary root (from 10 days on) and, later in development, in older parts of side roots, and the root cap. No GUS activity was detected in flower organs. PIIA-dependent GUS expression in 12-day-old plants was up-regulated after treatment by salicylic acid, Bion, wounding, 1-aminocyclopropane-1-carboxylic acid (ACC) and jasmonic acid. When transgenic PIIA:: uidA plants were grown devoid of iron, 9-day-old plants exhibited increased GUS activity in the leaves and, when devoid of phosphate, 11-day-old plants had increased GUS activity in the roots. In conclusion, this member of the patatin-related phospholipase A gene family showed properties of a defence and iron-stress and phosphate-stress gene, being transcriptionally up-regulated within hours or days.

Silencing of an α-dioxygenase gene, Ca-DOX, retards growth and suppresses basal disease resistance responses in Capsicum annum.

PubMed

Hong, Chi Eun; Ha, Young-Im; Choi, Hyoju; Moon, Ju Yeon; Lee, Jiyoung; Shin, Ah-Young; Park, Chang Jin; Yoon, Gyeong Mee; Kwon, Suk-Yoon; Jo, Ick-Hyun; Park, Jeong Mee

2017-03-01

Alpha-dioxygenases (α-DOX) catalyzing the primary oxygenation of fatty acids to oxylipins were recently found in plants. Here, the biological roles of the pepper α-DOX (Ca-DOX) gene, which is strongly induced during non-host pathogen infection in chili pepper, were examined. Virus-induced gene silencing demonstrated that down-regulation of Ca-DOX enhanced susceptibility to bacterial pathogens and suppressed the hypersensitive response via the suppression of pathogenesis-related genes such as PR4, proteinase inhibitor II and lipid transfer protein (PR14). Ca-DOX-silenced pepper plants also exhibited more retarded growth with lower epidermal cell numbers and reduced cell wall thickness than control plants. To better understand regulation of Ca-DOX, transgenic Arabidopsis plants harboring the β-glucuronidase (GUS) reporter gene driven from a putative Ca-DOX promoter were generated. GUS expression was significantly induced upon avirulent pathogen infection in transgenic Arabidopsis leaves, whereas GUS induction was relatively weak upon virulent pathogen treatment. After treatment with plant hormones, early and strong GUS expression was seen after treatment of salicylic acid, whereas ethylene and methyl jasmonate treatments produced relatively weak and late GUS signals. These results will enable us to further understand the role of α-DOX, which is important in lipid metabolism, defense responses, and growth development in plants.

Sobetirome and its Amide Prodrug Sob-AM2 Exert Thyromimetic Actions in Mct8-Deficient Brain.

PubMed

Bárez-López, Soledad; Hartley, Meredith D; Grijota-Martínez, Carmen; Scanlan, Thomas S; Guadaño-Ferraz, Ana

2018-06-29

Loss of function mutations in the thyroid hormone (TH)-specific cell membrane transporter, the monocarboxylate transporter 8 (MCT8), lead to profound psychomotor retardation and abnormal TH serum levels, with low thyroxine (T4) and high triiodothyronine (T3). Several studies point to impaired TH transport across brain barriers as a crucial pathophysiological mechanism resulting in cerebral hypothyroidism. Treatment options for MCT8-deficient patients are limited and are focused on overcoming the brain barriers. The aim of this study was to evaluate the ability of the TH analog sobetirome and its prodrug Sob-AM2 to access the brain and exert thyromimetic actions in the absence of Mct8. Juvenile wild-type (Wt) mice and mice lacking Mct8 and deiodinase type 2 (Mct8/Dio2KO) were treated systemically with daily injections of vehicle, 1 mg of sobetirome/kg body weight/day, or 0.3 mg of Sob-AM2/kg body weight/day for seven days. Sobetirome content was measured using liquid chromatography-tandem mass spectrometry, and T4 and T3 levels by specific radioimmunoassays. The effect of sobetirome treatment in the expression of T3-dependent genes was measured in the heart, liver, and cerebral cortex by real-time polymerase chain reaction. Sob-AM2 treatment in Mct8/Dio2KO animals led to 1.8-fold more sobetirome content in the brain and 2.5-fold less in plasma in comparison to the treatment with the parent drug sobetirome. Both sobetirome and Sob-AM2 treatments in Mct8/Dio2KO mice greatly decreased plasma T4 and T3 levels. Dio1 and Ucp2 gene expression was altered in the liver of Mct8/Dio2KO mice and was not affected by the treatments. In the heart, Hcn2 but not Atp2a2 expression was increased after treatment with the analogs. Interestingly, both sobetirome and Sob-AM2 treatments increased the expression of several T3-dependent genes in the brain such as Hr, Abcd2, Mme, and Flywch2 in Mct8/Dio2KO mice. Sobetirome and its amide prodrug Sob-AM2 can access the brain in the absence

Orgyia pseudotsugata baculovirus p10 and polyhedron envelope protein genes: analysis of their relative expression levels and role in polyhedron structure.

PubMed

Gross, C H; Russell, R L; Rohrmann, G F

1994-05-01

To investigate the regulation of p10 and polyhedron envelope protein (PEP) gene expression and their role in polyhedron development, Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis viruses lacking these genes were constructed. Recombinant viruses were produced, in which the p10 gene, the PEP gene or both genes were disrupted with the beta-glucuronidase (GUS) or beta-galactosidase (lacZ) genes. GUS activity under the control of the PEP protein promoter was observed later in infection and its maximal expression was less than 10% the level for p10 promoter-GUS constructs. Tissues from O. pseudotsugata larvae infected with these recombinants were examined by electron microscopy. Cells from insects infected with the p10- viruses lacked p10-associated fibrillar structures, but fragments of polyhedron envelope-like structures were observed on the surface of some polyhedra. Immunogold labelling of cells infected with the p10-GUS+ virus with an antibody directed against PEP showed that the PEP was concentrated at the surface of polyhedra. Although polyhedra produced by p10 and PEP gene deletion mutants demonstrated what appeared to be a polyhedron envelope by transmission electron microscopy, scanning electron microscopy showed that they had irregular, pitted surfaces that were different from wild-type polyhedra. These data suggested that both p10 and PEP are important for the proper formation of the periphery of polyhedra.

Identification of cis-elements and evaluation of upstream regulatory region of a rice anther-specific gene, OSIPP3, conferring pollen-specific expression in Oryza sativa (L.) ssp. indica.

PubMed

Manimaran, P; Raghurami Reddy, M; Bhaskar Rao, T; Mangrauthia, Satendra K; Sundaram, R M; Balachandran, S M

2015-12-01

Pollen-specific expression. Promoters comprise of various cis-regulatory elements which control development and physiology of plants by regulating gene expression. To understand the promoter specificity and also identification of functional cis-acting elements, progressive 5' deletion analysis of the promoter fragments is widely used. We have evaluated the activity of regulatory elements of 5' promoter deletion sequences of anther-specific gene OSIPP3, viz. OSIPP3-∆1 (1504 bp), OSIPP3-∆2 (968 bp), OSIPP3-∆3 (388 bp) and OSIPP3-∆4 (286 bp) through the expression of transgene GUS in rice. In silico analysis of 1504-bp sequence harboring different copy number of cis-acting regulatory elements such as POLLENLELAT52, GTGANTG10, enhancer element of LAT52 and LAT56 indicated that they were essential for high level of expression in pollen. Histochemical GUS analysis of the transgenic plants revealed that 1504- and 968-bp fragments directed GUS expression in roots and anthers, while the 388- and 286-bp fragments restricted the GUS expression to only pollen, of which 388 bp conferred strong GUS expression. Further, GUS staining analysis of different panicle development stages (P1-P6) confirmed that the GUS gene was preferentially expressed only at P6 stage (late pollen stage). The qRT-PCR analysis of GUS transcript revealed 23-fold higher expression of GUS transcript in OSIPP3-Δ1 followed by OSIPP3-Δ2 (eightfold) and OSIPP3-Δ3 (threefold) when compared to OSIPP3-Δ4. Based on our results, we proposed that among the two smaller fragments, the 388-bp upstream regulatory region could be considered as a promising candidate for pollen-specific expression of agronomically important transgenes in rice.

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

PubMed

Kraan, Claudine M; Cornish, Kim M; Bui, Quang M; Li, Xin; Slater, Howard R; Godler, David E

2018-01-01

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

A new role for the Arabidopsis AP2 transcription factor, LEAFY PETIOLE, in gibberellin-induced germination is revealed by the misexpression of a homologous gene, SOB2/DRN-LIKE.

PubMed

Ward, Jason M; Smith, Alison M; Shah, Purvi K; Galanti, Sarah E; Yi, Hankuil; Demianski, Agnes J; van der Graaff, Eric; Keller, Beat; Neff, Michael M

2006-01-01

Gibberellic acid (GA) promotes germination, stem/hypocotyl elongation, and leaf expansion during seedling development. Using activation-tagging mutagenesis, we identified a mutation, sob2-D (for suppressor of phytochromeB-4 [phyB-4]#2 dominant), which suppresses the long-hypocotyl phenotype of a phyB missense allele, phyB-4. This mutant phenotype is caused by the overexpression of an APETALA2 transcription factor, SOB2, also called DRN-like. SOB2/DRN-like transcript is not detectable in wild-type seedling or adult tissues via RT-PCR analysis, suggesting that SOB2/DRN-like may not be involved in seedling development under normal conditions. Adult sob2-D phyB-4 plants have curled leaves and club-like siliques, resembling plants that overexpress a closely related gene, LEAFY PETIOLE (LEP). Hypocotyls of a LEP-null allele, lep-1, are shorter in the light and dark, suggesting LEP involvement in seedling development. This aberrant hypocotyl phenotype is due at least in part to a delay in germination. In addition, lep-1 is less responsive to GA and more sensitive to the GA biosynthesis inhibitor paclobutrazol, indicating that LEP is a positive regulator of GA-induced germination. RT-PCR shows that LEP transcript accumulates in wild-type seeds during imbibition and germination, and the transcript levels of REPRESSOR OF ga1-3-LIKE2 (RGL2), a negative regulator of GA signaling during germination, is unaffected in lep-1. These results suggest LEP is a positive regulator of GA-induced germination acting independently of RGL2. An alternative model places LEP downstream of RGL2 in the GA-signaling cascade.

Mature Luffa Leaves (Luffa cylindrica L.) as a Tool for Gene Expression Analysis by Agroinfiltration

PubMed Central

Błażejewska, Kamila; Kapusta, Małgorzata; Zielińska, Elżbieta; Tukaj, Zbigniew; Chincinska, Izabela A.

2017-01-01

We exploited the potential of cucurbits for ectopic gene expression. Agroinfiltration is a simple and commonly used method to obtain transient expression of foreign genes in plants. In contrast to in vitro transformation techniques, agroinfiltration can be used for genetic modification of mature plant tissues. Although the cucurbits are commonly used as model plants for molecular biology and biotechnology studies, to date there are no literature sources on the possibility of transient gene expression in mature cucurbit tissues. Our research has shown that mature leaves of Luffa cylindrica L. (luffa), in contrast to other cucurbit species, can be successfully transiently transformed with Agrobacterium tumefaciens. We efficiently transformed luffa leaves with a reporter gene encoding β-glucuronidase (GUS). The GUS activity in transiently transformed leaf tissues was detected within 24 h after the infiltration with bacteria. Additionally, we have shown that the activity of a transiently expressed the GUS gene can be monitored directly in the EDTA-exudates collected from the cut petioles of the agroinfiltrated leaves. The results suggest that luffa leaves can be useful as a plant expression system for studies of physiological and biochemical processes in cucurbits. PMID:28270826

Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

USDA-ARS?s Scientific Manuscript database

Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

Identification and expression of three new Nicotiana plumbaginifolia genes which encode isoforms of a plasma-membrane H(+)-ATPase, and one of which is induced by mechanical stress.

PubMed

Oufattole, M; Arango, M; Boutry, M

2000-04-01

To analyze in detail the multigene family encoding the plasma-membrane H(+)-ATPase (pma) in Nicotiana plumbaginifolia Viv., five new pma genes (pma 5-9) were isolated. Three of these (pma 6, 8, 9) were fully characterized and classified into new and independent subfamilies. Their cell-type expression was followed by the beta-glucuronidase (gusA) reporter-gene method. While the pma8-gusA transgene was not expressed in transgenic tobacco, expression of the two other transgenes (pma6- and pma9-gusA) was found to be restricted to particular cell types. In the vegetative tissues, pma6-gusA expression was limited to the head cells of the leaf short trichomes, involved in secretion, and to the cortical parenchyma of the young nodes where the developing leaves and axillary flowering stalks join the stem. In the latter tissues, gene expression was enhanced by mechanical stress, suggesting that H(+)-ATPase might be involved in the strength of the tissues and their resistance to mechanical trauma. The pma9-gusA transgene was mainly expressed in the apical meristem of adventitious roots and axillary buds as well as in the phloem tissues of the stem, in which expression depended on the developmental stage. In flowers, pma9-gusA expression was limited to the mature pollen grains and the young fertilized ovules, while that of pma6-gusA was identified in most of the organs. Reverse transcription-polymerase chain reaction of leaf and stem RNA confirmed the expression of pma 6 and 9, while pma8 was found to be expressed in both organs at a lower level. In conclusion, although pma 6 and 9 had a more restricted expression pattern than the previously characterized pma genes, they were nevertheless expressed in cell types in which H(+)-ATPase had not been previously detected.

Calmodulin Gene Family in Potato: Developmental and Touch-Induced Expression of the mRNA Encoding a Novel Isoform

NASA Technical Reports Server (NTRS)

Takezawa, D.; Liu, Z. H.; An, G.; Poovaiah, B. W.

1995-01-01

Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca(2+)-binding area. The expression patterns of different genes were studied by northern analysis using the 3'-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the beta-glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5'-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.

A Plant Gene Up-Regulated at Rust Infection Sites

PubMed Central

Ayliffe, Michael A.; Roberts, James K.; Mitchell, Heidi J.; Zhang, Ren; Lawrence, Gregory J.; Ellis, Jeffrey G.; Pryor, Tony J.

2002-01-01

Expression of the fis1 gene from flax (Linum usitatissimum) is induced by a compatible rust (Melampsora lini) infection. Infection of transgenic plants containing a β-glucuronidase (GUS) reporter gene under the control of the fis1 promoter showed that induction is highly localized to those leaf mesophyll cells within and immediately surrounding rust infection sites. The level of induction reflects the extent of fungal growth. In a strong resistance reaction, such as the hypersensitive fleck mediated by the L6 resistance gene, there is very little fungal growth and a microscopic level of GUS expression. Partially resistant flax leaves show levels of GUS expression that were intermediate to the level observed in the fully susceptible infection. Sequence and deletion analysis using both transient Agrobacterium tumefaciens expression and stable transformation assays have shown that the rust-inducible fis1 promoter is contained within a 580-bp fragment. Homologs of fis1 were identified in expressed sequence tag databases of a range of plant species including dicots, monocots, and a gymnosperm. Homologous genes isolated from maize (Zea mays; mis1), barley (Hordeum vulgare; bis1), wheat (Triticum aestivum; wis1), and Arabidopsis encode proteins that are highly similar (76%–82%) to the FIS1 protein. The Arabidopsis homologue has been reported to encode a Δ1-pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate. RNA-blot analysis showed that mis1 in maize and the bis1 homolog in barley are both up-regulated by a compatible infection with the corresponding species-specific rust. The rust-induced genes homologous to fis1 are present in many plants. The promoters of these genes have potential roles for the engineering of synthetic rust resistance genes by targeting transgene expression to the sites of rust infection. PMID:12011348

Arabidopsis gene expression patterns during spaceflight

NASA Astrophysics Data System (ADS)

Paul, A.-L.; Ferl, R. J.

The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

A urate gene-by-diuretic interaction and gout risk in participants with hypertension: results from the ARIC study.

PubMed

McAdams-DeMarco, Mara A; Maynard, Janet W; Baer, Alan N; Kao, Linda W; Kottgen, Anna; Coresh, Josef

2013-05-01

To test for a urate gene-by-diuretic interaction on incident gout. The Atherosclerosis Risk in Communities Study is a prospective population-based cohort of 15 792 participants recruited from four US communities (1987-1989). Participants with hypertension and available single nucleotide polymorphism (SNP) genotype data were included. A genetic urate score (GUS) was created from common urate-associated SNPs for eight genes. Gout incidence was self-reported. Using logistic regression, the authors estimated the adjusted OR of incident gout by diuretic use, stratified by GUS median. Of 3524 participants with hypertension, 33% used a diuretic and 3.1% developed gout. The highest 9-year cumulative incidence of gout was in those with GUS above the median and taking a thiazide or loop diuretic (6.3%). Compared with no thiazide or loop diuretic use, their use was associated with an OR of 0.40 (95% CI 0.14 to 1.15) among those with a GUS below the median and 2.13 (95% CI 1.23 to 3.67) for those with GUS above the median; interaction p=0.006. When investigating the genes separately, SLC22A11 and SLC2A9 showed a significant interaction, consistent with the former encoding an organic anion/dicarboxylate exchanger, which mediates diuretic transport in the kidney. Participants who were genetically predisposed to hyperuricaemia were susceptible to developing gout when taking thiazide or loop diuretics, an effect not evident among those without a genetic predisposition. These findings argue for a potential benefit of genotyping individuals with hypertension to assess gout risk, relative in part to diuretic use.

A urate gene-by-diuretic interaction and gout risk in participants with hypertension: results from the ARIC study

PubMed Central

McAdams-DeMarco, Mara A; Maynard, Janet W; Baer, Alan N; Kao, Linda W; Kottgen, Anna; Coresh, Josef

2015-01-01

Objective To test for a urate gene-by-diuretic interaction on incident gout. Methods The Atherosclerosis Risk in Communities Study is a prospective population-based cohort of 15 792 participants recruited from four US communities (1987–1989). Participants with hypertension and available single nucleotide polymorphism (SNP) genotype data were included. A genetic urate score (GUS) was created from common urate-associated SNPs for eight genes. Gout incidence was self-reported. Using logistic regression, the authors estimated the adjusted OR of incident gout by diuretic use, stratified by GUS median. Results Of 3524 participants with hypertension, 33% used a diuretic and 3.1% developed gout. The highest 9-year cumulative incidence of gout was in those with GUS above the median and taking a thiazide or loop diuretic (6.3%). Compared with no thiazide or loop diuretic use, their use was associated with an OR of 0.40 (95% CI 0.14 to 1.15) among those with a GUS below the median and 2.13 (95% CI 1.23 to 3.67) for those with GUS above the median; interaction p=0.006. When investigating the genes separately, SLC22A11 and SLC2A9 showed a significant interaction, consistent with the former encoding an organic anion/dicarboxylate exchanger, which mediates diuretic transport in the kidney. Conclusions Participants who were genetically predisposed to hyperuricaemia were susceptible to developing gout when taking thiazide or loop diuretics, an effect not evident among those without a genetic predisposition. These findings argue for a potential benefit of genotyping individuals with hypertension to assess gout risk, relative in part to diuretic use. PMID:22753387

RNA-mediated gene silencing signals are not graft transmissible from the rootstock to the scion in greenhouse-grown apple plants Malus sp.

PubMed

Flachowsky, Henryk; Tränkner, Conny; Szankowski, Iris; Waidmann, Sascha; Hanke, Magda-Viola; Treutter, Dieter; Fischer, Thilo C

2012-01-01

RNA silencing describes the sequence specific degradation of RNA targets. Silencing is a non-cell autonomous event that is graft transmissible in different plant species. The present study is the first report on systemic acquired dsRNA-mediated gene silencing of transgenic and endogenous gene sequences in a woody plant like apple. Transgenic apple plants overexpressing a hairpin gene construct of the gusA reporter gene were produced. These plants were used as rootstocks and grafted with scions of the gusA overexpressing transgenic apple clone T355. After grafting, we observed a reduction of the gusA gene expression in T355 scions in vitro, but not in T355 scions grown in the greenhouse. Similar results were obtained after silencing of the endogenous Mdans gene in apple that is responsible for anthocyanin biosynthesis. Subsequently, we performed grafting experiments with Mdans silenced rootstocks and red leaf scions of TNR31-35 in order to evaluate graft transmitted silencing of the endogenous Mdans. The results obtained suggested a graft transmission of silencing signals in in vitro shoots. In contrast, no graft transmission of dsRNA-mediated gene silencing signals was detectable in greenhouse-grown plants and in plants grown in an insect protection tent.

RNA-Mediated Gene Silencing Signals Are Not Graft Transmissible from the Rootstock to the Scion in Greenhouse-Grown Apple Plants Malus sp

PubMed Central

Flachowsky, Henryk; Tränkner, Conny; Szankowski, Iris; Waidmann, Sascha; Hanke, Magda-Viola; Treutter, Dieter; Fischer, Thilo C.

2012-01-01

RNA silencing describes the sequence specific degradation of RNA targets. Silencing is a non-cell autonomous event that is graft transmissible in different plant species. The present study is the first report on systemic acquired dsRNA-mediated gene silencing of transgenic and endogenous gene sequences in a woody plant like apple. Transgenic apple plants overexpressing a hairpin gene construct of the gusA reporter gene were produced. These plants were used as rootstocks and grafted with scions of the gusA overexpressing transgenic apple clone T355. After grafting, we observed a reduction of the gusA gene expression in T355 scions in vitro, but not in T355 scions grown in the greenhouse. Similar results were obtained after silencing of the endogenous Mdans gene in apple that is responsible for anthocyanin biosynthesis. Subsequently, we performed grafting experiments with Mdans silenced rootstocks and red leaf scions of TNR31-35 in order to evaluate graft transmitted silencing of the endogenous Mdans. The results obtained suggested a graft transmission of silencing signals in in vitro shoots. In contrast, no graft transmission of dsRNA-mediated gene silencing signals was detectable in greenhouse-grown plants and in plants grown in an insect protection tent. PMID:22949844

Transferring cucumber mosaic virus-white leaf strain coat protein gene into Cucumis melo L. and evaluating transgenic plants for protection against infections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonsalves, C.; Xue, B.; Yepes, M.

1994-03-01

A single regeneration procedure using cotyledon examples effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens or microprojectile bombardment methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), [beta]-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 [mu]m 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg[center dot]liter[sup [minus]1] and carbenicillin (Cb) at 500more » mg[center dot]liter[sup [minus]1]. The authors' comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R[sub 0] plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes.« less

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

Origin, timing, and gene expression profile of adventitious rooting in Arabidopsis hypocotyls and stems.

PubMed

Welander, Margareta; Geier, Thomas; Smolka, Anders; Ahlman, Annelie; Fan, Jing; Zhu, Li-Hua

2014-02-01

Adventitious root (AR) formation is indispensable for vegetative propagation, but difficult to achieve in many crops. Understanding its molecular mechanisms is thus important for such species. Here we aimed at developing a rooting protocol for direct AR formation in stems, locating cellular AR origins in stems and exploring molecular differences underlying adventitious rooting in hypocotyls and stems. In-vitro-grown hypocotyls or stems of wild-type and transgenic ecotype Columbia (Col-0) of Arabidopsis thaliana were rooted on rooting media. Anatomy of AR formation, qRT-PCR of some rooting-related genes and in situ GUS expression were carried out during rooting from hypocotyls and stems. We developed a rooting protocol for AR formation in stems and traced back root origins in stems by anatomical and in situ expression studies. Unlike rooting in hypocotyls, rooting in stems was slower, and AR origins were mainly from lateral parenchyma of vascular bundles and neighboring starch sheath cells as well as, to a lesser extent, from phloem cap and xylem parenchyma. Transcript levels of GH3-3, LBD16, LBD29, and LRP1 in hypocotyls and stems were similar, but transcript accumulation was delayed in stems. In situ expression signals of DR5::GUS, LBD16::GUS, LBD29::GUS, and rolB::GUS reporters in stems mainly occurred at the root initiation sites, suggesting their involvement in AR formation. We have developed an efficient rooting protocol using half-strength Lepoivre medium for studying AR formation in stems, traced back the cellular AR origins in stems, and correlated expression of rooting-related genes with root initiation sites.

LuFLA1PRO and LuBGAL1PRO promote gene expression in the phloem fibres of flax (Linum usitatissimum).

PubMed

Hobson, Neil; Deyholos, Michael K

2013-04-01

Cell type-specific promoters were identified that drive gene expression in an industrially important product. To identify flax (Linum usitatissimum) gene promoters, we analyzed the genomic regions upstream of a fasciclin-like arabinogalactan protein (LuFLA1) and a beta-galactosidase (LuBGAL1). Both of these genes encode transcripts that have been found to be highly enriched in tissues bearing phloem fibres. Using a beta-glucuronidase (GUS) reporter construct, we found that a 908-bp genomic sequence upstream of LuFLA1 (LuFLA1PRO) directed GUS expression with high specificity to phloem fibres undergoing secondary cell wall development. The DNA sequence upstream of LuBGAL1 (LuBGAL1PRO) likewise produced GUS staining in phloem fibres with developing secondary walls, as well as in tissues of developing flowers and seed bolls. These data provide further evidence of a specific role for LuFLA1 in phloem fibre development, and demonstrate the utility of LuFLA1PRO and LuBGAL1PRO as tools for biotechnology and further investigations of phloem fibre development.

Cloning and characterization of the promoter of the 9-cis-epoxycarotenoid dioxygenase gene in Arachis hypogaea L.

PubMed

Liang, Jianhua; Yang, Lixia; Chen, Xiong; Li, Ling; Guo, Dongliang; Li, Haihang; Zhang, Biyu

2009-09-01

We cloned the promoter of the 9-cis-epoxycarotenoid dioxygenase gene from Arachis hypogaea L. beta-Glucuronidase (GUS) histochemical staining and GUS activity assay indicated that the activity of the promoter was exhibited predominantly in the leaves and enhanced by water and NaCl stresses, and by application of abscisic acid (ABA) and salicylic acid (SA) in transgenic Arabidopsis. Moreover, two novel ABRE-like (abscisic acid response element) elements were identified in the promoter region.

Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia beta-1,3-glucanase gene.

PubMed Central

Castresana, C; de Carvalho, F; Gheysen, G; Habets, M; Inzé, D; Van Montagu, M

1990-01-01

The Nicotiana plumbaginifolia gn1 gene encoding a beta-1,3-glucanase isoform has been characterized. The gn1 product represents an isoform distinct from the previously identified tobacco beta-1,3-glucanases. By expressing gn1 in Escherichia coli, we have determined directly that the encoded protein does, indeed, correspond to a beta-1,3-glucanase. In N. plumbaginifolia, gn1 was found to be expressed in roots and older leaves. Transgenic tobacco plants containing the 5'-noncoding region of gn1 fused to the beta-glucuronidase (GUS) reporter gene also showed maximum levels of GUS activity in roots and older leaves. No detectable activity was present in the upper part of the transgenic plants with the exception of stem cells at the bases of emerging shoots. The expression conferred by the gn1 promoter was differentially induced in response to specific plant stress treatments. Studies of three plant-bacteria interactions showed high levels of GUS activity when infection resulted in a hypersensitive reaction. Increased gene expression was confined to cells surrounding the necrotic lesions. The observed expression pattern suggests that the characterized beta-1,3-glucanase plays a role both in plant development and in the defense response against pathogen infection. PMID:2152158

The mouse genome displays highly dynamic populations of KRAB-zinc finger protein genes and related genetic units

PubMed Central

Kauzlaric, Annamaria; Ecco, Gabriela; Cassano, Marco; Duc, Julien; Imbeault, Michael; Trono, Didier

2017-01-01

KRAB-containing poly-zinc finger proteins (KZFPs) constitute the largest family of transcription factors encoded by mammalian genomes, and growing evidence indicates that they fulfill functions critical to both embryonic development and maintenance of adult homeostasis. KZFP genes underwent broad and independent waves of expansion in many higher vertebrates lineages, yet comprehensive studies of members harbored by a given species are scarce. Here we present a thorough analysis of KZFP genes and related units in the murine genome. We first identified about twice as many elements than previously annotated as either KZFP genes or pseudogenes, notably by assigning to this family an entity formerly considered as a large group of Satellite repeats. We then could delineate an organization in clusters distributed throughout the genome, with signs of recombination, translocation, duplication and seeding of new sites by retrotransposition of KZFP genes and related genetic units (KZFP/rGUs). Moreover, we harvested evidence indicating that closely related paralogs had evolved through both drifting and shifting of sequences encoding for zinc finger arrays. Finally, we could demonstrate that the KAP1-SETDB1 repressor complex tames the expression of KZFP/rGUs within clusters, yet that the primary targets of this regulation are not the KZFP/rGUs themselves but enhancers contained in neighboring endogenous retroelements and that, underneath, KZFPs conserve highly individualized patterns of expression. PMID:28334004

The mouse genome displays highly dynamic populations of KRAB-zinc finger protein genes and related genetic units.

PubMed

Kauzlaric, Annamaria; Ecco, Gabriela; Cassano, Marco; Duc, Julien; Imbeault, Michael; Trono, Didier

2017-01-01

KRAB-containing poly-zinc finger proteins (KZFPs) constitute the largest family of transcription factors encoded by mammalian genomes, and growing evidence indicates that they fulfill functions critical to both embryonic development and maintenance of adult homeostasis. KZFP genes underwent broad and independent waves of expansion in many higher vertebrates lineages, yet comprehensive studies of members harbored by a given species are scarce. Here we present a thorough analysis of KZFP genes and related units in the murine genome. We first identified about twice as many elements than previously annotated as either KZFP genes or pseudogenes, notably by assigning to this family an entity formerly considered as a large group of Satellite repeats. We then could delineate an organization in clusters distributed throughout the genome, with signs of recombination, translocation, duplication and seeding of new sites by retrotransposition of KZFP genes and related genetic units (KZFP/rGUs). Moreover, we harvested evidence indicating that closely related paralogs had evolved through both drifting and shifting of sequences encoding for zinc finger arrays. Finally, we could demonstrate that the KAP1-SETDB1 repressor complex tames the expression of KZFP/rGUs within clusters, yet that the primary targets of this regulation are not the KZFP/rGUs themselves but enhancers contained in neighboring endogenous retroelements and that, underneath, KZFPs conserve highly individualized patterns of expression.

Virgil Gus Grissom's Visit to LaRC

NASA Image and Video Library

1963-02-22

Astronaut Virgil "Gus" Grissom at the controls of the Visual Docking Simulator. From A.W. Vogeley, "Piloted Space-Flight Simulation at Langley Research Center," Paper presented at the American Society of Mechanical Engineers 1966 Winter Meeting, New York, NY, November 27-December 1, 1966. "This facility was [later known as the Visual-Optical Simulator.] It presents to the pilot an out-the-window view of his target in correct 6 degrees of freedom motion. The scene is obtained by a television camera pick-up viewing a small-scale gimbaled model of the target." "For docking studies, the docking target picture was projected onto the surface of a 20-foot-diameter sphere and the pilot could, effectively, maneuver into contract. this facility was used in a comparison study with the Rendezvous Docking Simulator - one of the few comparison experiments in which conditions were carefully controlled and a reasonable sample of pilots used. All pilots preferred the more realistic RDS visual scene. The pilots generally liked the RDS angular motion cues although some objected to the false gravity cues that these motions introduced. Training time was shorter on the RDS, but final performance on both simulators was essentially equal. " "For station-keeping studies, since close approach is not required, the target was presented to the pilot through a virtual-image system which projects his view to infinity, providing a more realistic effect. In addition to the target, the system also projects a star and horizon background. "

Characterization of Pseudomonas putida Genes Responsive to Nutrient Limitation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Syn, Chris K.; Magnuson, Jon K.; Kingsley, Mark T.

2004-06-01

The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation/biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high nutrient- and oxygen-availability. Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 was examined under various conditions chosen to mimic the soil environment: low carbon, phosphate, nitrate, or oxygen, and in the rhizosphere. Based on their expression profiles, three nutrient-responsive mutant (NRM) strains, NRM5, NRM7, and NRM17, were selected for identification of the tagged genes. In themore » mutant strain NRM5, expression of the glutamate dehydrogenase (gdhA) gene was increased between 4.9- to 26.4-fold under various low nutrient conditions. In NRM7, expression of the novel NADPH:quinone oxidoreductase-like (nql) gene was consistently amongst the highest and was synergistically upregulated by low nutrient and anoxic conditions. The cyoD gene in NRM17, which encodes the fourth subunit of the cytochrome o ubiquinol oxidase complex, had decreased expression in low nutrient conditions but its absolute expression levels was still amongst the highest. Additionally, it was independent of oxygen availability, in contrast to that in E. coli.« less

Overexpression of a MADS-box gene from birch (Betula platyphylla) promotes flowering and enhances chloroplast development in transgenic tobacco.

PubMed

Qu, Guan-Zheng; Zheng, Tangchun; Liu, Guifeng; Wang, Wenjie; Zang, Lina; Liu, Huanzhen; Yang, Chuanping

2013-01-01

In this study, a MADS-box gene (BpMADS), which is an ortholog of AP1 from Arabidopsis, was isolated from birch (Betula platyphylla). Transgenic Arabidopsis containing a BpMADS promoter::GUS construct was produced, which exhibited strong GUS staining in sepal tissues. Ectopic expression of BpMADS significantly enhanced the flowering of tobacco (35S::BpMADS). In addition, the chloroplasts of transgenic tobacco exhibited much higher growth and division rates, as well rates of photosynthesis, than wild-type. A grafting experiment demonstrated that the flowering time of the scion was not affected by stock that overexpressed BpMADS. In addition, the overexpression of BpMADS resulted in the upregulation of some flowering-related genes in tobacco.

Isolation and characterization of a water stress-specific genomic gene, pwsi 18, from rice.

PubMed

Joshee, N; Kisaka, H; Kitagawa, Y

1998-01-01

One of the water stress-specific cDNA clones of rice characterised previously, wsi18, was selected for further study. The wsi18 gene can be induced by water stress conditions such as mannitol, NaCl, and dryness, but not by ABA, cold, or heat. A genomic clone for wsi18, pwsi18, contained about 1.7 kbp of the 5' upstream sequence, two introns, and the full coding sequence. The 5'-upstream sequence of pwsi18 contained putative cis-acting elements, namely an ABA-responsive element (ABRE), three G-boxes, three E-boxes, a MEF-2 sequence, four direct and two inverted repeats, and four sequences similar to DRE, which is involved in the dehydration response of Arabidopsis genes. The gusA reporter gene under the control of the pwsi18 promoter showed transient expression in response to water stress. Deletion of the downstream DRE-like sequence between the distal G-boxes-2 and -3 resulted in rather low GUS expression.

Comparison of the Structure and Expression of Odd-Skipped and Two Related Genes That Encode a New Family of Zinc Finger Proteins in Drosophila

PubMed Central

Hart, M. C.; Wang, L.; Coulter, D. E.

1996-01-01

The odd-skipped (odd) gene, which was identified on the basis of a pair-rule segmentation phenotype in mutant embryos, is initially expressed in the Drosophila embryo in seven pair-rule stripes, but later exhibits a segment polarity-like pattern for which no phenotypic correlate is apparent. We have molecularly characterized two embryonically expressed odd-cognate genes, sob and bowel (bowl), that encode proteins with highly conserved C(2)H(2) zinc fingers. While the Sob and Bowl proteins each contain five tandem fingers, the Odd protein lacks a fifth (C-terminal) finger and is also less conserved among the four common fingers. Reminiscent of many segmentation gene paralogues, the closely linked odd and sob genes are expressed during embryogenesis in similar striped patterns; in contrast, the less-tightly linked bowl gene is expressed in a distinctly different pattern at the termini of the early embryo. Although our results indicate that odd and sob are more likely than bowl to share overlapping developmental roles, some functional divergence between the Odd and Sob proteins is suggested by the absence of homology outside the zinc fingers, and also by amino acid substitutions in the Odd zinc fingers at positions that appear to be constrained in Sob and Bowl. PMID:8878683

Production of MPS VII mouse (Gustm(hE540A·mE536A)Sly) doubly tolerant to human and mouse β-glucuronidase

PubMed Central

Tomatsu, Shunji; Orii, Koji O.; Vogler, Carole; Grubb, Jeffrey H.; Snella, Elizabeth M.; Gutierrez, Monica; Dieter, Tatiana; Holden, Christopher C.; Sukegawa, Kazuko; Orii, Tadao; Kondo, Naomi; Sly, William S.

2006-01-01

Mucopolysaccharidosis VII (MPS VII, Sly syndrome) is an autosomal recessive lysosomal storage disease caused by β-glucuronidase (GUS) deficiency. A naturally occurring mouse model of that disease has been very useful for studying experimental approaches to therapy. However, immune responses can complicate evaluation of the long-term benefits of enzyme replacement or gene therapy delivered to adult MPS VII mice. To make this model useful for studying the long-term effectiveness and side effects of experimental therapies delivered to adult mice, we developed a new MPS VII mouse model, which is tolerant to both human and murine GUS. To achieve this, we used homologous recombination to introduce simultaneously a human cDNA transgene expressing inactive human GUS into intron 9 of the murine Gus gene and a targeted active site mutation (E536A) into the adjacent exon 10. When the heterozygote products of germline transmission were bred to homozygosity, the homozygous mice expressed no GUS enzyme activity but expressed inactive human GUS protein highly and were tolerant to immune challenge with human enzyme. Expression of the mutant murine Gus gene was reduced to about 10% of normal levels, but the inactive murine GUS enzyme also conferred tolerance to murine GUS. This MPS VII mouse model should be useful to evaluate therapeutic responses in adult mice receiving repetitive doses of enzyme or mice receiving gene therapy as adults. Heterozygotes expressed only 9.5–26% of wild-type levels of murine GUS instead of the expected 50%, indicating a dominant-negative effect of the mutant enzyme monomers on the activity of GUS tetramers in different tissues. Corrective gene therapy in this model should provide high enough levels of expression of normal GUS monomers to overcome the dominant negative effect of mutant monomers on newly synthesized GUS tetramers in most tissues. PMID:12700165

Arabidopsis gene expression patterns are altered during spaceflight

NASA Astrophysics Data System (ADS)

Paul, Anna-Lisa; Popp, Michael P.; Gurley, William B.; Guy, Charles; Norwood, Kelly L.; Ferl, Robert J.

The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments results in differential gene expression. A 5-day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β-Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on gene expression patterns initially by using the Adh/GUS transgene to address specifically the possibility that spaceflight induces a hypoxic stress response (Paul, A.L., Daugherty, C.J., Bihn, E.A., Chapman, D.K., Norwood, K.L., Ferl, R.J., 2001. Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis, Plant Physiol. 126, 613-621). As a follow-on to the reporter gene analysis, we report here the evaluation of genome-wide patterns of native gene expression within Arabidopsis shoots utilizing the Agilent DNA array of 21,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes was further characterized with quantitative Real-Time RT PCR (ABI - Taqman®). Comparison of the patterns of expression for arrays probed with RNA isolated from plants exposed to spaceflight compared to RNA isolated from ground control plants revealed 182 genes that were differentially expressed in response to the spaceflight mission by more than 4-fold, and of those only 50 genes were expressed at levels chosen to support a conservative change call. None of the genes that are hallmarks of hypoxic stress were induced to this level. However, genes related to heat shock were dramatically induced - but in a pattern and under growth conditions that are not easily explained by elevated temperatures. These gene expression data are discussed in light of current models for plant responses to the spaceflight environment and with regard to potential future spaceflight experiment

The promoter of a plant defensin gene directs specific expression in nematode-induced syncytia in Arabidopsis roots.

PubMed

Siddique, Shahid; Wieczorek, Krzysztof; Szakasits, Dagmar; Kreil, David P; Bohlmann, Holger

2011-10-01

The beet cyst nematode Heterodera schachtii induces a feeding site, called syncytium, in roots of host plants. In Arabidopsis, one of the genes whose expression is strongly induced in these structures is Pdf2.1 which codes for an antimicrobial plant defensin. Arabidopsis has 13 plant defensin genes. Besides Pdf2.1, the Pdf2.2 and Pdf2.3 genes were strongly expressed in syncytia and therefore the expression of all three Pdf genes was studied in detail. The promoter of the Pdf2.1 gene turned out to be an interesting candidate to drive a syncytium-specific expression of foreign genes as RT-PCR showed that apart from the feeding site it was only expressed in siliques (seeds). The Pdf2.2 and Pdf2.3 genes were in addition expressed in seedlings, roots, leaves, stems, and flowers. These results were supported by the analysis of promoter::GUS lines. After infection with H. schachtii all GUS lines showed a strong staining in syncytia at 5 and 15 dpi. This expression pattern was confirmed by in situ RT-PCR. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Sugar beet proteinase inhibitor (BvSTI) gene promoter is regulated by insects and wounding in transgenic Nicotiana benthamiana

USDA-ARS?s Scientific Manuscript database

A regulatory sequence from a serine proteinase inhibitor gene (BvSTIpro) shown to be up-regulated in resistant interactions with a root pest of sugar beet, the sugar beet root maggot, was fused to the ß-glucuronidase (GUS) reporter gene to characterize its expression patterns in transgenic Nicotiana...

Identification of a cis-regulatory region of a gene in Arabidopsis thaliana whose induction by dehydration is mediated by abscisic acid and requires protein synthesis.

PubMed

Iwasaki, T; Yamaguchi-Shinozaki, K; Shinozaki, K

1995-05-20

In Arabidopsis thaliana, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA) but the gene does not include any sequence corresponding to the consensus ABA-responsive element (ABRE), RYACGTGGYR, in its promoter region. The cis-regulatory region of the rd22 promoter was identified by monitoring the expression of beta-glucuronidase (GUS) activity in leaves of transgenic tobacco plants transformed with chimeric gene fusions constructed between 5'-deleted promoters of rd22 and the coding region of the GUS reporter gene. A 67-bp nucleotide fragment corresponding to positions -207 to -141 of the rd22 promoter conferred responsiveness to dehydration and ABA on a non-responsive promoter. The 67-bp fragment contains the sequences of the recognition sites for some transcription factors, such as MYC, MYB, and GT-1. The fact that accumulation of rd22 mRNA requires protein synthesis raises the possibility that the expression of rd22 might be regulated by one of these trans-acting protein factors whose de novo synthesis is induced by dehydration or ABA. Although the structure of the RD22 protein is very similar to that of a non-storage seed protein, USP, of Vicia faba, the expression of the GUS gene driven by the rd22 promoter in non-stressed transgenic Arabidopsis plants was found mainly in flowers and bolted stems rather than in seeds.

Overexpression of a MADS-Box Gene from Birch (Betula platyphylla) Promotes Flowering and Enhances Chloroplast Development in Transgenic Tobacco

PubMed Central

Qu, Guan-Zheng; Zheng, Tangchun; Liu, Guifeng; Wang, Wenjie; Zang, Lina; Liu, Huanzhen; Yang, Chuanping

2013-01-01

In this study, a MADS-box gene (BpMADS), which is an ortholog of AP1 from Arabidopsis, was isolated from birch (Betula platyphylla). Transgenic Arabidopsis containing a BpMADS promoter::GUS construct was produced, which exhibited strong GUS staining in sepal tissues. Ectopic expression of BpMADS significantly enhanced the flowering of tobacco (35S::BpMADS). In addition, the chloroplasts of transgenic tobacco exhibited much higher growth and division rates, as well rates of photosynthesis, than wild-type. A grafting experiment demonstrated that the flowering time of the scion was not affected by stock that overexpressed BpMADS. In addition, the overexpression of BpMADS resulted in the upregulation of some flowering-related genes in tobacco. PMID:23691043

Cloning and Functional Analysis of the Promoter of an Ascorbate Oxidase Gene from Gossypium hirsutum

PubMed Central

Xin, Shan; Tao, Chengcheng; Li, Hongbin

2016-01-01

Apoplastic ascorbate oxidase (AO) plays significant roles in plant cell growth. However, the mechanism of underlying the transcriptional regulation of AO in Gossypium hirsutum remains unclear. Here, we obtained a 1,920-bp promoter sequence from the Gossypium hirsutum ascorbate oxidase (GhAO1) gene, and this GhAO1 promoter included a number of known cis-elements. Promoter activity analysis in overexpressing pGhAO1::GFP-GUS tobacco (Nicotiana benthamiana) showed that the GhAO1 promoter exhibited high activity, driving strong reporter gene expression in tobacco trichomes, leaves and roots. Promoter 5’-deletion analysis demonstrated that truncated GhAO1 promoters with serial 5’-end deletions had different GUS activities. A 360-bp fragment was sufficient to activate GUS expression. The P-1040 region had less GUS activity than the P-720 region, suggesting that the 320-bp region from nucleotide -720 to -1040 might include a cis-element acting as a silencer. Interestingly, an auxin-responsive cis-acting element (TGA-element) was uncovered in the promoter. To analyze the function of the TGA-element, tobacco leaves transformed with promoters with different 5’ truncations were treated with indole-3-acetic acid (IAA). Tobacco leaves transformed with the promoter regions containing the TGA-element showed significantly increased GUS activity after IAA treatment, implying that the fragment spanning nucleotides -1760 to -1600 (which includes the TGA-element) might be a key component for IAA responsiveness. Analyses of the AO promoter region and AO expression pattern in Gossypium arboreum (Ga, diploid cotton with an AA genome), Gossypium raimondii (Gr, diploid cotton with a DD genome) and Gossypium hirsutum (Gh, tetraploid cotton with an AADD genome) indicated that AO promoter activation and AO transcription were detected together only in D genome/sub-genome (Gr and Gh) cotton. Taken together, these results suggest that the 1,920-bp GhAO1 promoter is a functional sequence

Cloning and Functional Analysis of the Promoter of an Ascorbate Oxidase Gene from Gossypium hirsutum.

PubMed

Xin, Shan; Tao, Chengcheng; Li, Hongbin

2016-01-01

Apoplastic ascorbate oxidase (AO) plays significant roles in plant cell growth. However, the mechanism of underlying the transcriptional regulation of AO in Gossypium hirsutum remains unclear. Here, we obtained a 1,920-bp promoter sequence from the Gossypium hirsutum ascorbate oxidase (GhAO1) gene, and this GhAO1 promoter included a number of known cis-elements. Promoter activity analysis in overexpressing pGhAO1::GFP-GUS tobacco (Nicotiana benthamiana) showed that the GhAO1 promoter exhibited high activity, driving strong reporter gene expression in tobacco trichomes, leaves and roots. Promoter 5'-deletion analysis demonstrated that truncated GhAO1 promoters with serial 5'-end deletions had different GUS activities. A 360-bp fragment was sufficient to activate GUS expression. The P-1040 region had less GUS activity than the P-720 region, suggesting that the 320-bp region from nucleotide -720 to -1040 might include a cis-element acting as a silencer. Interestingly, an auxin-responsive cis-acting element (TGA-element) was uncovered in the promoter. To analyze the function of the TGA-element, tobacco leaves transformed with promoters with different 5' truncations were treated with indole-3-acetic acid (IAA). Tobacco leaves transformed with the promoter regions containing the TGA-element showed significantly increased GUS activity after IAA treatment, implying that the fragment spanning nucleotides -1760 to -1600 (which includes the TGA-element) might be a key component for IAA responsiveness. Analyses of the AO promoter region and AO expression pattern in Gossypium arboreum (Ga, diploid cotton with an AA genome), Gossypium raimondii (Gr, diploid cotton with a DD genome) and Gossypium hirsutum (Gh, tetraploid cotton with an AADD genome) indicated that AO promoter activation and AO transcription were detected together only in D genome/sub-genome (Gr and Gh) cotton. Taken together, these results suggest that the 1,920-bp GhAO1 promoter is a functional sequence with a

Phylogenetic distribution of genes encoding β-glucuronidase activity in human colonic bacteria and the impact of diet on faecal glycosidase activities.

PubMed

McIntosh, Freda M; Maison, Nathalie; Holtrop, Grietje; Young, Pauline; Stevens, Valerie J; Ince, Jennifer; Johnstone, Alexandra M; Lobley, Gerald E; Flint, Harry J; Louis, Petra

2012-08-01

Bacterial β-glucuronidase in the human colon plays an important role in cleaving liver conjugates of dietary compounds and xenobiotics, while other glycosidase activities are involved in the conversion of dietary plant glycosides. Here we detected an increase in β-glucuronidase activity in faecal samples from obese volunteers following a high-protein moderate carbohydrate weight-loss diet, compared with a weight maintenance diet, but little or no changes were observed when the type of fermentable carbohydrate was varied. Other faecal glycosidase activities showed little or no change over a fivefold range of dietary NSP intake, although α-glucosidase increased on a resistant starch-enriched diet. Two distinct groups of gene, gus and BG, have been reported to encode β-glucuronidase activity among human colonic bacteria. Degenerate primers were designed against these genes. Overall, Firmicutes were found to account for 96% of amplified gus sequences, with three operational taxonomic units particularly abundant, whereas 59% of amplified BG sequences belonged to Bacteroidetes and 41% to Firmicutes. A similar distribution of operational taxonomic units was found in a published metagenome dataset involving a larger number of volunteers. Seven cultured isolates of human colonic bacteria that carried only the BG gene gave relatively low β-glucuronidase activity that was not induced by 4-nitrophenyl-β-D-glucuronide. By comparison, in three of five isolates that possessed only the gus gene, β-glucuronidase activity was induced. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

An auxin responsive CLE gene regulates shoot apical meristem development in Arabidopsis

PubMed Central

Guo, Hongyan; Zhang, Wei; Tian, Hainan; Zheng, Kaijie; Dai, Xuemei; Liu, Shanda; Hu, Qingnan; Wang, Xianling; Liu, Bao; Wang, Shucai

2015-01-01

Plant hormone auxin regulates most, if not all aspects of plant growth and development, including lateral root formation, organ pattering, apical dominance, and tropisms. Peptide hormones are peptides with hormone activities. Some of the functions of peptide hormones in regulating plant growth and development are similar to that of auxin, however, the relationship between auxin and peptide hormones remains largely unknown. Here we report the identification of OsCLE48, a rice (Oryza sativa) CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION) gene, as an auxin response gene, and the functional characterization of OsCLE48 in Arabidopsis and rice. OsCLE48 encodes a CLE peptide hormone that is similar to Arabidopsis CLEs. RT-PCR analysis showed that OsCLE48 was induced by exogenously application of IAA (indole-3-acetic acid), a naturally occurred auxin. Expression of integrated OsCLE48p:GUS reporter gene in transgenic Arabidopsis plants was also induced by exogenously IAA treatment. These results indicate that OsCLE48 is an auxin responsive gene. Histochemical staining showed that GUS activity was detected in all the tissue and organs of the OsCLE48p:GUS transgenic Arabidopsis plants. Expression of OsCLE48 under the control of the 35S promoter in Arabidopsis inhibited shoot apical meristem development. Expression of OsCLE48 under the control of the CLV3 native regulatory elements almost completely complemented clv3-2 mutant phenotypes, suggesting that OsCLE48 is functionally similar to CLV3. On the other hand, expression of OsCLE48 under the control of the 35S promoter in Arabidopsis has little, if any effects on root apical meristem development, and transgenic rice plants overexpressing OsCLE48 are morphologically indistinguishable from wild type plants, suggesting that the functions of some CLE peptides may not be fully conserved in Arabidopsis and rice. Taken together, our results showed that OsCLE48 is an auxin responsive peptide hormone gene, and it regulates shoot apical

Assessment of RNAi-induced silencing in banana (Musa spp.).

PubMed

Dang, Tuong Vi T; Windelinckx, Saskia; Henry, Isabelle M; De Coninck, Barbara; Cammue, Bruno P A; Swennen, Rony; Remy, Serge

2014-09-18

In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana. Using embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6-8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target

Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme.

PubMed

Moriau, L; Michelet, B; Bogaerts, P; Lambert, L; Michel, A; Oufattole, M; Boutry, M

1999-07-01

The plasma membrane H+-ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+-ATPase) genes. Four of these are classified into two gene subfamilies, pma1-2-3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5' end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma-gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell-type specific manner. Expression in these organs was confirmed at the protein level, using subfamily-specific antibodies. (ii) pma4-gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+-ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+-ATPases with different enzymatic properties.

The role of peu-miR164 and its target PeNAC genes in response to abiotic stress in Populus euphratica.

PubMed

Lu, Xin; Dun, Hui; Lian, Conglong; Zhang, Xiaofei; Yin, Weilun; Xia, Xinli

2017-06-01

Plant miR164 family is highly conserved and miR164 members regulate conserved targets belonging to NAC transcription factors. Our previous studies have revealed that peu-miR164a-e and its target gene POPTR_0007s08420 participate in abiotic stress response in Populus euphratica according to deep sequencing and degradome sequencing. In this study, miR164 family comprises six members that generate two mature products (miR164a-e and miR164f) and target seven NAC genes in P. euphratica. Co-expression in Nicotiana benthamiana and 5' RACE confirmed that peu-miR164 directs PeNAC070, PeNAC012 and PeNAC028 mRNAs cleavage. Expression profiles of primary peu-miR164 a/b/c/d/e bear similarity to those of peu-miR164a-e, whereas PeNAC070 and PeNAC081 showed inverse expression patterns with peu-miR164a-e under abiotic stresses. Existence of cis-acting elements in PeNAC070 promoter (ABRE,MBs, Box-W1, GC-motif, and W-box) and in peu-MIR164b promoter (HSE) further confirmed different responses of peu-miR164 and PeNAC070 to abiotic stresses. Histochemical β-glucuronidase (GUS) staining revealed that GUS activities increased when Pro PeNAC070 ::GUS transgenic Arabidopsis plants were exposed to NaCl, mannitol and abscisic acid (ABA), whereas GUS activity of Pro peu-MIR164b ::GUS plants decreased under ABA treatment. Subcellular localization and transactivation assays showed that PeNAC070 protein was localized to the nucleus and exhibited transactivation activity at the C-terminal. Overexpression of PeNAC070 in Arabidopsis promoted lateral root development, delayed stem elongation, and increased sensitivity of transgenic plants to drought and salt stresses. This study aids in understanding the adaptability of P. euphratica to extreme drought and salt environment by analysing tissue-specific expression patterns of miR164-regulated and specific promoter-regulated PeNAC genes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1: analysis of cis-acting promoter elements required for regulation by abscisic acid and VP1.

PubMed

Hattori, T; Terada, T; Hamasuna, S

1995-06-01

Osem, a rice gene homologous to the wheat Em gene, which encodes one of the late-embryogenesis abundant proteins was isolated. The gene was characterized with respect to control of transcription by abscisic acid (ABA) and the transcriptional activator VP1, which is involved in the ABA-regulated gene expression during late embryo-genesis. A fusion gene (Osem-GUS) consisting of the Osem promoter and the bacterial beta-glucuronidase (GUS) gene was constructed and tested in a transient expression system, using protoplasts derived from a suspension-cultured line of rice cells, for activation by ABA and by co-transfection with an expression vector (35S-Osvp1) for the rice VP1 (OSVP1) cDNA. The expression of Osem-GUS was strongly (40- to 150-fold) activated by externally applied ABA and by over-expression of (OS)VP1. The Osem promoter has three ACGTG-containing sequences, motif A, motif B and motif A', which resemble the abscisic acid-responsive element (ABRE) that was previously identified in the wheat Em and the rice Rab16. There is also a CATGCATG sequence, which is known as the Sph box and is shown to be essential for the regulation by VP1 of the maize anthocyanin regulatory gene C1. Focusing on these sequence elements, various mutant derivatives of the Osem promoter in the transient expression system were assayed. The analysis revealed that motif A functions not only as an ABRE but also as a sequence element required for the regulation by (OS)VP1.

Links between sulphur oxidation and sulphur-oxidising bacteria abundance and diversity in soil microcosms based on soxB functional gene analysis.

PubMed

Tourna, Maria; Maclean, Paul; Condron, Leo; O'Callaghan, Maureen; Wakelin, Steven A

2014-06-01

Sulphur-oxidising bacteria (SOB) play a key role in the biogeochemical cycling of sulphur in soil ecosystems. However, the ecology of SOB is poorly understood, and there is little knowledge about the taxa capable of sulphur oxidation, their distribution, habitat preferences and ecophysiology. Furthermore, as yet there are no conclusive links between SOB community size or structure and rates of sulphur oxidation. We have developed a molecular approach based on primer design targeting the soxB functional gene of nonfilamentous chemolithotrophic SOB that allows assessment of both abundance and diversity. Cloning and sequencing revealed considerable diversity of known soxB genotypes from agricultural soils and also evidence for previously undescribed taxa. In a microcosm experiment, abundance of soxB genes increased with sulphur oxidation rate in soils amended with elemental sulphur. Addition of elemental sulphur to soil had a significant effect in the soxB gene diversity, with the chemolithotrophic Thiobacillus-like Betaproteobacteria sequences dominating clone libraries 6 days after sulphur application. Using culture-independent methodology, the study provides evidence for links between abundance and diversity of SOB and sulphur oxidation. The methodology provides a new tool for investigation of the ecology and role of SOB in soil sulphur biogeochemistry. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Analysis of tandem repeat units of the promoter of capsanthin/capsorubin synthase (Ccs) gene in pepper fruit.

PubMed

Tian, Shi-Lin; Li, Zheng; Li, Li; Shah, S N M; Gong, Zhen-Hui

2017-07-01

Capsanthin/capsorubin synthase ( Ccs ) gene is a key gene that regulates the synthesis of capsanthin and the development of red coloration in pepper fruits. There are three tandem repeat units in the promoter region of Ccs , but the potential effects of the number of repetitive units on the transcriptional regulation of Ccs has been unclear. In the present study, expression vectors carrying different numbers of repeat units of the Ccs promoter were constructed, and the transient expression of the β-glucuronidase ( GUS ) gene was used to detect differences in expression levels associated with the promoter fragments. These repeat fragments and the plant expression vector PBI121 containing the 35s CaMV promoter were ligated to form recombinant vectors that were transfected into Agrobacterium tumefaciens GV3101. A fluorescence spectrophotometer was used to analyze the expression associated with the various repeat units. It was concluded that the constructs containing at least one repeat were associated with GUS expression, though they did not differ from one another. This repeating unit likely plays a role in transcription and regulation of Ccs expression.

Laser Microdissection and Spatiotemporal Pinoresinol-Lariciresinol Reductase Gene Expression Assign the Cell Layer-Specific Accumulation of Secoisolariciresinol Diglucoside in Flaxseed Coats.

PubMed

Fang, Jingjing; Ramsay, Aïna; Renouard, Sullivan; Hano, Christophe; Lamblin, Frédéric; Chabbert, Brigitte; Mesnard, François; Schneider, Bernd

2016-01-01

The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed ( Linum usitatissimum L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of L. usitatissimum ( LuPLR1 ), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase ( GUS ) reporter gene, and the spatio-temporal regulation of LuPLR1 gene expression in flaxseed was determined by histochemical and activity assays of GUS . The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats.

Laser Microdissection and Spatiotemporal Pinoresinol-Lariciresinol Reductase Gene Expression Assign the Cell Layer-Specific Accumulation of Secoisolariciresinol Diglucoside in Flaxseed Coats

PubMed Central

Fang, Jingjing; Ramsay, Aïna; Renouard, Sullivan; Hano, Christophe; Lamblin, Frédéric; Chabbert, Brigitte; Mesnard, François; Schneider, Bernd

2016-01-01

The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed (Linum usitatissimum L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of L. usitatissimum (LuPLR1), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase (GUS) reporter gene, and the spatio-temporal regulation of LuPLR1 gene expression in flaxseed was determined by histochemical and activity assays of GUS. The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats. PMID:27917190

From pollen tubes to infection threads: recruitment of Medicago floral pectic genes for symbiosis.

PubMed

Rodríguez-Llorente, Ignacio D; Pérez-Hormaeche, Javier; El Mounadi, Kaoutar; Dary, Mohammed; Caviedes, Miguel A; Cosson, Viviane; Kondorosi, Adam; Ratet, Pascal; Palomares, Antonio J

2004-08-01

While the biology of nitrogen-fixing root nodules has been extensively studied, little is known about the evolutionary events that predisposed legume plants to form symbiosis with rhizobia. We have studied the presence and the expression of two pectic gene families in Medicago, polygalacturonases (PGs) and pectin methyl esterases (PMEs) during the early steps of the Sinorhizobium meliloti-Medicago interaction and compared them with related pollen-specific genes. First, we have compared the expression of MsPG3, a PG gene specifically expressed during the symbiotic interaction, with the expression of MsPG11, a highly homologous pollen-specific gene, using promoter-gus fusions in transgenic M. truncatula and tobacco plants. These results demonstrated that the symbiotic promoter functions as a pollen-specific promoter in the non-legume host. Second, we have identified the presence of a gene family of at least eight differentially expressed PMEs in Medicago. One subfamily is represented by one symbiotic gene (MtPER) and two pollen-expressed genes (MtPEF1 and MtPEF2) that are clustered in the M. truncatula genome. The promoter-gus studies presented in this work and the homology between plant PGs, together with the analysis of the PME locus structure and MtPER expression studies, suggest that the symbiotic MsPG3 and MtPER could have as ancestors pollen-expressed genes involved in polar tip growth processes during pollen tube elongation. Moreover, they could have been recruited after gene duplication in the symbiotic interaction to facilitate polar tip growth during infection thread formation.

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system.

PubMed

Inui, Hideyuki; Gion, Keiko; Utani, Yasushi; Wakai, Taketo; Kodama, Susumu; Eun, Heesoo; Kim, Yun-Seok; Ohkawa, Hideo

2012-01-01

The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated β-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorinated dibenzeno-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of residential and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g(-1) of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system.

PubMed

Inui, Hideyuki; Gion, Keiko; Utani, Yasushi; Wakai, Taketo; Kodama, Susumu; Eun, Heesoo; Kim, Yun-Seok; Ohkawa, Hideo

2012-01-01

The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated β-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorodibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of life and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g(-1) of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

Persistence, population dynamics and competitiveness for nodulation of marker gene-tagged Rhizobium galegae strains in field lysimeters in the boreal climatic zone.

PubMed

Pitkäjärvi, Jyrki; Räsänen, Leena A; Langenskiöld, Jenny; Wallenius, Kaisa; Niemi, Maarit; Lindström, Kristina

2003-10-01

Abstract A non-indigenous wild-type strain Rhizobium galegae HAMBI 540, which specifically nodulates perennial goat's rue (Galega orientalis), and its marker gene-tagged derivatives R. galegae HAMBI 2363(luc), R. galegae HAMBI 2368(gusA21) and R. galegae HAMBI 2364(gusA30) were used to evaluate the persistence, population dynamics and competitiveness for nodulation of rhizobia under field conditions in Finland. The lysimeters were filled with clean or diesel oil-polluted (3000 mug g(-1)) agricultural soil. During the first 2 years of the field release luc- and gusA21-tagged strains could be effectively detected by cultivation, reinforced with colony polymerase chain reaction. The population densities remained relatively stable from 10(4) to 10(5) cfu g(-1) dry soil from spring until late autumn. Replicate limiting dilution polymerase chain reaction analysis gave comparable results with cultivation with strain HAMBI 2363 until 49 weeks after inoculation. GUS activity of strain HAMBI 2368 could be stably detected in nodules and soil. On the other hand, luc activity weakened clearly in cold conditions along with decreased metabolic activity of rhizobia. The competitive ability for nodulation of the gusA30-tagged strain decreased slowly with time compared to the wild-type strain. Moderate soil pollution did not have significant effects on target bacteria or plant growth. Limited vertical movement of target bacteria outside the rhizosphere was detected from percolated water.

Season of Birth and Dopamine Receptor Gene Associations with Impulsivity, Sensation Seeking and Reproductive Behaviors

PubMed Central

Eisenberg, Dan T. A.; Campbell, Benjamin; MacKillop, James; Lum, J. Koji; Wilson, David S.

2007-01-01

Background Season of birth (SOB) has been associated with many physiological and psychological traits including novelty seeking and sensation seeking. Similar traits have been associated with genetic polymorphisms in the dopamine system. SOB and dopamine receptor genetic polymorphisms may independently and interactively influence similar behaviors through their common effects on the dopaminergic system. Methodology/Principal Findings Based on a sample of 195 subjects, we examined whether SOB was associated with impulsivity, sensation seeking and reproductive behaviors. Additionally we examined potential interactions of dopamine receptor genes with SOB for the same set of traits. Phenotypes were evaluated using the Sociosexual Orientation Inventory, the Barratt Impulsivity Scale, the Eysenck Impulsivity Questionnaire, the Sensation Seeking Scale, and the Delay Discounting Task. Subjects were also asked about their age at first sex as well as their desired age at the birth of their first child. The dopamine gene polymorphisms examined were Dopamine Receptor D2 (DRD2) TaqI A and D4 (DRD4) 48 bp VNTR. Primary analyses included factorial gender×SOB ANOVAs or binary logistic regression models for each dependent trait. Secondary analysis extended the factorial models by also including DRD2 and DRD4 genotypes as independent variables. Winter-born males were more sensation seeking than non-winter born males. In factorial models including both genotype and season of birth as variables, two previously unobserved effects were discovered: (1) a SOB×DRD4 interaction effect on venturesomeness and (2) a DRD2×DRD4 interaction effect on sensation seeking. Conclusion These results are consistent with past findings that SOB is related to sensation seeking. Additionally, these results provide tentative support for the hypothesis that SOB modifies the behavioral expression of dopaminergic genetic polymorphism. These findings suggest that SOB should be included in future studies of

Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

NASA Technical Reports Server (NTRS)

Jackson, Jacquelyn; Egnin, Marceline; Xue, Qi-Han; Prakash, C. S.

1997-01-01

Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

Abscisic Acid-Dependent and -Independent Expression of the Carrot Late-Embryogenesis-Abundant-Class Gene Dc3 in Transgenic Tobacco Seedlings1

PubMed Central

Siddiqui, Najeeb U.; Chung, Hwa-Jee; Thomas, Terry L.; Drew, Malcolm C.

1998-01-01

We studied the expression of three promoter 5′ deletion constructs (−218, −599, and −1312) of the LEA (late embryogenesis abundant)-class gene Dc3 fused to β-glucuronidase (GUS), where each construct value refers to the number of base pairs upstream of the transcription start site at which the deletion occurred. The Dc3 gene is noted for its induction by abscisic acid (ABA), but its response to other plant hormones and various environmental stresses has not been reported previously for vegetative cells. Fourteen-day-old transgenic tobacco (Nicotiana tabacum L.) seedlings were exposed to dehydration, hypoxia, salinity, exogenous ethylene, or exogenous methyl jasmonate (MeJa). GUS activity was quantified fluorimetrically and expression was observed by histochemical staining of the seedlings. An increase in GUS activity was observed in plants with constructs −599 and −1312 in response to dehydration and salinity within 6 h of stress, and at 12 h in response to hypoxia. No increase in endogenous ABA was found in any of the three lines, even after 72 h of hypoxia. An ABA-independent increase in GUS activity was observed when endogenous ABA biosynthesis was blocked by fluridone and plants were exposed to 5 μL L−1 ethylene in air or 100 μm MeJa. Virtually no expression was observed in construct −218 in response to dehydration, salinity, or MeJa, but there was a moderate response to ethylene and hypoxia. This suggests that the region between −218 and −599 is necessary for ABA (dehydration and salinity)- and MeJa-dependent expression, whereas ethylene-mediated expression does not require this region of the promoter. PMID:9847092

The Competence of Maize Shoot Meristems for Integrative Transformation and Inherited Expression of Transgenes.

PubMed Central

Zhong, H.; Sun, B.; Warkentin, D.; Zhang, S.; Wu, R.; Wu, T.; Sticklen, M. B.

1996-01-01

We have developed a novel and reproducible system for recovery of fertile transgenic maize (Zea mays L.) plants. The transformation was performed using microprojectile bombardment of cultured shoot apices of maize with a plasmid carrying two linked genes, the Streptomyces hygroscopicus phosphinothricin acetyltransferase gene (bar) and the potato proteinase inhibitor II gene, either alone or in combination with another plasmid containing the 5[prime] region of the rice actin 1 gene fused to the Escherichia coli [beta]-glucuronidase gene (gus). Bombarded shoot apices were subsequently multiplied and selected under 3 to 5 mg/L glufosinate ammonium. Co-transformation frequency was 100% (146/146) for linked genes and 80% (41/51) for unlinked genes. Co-expression frequency of the bar and gus genes was 57% (29/51). The co-integration, co-inheritance, and co-expression of bar, the potato proteinase inhibitor II gene, and gus in transgenic R0, R1, and R2 plants were confirmed. Localized expression of the actin 1-GUS protein in the R0 and R1 plants was extensively analyzed by histochemical and fluorometric assays. PMID:12226244

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated β-glucuronidase reporter gene expression system

PubMed Central

Inui, Hideyuki; Gion, Keiko; Utani, Yasushi; Wakai, Taketo; Kodama, Susumu; Eun, Heesoo; Kim, Yun-Seok; Ohkawa, Hideo

2012-01-01

The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated β-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorodibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of life and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g−1 of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples. PMID:22428884

Cloning and Characterization of 5′ Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

PubMed Central

Tang, Guiying; Xu, Pingli; Liu, Wei; Liu, Zhanji; Shan, Lei

2015-01-01

LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5′ flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5′ terminal and/or 3′ terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65bp proximal promoter region and the 52bp 5′ UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements. PMID:26426444

Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum.

PubMed

Chen, Ke; de Borne, François Dorlhac; Julio, Emilie; Obszynski, Julie; Pale, Patrick; Otten, Léon

2016-08-01

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus

PubMed Central

Woolston, Benjamin M.; Schlagnhaufer, Carl; Wilkinson, Jack; Larsen, Jeffrey; Shi, Zhixin; Mayer, Kimberly M.; Walters, Donald S.; Curtis, Wayne R.; Romaine, C. Peter

2011-01-01

Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologous protein expression system have revealed unexpected contributions of the mycelial inoculants in the morphogenesis of the fruiting body. These contributions were elucidated using a dual-inoculant method whereby the two layers were differientially inoculated with transgenic β-glucuronidase (GUS) and wild-type (WT) lines. Surprisingly, use of a transgenic GUS line in the lower substrate and a WT line in the upper substrate yielded fruiting bodies expressing GUS activity while lacking the GUS transgene. Results of PCR and RT-PCR analyses for the GUS transgene and RNA transcript, respectively, suggested translocation of the GUS protein from the transgenic mycelium colonizing the lower layer into the fruiting body that developed exclusively from WT mycelium colonizing the upper layer. Effective translocation of the GUS protein depended on the use of a transgenic line in the lower layer in which the GUS gene was controlled by a vegetative mycelium-active promoter (laccase 2 and β-actin), rather than a fruiting body-active promoter (hydrophobin A). GUS-expressing fruiting bodies lacking the GUS gene had a bonafide WT genotype, confirmed by the absence of stably inherited GUS and hygromycin phosphotransferase selectable marker activities in their derived basidiospores and mycelial tissue cultures. Differientially inoculating the two substrate layers with individual lines carrying the GUS gene controlled by different tissue-preferred promoters resulted in up to a ∼3.5-fold increase in GUS activity over that obtained with a single inoculant. Our findings support the existence of a previously undescribed phenomenon of long

Reversible Heat-Induced Inactivation of Chimeric β-Glucuronidase in Transgenic Plants1

PubMed Central

Almoguera, Concepción; Rojas, Anabel; Jordano, Juan

2002-01-01

We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to β-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5′-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions. PMID:12011363

Evidence for the negative regulation of phytase gene expression in Streptomyces lividans and Streptomyces coelicolor.

PubMed

Boukhris, Ines; Dulermo, Thierry; Chouayekh, Hichem; Virolle, Marie-Joëlle

2016-01-01

Sco7697, a gene encoding a phytase, enzyme able to degrade phytate (myo-inositol 1,2,3,4,5,6-hexakis phosphate), the most abundant phosphorus storing compound in plants is present in the genome of S. coelicolor, a soil born bacteria with a saprophytic lifestyle. The expression of this gene was previously shown to be induced in conditions of Pi limitation by the response regulator PhoP binding to an operator sequence, the PHO box, located upstream of the -35 promoter sequence. A close examination of the promoter region of sco7697 revealed the presence of another putative operator site, a Direct Repeat (DR), located downstream of the -10 promoter sequence. In order to determine whether this DR played a role in regulation of sco7697 expression, different variants of the phytase gene promoter region were transcriptionally fused to the ß-glucuronidase reporter gene (GUS). As expected, deletion of the PHO box led to abolition of sco7697 induction in conditions of Pi limitation. Interestingly, alteration of the DR correlated with a dramatic increase of GUS expression but only when PhoP was present. These results demonstrated that this DR is the site of strong negative regulation by an unknown repressor. The latter would impede the necessary activation of phytase expression by PhoP. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nuclear targeting of the maize R protein requires two nuclear localization sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shieh, M.W.; Raikhel, N.V.; Wessler, S.R.

1993-02-01

Previous genetic and structural evidence indicates that the maize R gene encodes a nuclear transcriptional activating factor. In-frame carboxyl- and amino-terminal fusions of the R gene to the reporter gene encoding [beta]-glucuronidase (GUS) were sufficient to direct GUS to the nucleus of the transiently transformed onion (Allium cepa) epidermal cells. Further analysis of chimeric constructs containing regions of the R gene fused to the GUS cDNA revealed three specific nuclear localization sequences (NLSs) that were capable of redirecting the GUS protein to the nucleus. Amino-terminal NLS-A (amino acids 100-109, GDRRAAPARP) contained several arginine residues; a similar localization signal is foundmore » in only a few viral proteins. The medial NLS-M (amino acids 419-428, MSERKRREKL) is a simian virus 40 large T antigen-type NLS, and the carboxyl-terminal NLS-C (amino acids 598-610, MISESLRKAIGKR) is a mating type [alpha]2 type. NLSs M and C are independently sufficient to direct the GUS protein to the nucleus when it is fused at the amino terminus of GUS, whereas NLS-A fused to GUS partitioned between the nucleus and cytoplasm. Similar partitioning was observed when localization signals NLS-A and NLS-C were independently fused to the carboxy-terminal portion of GUS. A sequential deletion of the localization signals indicated that the amino-terminal and carboxyl-terminal fusions of R and GUS were redirected to the nucleus only when both NLS-A and -M, or NLS-C and -M, were present. These results indicate that multiple localization signals are necessary for nuclear targeting of this protein. The conservation of the localization signals within the alleles of R and similar proteins from other organisms is also discussed. 45 refs., 6 figs.« less

Flexible tools for gene expression and silencing in tomato.

PubMed

Fernandez, Ana I; Viron, Nicolas; Alhagdow, Moftah; Karimi, Mansour; Jones, Matthew; Amsellem, Ziva; Sicard, Adrien; Czerednik, Anna; Angenent, Gerco; Grierson, Donald; May, Sean; Seymour, Graham; Eshed, Yuval; Lemaire-Chamley, Martine; Rothan, Christophe; Hilson, Pierre

2009-12-01

As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources.

Ozone-induced gene expression occurs via ethylene-dependent and -independent signalling.

PubMed

Grimmig, Bernhard; Gonzalez-Perez, Maria N; Leubner-Metzger, Gerhard; Vögeli-Lange, Regina; Meins, Fred; Hain, Rüdiger; Penuelas, Josep; Heidenreich, Bernd; Langebartels, Christian; Ernst, Dieter; Sandermann, Heinrich

2003-03-01

Recent studies suggest that ethylene is involved in signalling ozone-induced gene expression. We show here that application of ozone increased glucuronidase (GUS) expression of chimeric reporter genes regulated by the promoters of the tobacco class I beta-1,3-glucanases (GLB and Gln2) and the grapevine resveratrol synthase (Vst1) genes in transgenic tobacco leaves. 5'-deletion analysis of the class I beta-1,3-glucanase promoter revealed that ozone-induced gene regulation is mainly mediated by the distal enhancer region containing the positively acting ethylene-responsive element (ERE). In addition, application of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, blocked ozone-induced class I beta-1,3-glucanase promoter activity. Enhancer activity and ethylene-responsiveness depended on the integrity of the GCC boxes, cis-acting elements present in the ERE of the class I beta-1,3-glucanase and the basic-type pathogenesis-related PR-1 protein (PRB-1b) gene promoters. The minimal PRB-1b promoter containing only the ERE with intact GCC boxes, was sufficient to confer 10-fold ozone inducibility to a GUS-reporter gene, while a substitution mutation in the GCC box abolished ozone responsiveness. The ERE region of the class I beta-1,3-glucanase promoter containing two intact GCC boxes confered strong ozone inducibility to a minimal cauliflower mosaic virus (CaMV) 35S RNA promoter, whereas two single-base substitution in the GCC boxes resulted in a complete loss of ozone inducibility. Taken together, these datastrongly suggest that ethylene is signalling ozone-induced expression of class I beta-l,3-glucanase and PRB-1b genes. Promoter analysis of the stilbene synthase Vst1 gene unravelled different regions for ozone and ethylene-responsiveness. Application of 1-MCP blocked ethylene-induced Vst1 induction, but ozone induction was not affected. This shows that ozone-induced gene expression occurs via at least two different signalling mechanisms and suggests an

Isolation and characterization of an ubiquitin extension protein gene (JcUEP) promoter from Jatropha curcas.

PubMed

Tao, Yan-Bin; He, Liang-Liang; Niu, Long-Jian; Xu, Zeng-Fu

2015-04-01

The JcUEP promoter is active constitutively in the bio-fuel plant Jatropha curcas , and is an alternative to the widely used CaMV35S promoter for driving constitutive overexpression of transgenes in Jatropha. Well-characterized promoters are required for transgenic breeding of Jatropha curcas, a biofuel feedstock with great potential for production of bio-diesel and bio-jet fuel. In this study, an ubiquitin extension protein gene from Jatropha, designated JcUEP, was identified to be ubiquitously expressed. Thus, we isolated a 1.2 kb fragment of the 5' flanking region of JcUEP and evaluated its activity as a constitutive promoter in Arabidopsis and Jatropha using the β-glucuronidase (GUS) reporter gene. As expected, histochemical GUS assay showed that the JcUEP promoter was active in all Arabidopsis and Jatropha tissues tested. We also compared the activity of the JcUEP promoter with that of the cauliflower mosaic virus 35S (CaMV35S) promoter, a well-characterized constitutive promoter conferring strong transgene expression in dicot species, in various tissues of Jatropha. In a fluorometric GUS assay, the two promoters showed similar activities in stems, mature leaves and female flowers; while the CaMV35S promoter was more effective than the JcUEP promoter in other tissues, especially young leaves and inflorescences. In addition, the JcUEP promoter retained its activity under stress conditions in low temperature, high salt, dehydration and exogenous ABA treatments. These results suggest that the plant-derived JcUEP promoter could be an alternative to the CaMV35S promoter for driving constitutive overexpression of transgenes in Jatropha and other plants.

A Novel WRKY transcription factor is required for induction of PR-1a gene expression by salicylic acid and bacterial elicitors.

PubMed

van Verk, Marcel C; Pappaioannou, Dimitri; Neeleman, Lyda; Bol, John F; Linthorst, Huub J M

2008-04-01

PR-1a is a salicylic acid-inducible defense gene of tobacco (Nicotiana tabacum). One-hybrid screens identified a novel tobacco WRKY transcription factor (NtWRKY12) with specific binding sites in the PR-1a promoter at positions -564 (box WK(1)) and -859 (box WK(2)). NtWRKY12 belongs to the class of transcription factors in which the WRKY sequence is followed by a GKK rather than a GQK sequence. The binding sequence of NtWRKY12 (WK box TTTTCCAC) deviated significantly from the consensus sequence (W box TTGAC[C/T]) shown to be recognized by WRKY factors with the GQK sequence. Mutation of the GKK sequence in NtWRKY12 into GQK or GEK abolished binding to the WK box. The WK(1) box is in close proximity to binding sites in the PR-1a promoter for transcription factors TGA1a (as-1 box) and Myb1 (MBSII box). Expression studies with PR-1a promoterbeta-glucuronidase (GUS) genes in stably and transiently transformed tobacco indicated that NtWRKY12 and TGA1a act synergistically in PR-1a expression induced by salicylic acid and bacterial elicitors. Cotransfection of Arabidopsis thaliana protoplasts with 35SNtWRKY12 and PR-1aGUS promoter fusions showed that overexpression of NtWRKY12 resulted in a strong increase in GUS expression, which required functional WK boxes in the PR-1a promoter.

Cotransformation of Trichoderma harzianum with β-Glucuronidase and Green Fluorescent Protein Genes Provides a Useful Tool for Monitoring Fungal Growth and Activity in Natural Soils†

PubMed Central

Bae, Yeoung-Seuk; Knudsen, Guy R.

2000-01-01

Trichoderma harzianum was cotransformed with genes encoding green fluorescent protein (GFP), β-glucuronidase (GUS), and hygromycin B (hygB) resistance, using polyethylene glycol-mediated transformation. One cotransformant (ThzID1-M3) was mitotically stable for 6 months despite successive subculturing without selection pressure. ThzID1-M3 morphology was similar to that of the wild type; however, the mycelial growth rate on agar was reduced. ThzID1-M3 was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of Sclerotinia sclerotiorum was compared with that of the wild-type strain. Wild-type and transformant strains both colonized sclerotia at levels above those of indigenous Trichoderma spp. in untreated controls. There were no significant differences in colonization levels between wild-type and cotransformant strains; however, the presence of the GFP and GUS marker genes permitted differentiation of introduced Trichoderma from indigenous strains. GFP activity was a useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. Addition of 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc) substrate to recovered glass slides confirmed the activity of GUS as well as GFP in soil. Our results suggest that cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil. PMID:10653755

Characterization of a Maize Wip1 Promoter in Transgenic Plants

PubMed Central

Zhang, Shengxue; Lian, Yun; Liu, Yan; Wang, Xiaoqing; Liu, Yunjun; Wang, Guoying

2013-01-01

The Maize Wip1 gene encodes a wound-induced Bowman-Birk inhibitor (BBI) protein which is a type of serine protease inhibitor, and its expression is induced by wounding or infection, conferring resistance against pathogens and pests. In this study, the maize Wip1 promoter was isolated and its function was analyzed. Different truncated Wip1 promoters were fused upstream of the GUS reporter gene and transformed into Arabidopsis, tobacco and rice plants. We found that (1) several truncated maize Wip1 promoters led to strong GUS activities in both transgenic Arabidopsis and tobacco leaves, whereas low GUS activity was detected in transgenic rice leaves; (2) the Wip1 promoter was not wound-induced in transgenic tobacco leaves, but was induced by wounding in transgenic rice leaves; (3) the truncated Wip1 promoter had different activity in different organs of transgenic tobacco plants; (4) the transgenic plant leaves containing different truncated Wip1 promoters had low GUS transcripts, even though high GUS protein level and GUS activities were observed; (5) there was one transcription start site of Wip1 gene in maize and two transcription start sites of GUS in Wip1::GUS transgenic lines; (6) the adjacent 35S promoter which is present in the transformation vectors enhanced the activity of the truncated Wip1 promoters in transgenic tobacco leaves, but did not influence the disability of truncated Wip1231 promoter to respond to wounding signals. We speculate that an ACAAAA hexamer, several CAA trimers and several elements similar to ACAATTAC octamer in the 5′-untranslated region might contribute to the strong GUS activity in Wip1231 transgenic lines, meanwhile, compared to the 5′-untranslated region from Wip1231 transgenic lines, the additional upstream open reading frames (uORFs) in the 5′-untranslated region from Wip1737 transgenic lines might contribute to the lower level of GUS transcript and GUS activity. PMID:24322445

Functional characterization of the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter in transgenic tomato plants.

PubMed

Yang, Qingjie; Yuan, Dawei; Shi, Lianxuan; Capell, Teresa; Bai, Chao; Wen, Nuan; Lu, Xiaodan; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

2012-10-01

The accumulation of carotenoids in plants depends critically on the spatiotemporal expression profiles of the genes encoding enzymes in the carotenogenic pathway. We cloned and characterized the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter to determine its role in the regulation of carotenogenesis, because the native gene is expressed at high levels in petals, which contain abundant chromoplasts. We transformed tomato (Solanum lycopersicum cv. Micro-Tom) plants with the gusA gene encoding the reporter enzyme β-glucuronidase (GUS) under the control of the GlZEP promoter, and investigated the reporter expression profile at the mRNA and protein levels. We detected high levels of gusA expression and GUS activity in chromoplast-containing flowers and fruits, but minimal levels in immature fruits containing green chloroplasts, in sepals, leaves, stems and roots. GlZEP-gusA expression was strictly associated with fruit development and chromoplast differentiation, suggesting an evolutionarily-conserved link between ZEP and the differentiation of organelles that store carotenoid pigments. The impact of our results on current models for the regulation of carotenogenesis in plants is discussed.

A study on the influence of different promoter and 5'UTR (URM) cassettes from Arabidopsis thaliana on the expression level of the reporter gene β glucuronidase in tobacco and cotton.

PubMed

Agarwal, Parul; Garg, Varsha; Gautam, Taru; Pillai, Beena; Kanoria, Shaveta; Burma, Pradeep Kumar

2014-04-01

Several reports of promoters from plants, viral and artificial origin that confer high constitutive expression are known. Among these the CaMV 35S promoter is used extensively for transgene expression in plants. We identified candidate promoters from Arabidopsis based on their transcript levels (meta-analysis of available microarray control datasets) to test their activity in comparison to the CaMV 35S promoter. A set of 11 candidate genes were identified which showed high transcript levels in the aerial tissue (i.e. leaf, shoot, flower and stem). In the initial part of the study binary vectors were developed wherein the promoter and 5'UTR region of these candidate genes (Upstream Regulatory Module, URM) were cloned upstream to the reporter gene β glucuronidase (gus). The promoter strengths were tested in transformed callus of Nicotiana tabacum and Gossypium hirsutum. On the basis of the results obtained from the callus, the influence of the URM cassettes on transgene expression was tested in transgenic tobacco. The URM regions of the genes encoding a subunit of photosystem I (PHOTO) and geranyl geranyl reductase (GGR) in A. thaliana genome showed significantly high levels of GUS activity in comparison to the CaMV 35S promoter. Further, when the 5'UTRs of both the genes were placed downstream to the CaMV 35S promoter it led to a substantial increase in GUS activity in transgenic tobacco lines and cotton callus. The enhancement observed was even higher to that observed with the viral leader sequences like Ω and AMV, known translational enhancers. Our results indicate that the two URM cassettes or the 5'UTR regions of PHOTO and GGR when placed downstream to the CaMV 35S promoter can be used to drive high levels of transgene expression in dicotyledons.

Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.).

PubMed

Herzog, Katja; Flachowsky, Henryk; Deising, Holger B; Hanke, Magda-Viola

2012-04-25

Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene. Copyright © 2012 Elsevier B.V. All rights reserved.

Isolation of the endosperm-specific LPAAT gene promoter from coconut (Cocos nucifera L.) and its functional analysis in transgenic rice plants.

PubMed

Xu, Li; Ye, Rongjian; Zheng, Yusheng; Wang, Zhekui; Zhou, Peng; Lin, Yongjun; Li, Dongdong

2010-09-01

As one of the key tropical crops, coconut (Cocos nucifera L.) is a member of the monocotyledonous family Aracaceae (Palmaceae). In this study, we amplified the upstream region of an endosperm-specific expression gene, Lysophosphatidyl acyltransferase (LPAAT), from the coconut genomic DNA by chromosome walking. In this sequence, we found several types of promoter-related elements including TATA-box, CAAT-box and Skn1-motif. In order to further examine its function, three different 5'-deletion fragments were inserted into pBI101.3, a plant expression vector harboring the LPAAT upstream sequence, leading to pBI101.3-L1, pBI101.3-L2 and pBI101.3-L3, respectively. We obtained transgenic plants of rice by Agrobacterium-mediated callus transformation and plant regeneration and detected the expression of gus gene by histochemical staining and fluorometric determination. We found that gus gene driven by the three deletion fragments was specifically expressed in the endosperm of rice seeds, but not in the empty vector of pBI101.3 and other tissues. The highest expression level of GUS was at 15 DAF in pBI101.3-L3 and pBI101.3-L2 transgenic lines, while the same level was detected at 10 DAF in pBI101.3-L1. The expression driven by the whole fragment was up to 1.76- and 2.8-fold higher than those driven by the -817 bp and -453 bp upstream fragments, and 10.7-fold higher than that driven by the vector without the promoter. Taken together, our results strongly suggest that these promoter fragments from coconut have a significant potential in genetically improving endosperm in main crops.

Drug-Encoded Biomarkers for Monitoring Biological Therapies

PubMed Central

Bedenk, Kristina; Zhang, Qian; Frentzen, Alexa; Cappello, Joseph; Fischer, Utz; Szalay, Aladar A.

2015-01-01

Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers. PMID:26348361

The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression.

PubMed

Zhou, Fei; Sun, Tian-Hu; Zhao, Lei; Pan, Xi-Wu; Lu, Shan

2015-01-01

The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, -421 bp from the translation initiation site) which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA), we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS) transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD) and constant dark (DD) conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC) driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant.

A minimal peach type II chlorophyll a/b-binding protein promoter retains tissue-specificity and light regulation in tomato

PubMed Central

Bassett, Carole L; Callahan, Ann M; Artlip, Timothy S; Scorza, Ralph; Srinivasan, Chinnathambi

2007-01-01

Background Promoters with tissue-specificity are desirable to drive expression of transgenes in crops to avoid accumulation of foreign proteins in edible tissues/organs. Several photosynthetic promoters have been shown to be strong regulators of expression of transgenes in light-responsive tissues and would be good candidates for leaf and immature fruit tissue-specificity, if expression in the mature fruit were minimized. Results A minimal peach chlorophyll a/b-binding protein gene (Lhcb2*Pp1) promoter (Cab19) was isolated and fused to an uidA (β-glucuronidase [GUS]) gene containing the PIV2 intron. A control vector carrying an enhanced mas35S CaMV promoter fused to uidA was also constructed. Two different orientations of the Cab19::GUS fusion relative to the left T-DNA border of the binary vector were transformed into tomato. Ten independent regenerants of each construct and an untransformed control line were assessed both qualitatively and quantitatively for GUS expression in leaves, fruit and flowers, and quantitatively in roots. Conclusion The minimal CAB19 promoter conferred GUS activity primarily in leaves and green fruit, as well as in response to light. GUS activity in the leaves of both Cab19 constructs averaged about 2/3 that observed with mas35S::GUS controls. Surprisingly, GUS activity in transgenic green fruit was considerably higher than leaves for all promoter constructs; however, in red, ripe fruit activities were much lower for the Cab19 promoter constructs than the mas35S::GUS. Although GUS activity was readily detectable in flowers and roots of mas35S::GUStransgenic plants, little activity was observed in plants carrying the Cab19 promoter constructs. In addition, the light-inducibility of the Cab19::GUS constructs indicated that all the requisite cis-elements for light responsiveness were contained on the Cab19 fragment. The minimal Cab19 promoter retains both tissue-specificity and light regulation and can be used to drive expression of

Interaction Effects of Season of Birth and Cytokine Genes on Schizotypal Traits in the General Population

PubMed Central

Korovaitseva, Galina I.; Lezheiko, Tatyana V.; Golimbet, Vera E.

2017-01-01

Literature suggests that the effect of winter birth on vulnerability to schizophrenia might be mediated by increased expression of proinflammatory cytokines due to prenatal infection and its inadequate regulation by anti-inflammatory factors. As the response of the immune system depends on genotype, this study assessed the interaction effects of cytokine genes and season of birth (SOB) on schizotypy measured with the Schizotypal Personality Questionnaire (SPQ-74). We searched for associations of IL1B rs16944, IL4 rs2243250, and IL-1RN VNTR polymorphisms, SOB, and their interactions with the SPQ-74 total score in a sample of 278 healthy individuals. A significant effect of the IL4 X SOB interaction was found, p = 0.007 and η2 = 0.028. We confirmed this effect using an extended sample of 373 individuals. Homozygotes CC born in winter showed the highest SPQ total score and differed significantly from winter-born T allele carriers, p = 0.049. This difference was demonstrated for cognitive-perceptual and disorganized but not interpersonal dimensions. The findings are consistent with the hypothesis that the cytokine genes by SOB interaction can influence variability of schizotypal traits in the general population. The IL4 T allele appeared to have a protective effect against the development of positive and disorganized schizotypal traits in winter-born individuals. PMID:29464121

Interaction Effects of Season of Birth and Cytokine Genes on Schizotypal Traits in the General Population.

PubMed

Alfimova, Margarita V; Korovaitseva, Galina I; Lezheiko, Tatyana V; Golimbet, Vera E

2017-01-01

Literature suggests that the effect of winter birth on vulnerability to schizophrenia might be mediated by increased expression of proinflammatory cytokines due to prenatal infection and its inadequate regulation by anti-inflammatory factors. As the response of the immune system depends on genotype, this study assessed the interaction effects of cytokine genes and season of birth (SOB) on schizotypy measured with the Schizotypal Personality Questionnaire (SPQ-74). We searched for associations of IL1B rs16944, IL4 rs2243250, and IL-1RN VNTR polymorphisms, SOB, and their interactions with the SPQ-74 total score in a sample of 278 healthy individuals. A significant effect of the IL4 X SOB interaction was found, p = 0.007 and η 2 = 0.028. We confirmed this effect using an extended sample of 373 individuals. Homozygotes CC born in winter showed the highest SPQ total score and differed significantly from winter-born T allele carriers, p = 0.049. This difference was demonstrated for cognitive-perceptual and disorganized but not interpersonal dimensions. The findings are consistent with the hypothesis that the cytokine genes by SOB interaction can influence variability of schizotypal traits in the general population. The IL4 T allele appeared to have a protective effect against the development of positive and disorganized schizotypal traits in winter-born individuals.

A role for the gene regulatory module microRNA172/TARGET OF EARLY ACTIVATION TAGGED 1/FLOWERING LOCUS T (miRNA172/TOE1/FT) in the feeding sites induced by Meloidogyne javanica in Arabidopsis thaliana.

PubMed

Díaz-Manzano, Fernando E; Cabrera, Javier; Ripoll, Juan-José; Del Olmo, Iván; Andrés, Mari Fe; Silva, Ana Cláudia; Barcala, Marta; Sánchez, María; Ruíz-Ferrer, Virginia; de Almeida-Engler, Janice; Yanofsky, Martin F; Piñeiro, Manuel; Jarillo, Jose Antonio; Fenoll, Carmen; Escobar, Carolina

2018-01-01

Root knot nematodes (RKNs) penetrate into the root vascular cylinder, triggering morphogenetic changes to induce galls, de novo formed 'pseudo-organs' containing several giant cells (GCs). Distinctive gene repression events observed in early gall/GCs development are thought to be mediated by post-transcriptional silencing via microRNAs (miRNAs), a process that is far from being fully characterized. Arabidopsis thaliana backgrounds with altered activities based on target 35S::MIMICRY172 (MIM172), 35S::TARGET OF EARLY ACTIVATION TAGGED 1 (TOE1)-miR172-resistant (35S::TOE1 R ) and mutant (flowering locus T-10 (ft-10)) lines were used for functional analysis of nematode infective and reproductive parameters. The GUS-reporter lines, MIR172A-E::GUS, treated with auxin (IAA) and an auxin-inhibitor (a-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA)), together with the MIR172C AuxRE::GUS line with two mutated auxin responsive elements (AuxREs), were assayed for nematode-dependent gene expression. Arabidopsis thaliana backgrounds with altered expression of miRNA172, TOE1 or FT showed lower susceptibility to the RKNs and smaller galls and GCs. MIR172C-D::GUS showed restricted promoter activity in galls/GCs that was regulated by auxins through auxin-responsive factors. IAA induced their activity in galls while PEO-IAA treatment and mutations in AuxRe motifs abolished it. The results showed that the regulatory module miRNA172/TOE1/FT plays an important role in correct GCs and gall development, where miRNA172 is modulated by auxins. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Expression of a polyubiquitin promoter isolated from Gladiolus.

PubMed

Joung, Young Hee; Kamo, Kathryn

2006-10-01

A polyubiquitin promoter (GUBQ1) including its 5'UTR and intron was isolated from the floral monocot Gladiolus because high levels of expression could not be obtained using publicly available promoters isolated from either cereals or dicots. Sequencing of the promoter revealed highly conserved 5' and 3' intron splicing sites for the 1.234 kb intron. The coding sequence of the first two ubiquitin genes showed the highest homology (87 and 86%, respectively) to the ubiquitin genes of Nicotiana tabacum and Oryza sativa RUBQ2. Transient expression following gene gun bombardment showed that relative levels of GUS activity with the GUBQ1 promoter were comparable to the CaMV 35S promoter in gladiolus, tobacco, rose, rice, and the floral monocot freesia. The highest levels of GUS expression with GUBQ1 were attained with Gladiolus. The full-length GUBQ1 promoter including 5'UTR and intron were necessary for maximum GUS expression in Gladiolus. The relative GUS activity for the promoter only was 9%, and the activity for the promoter with 5'UTR and 399 bp of the full-length 1.234 kb intron was 41%. Arabidopsis plants transformed with uidA under GUBQ1 showed moderate GUS expression throughout young leaves and in the vasculature of older leaves. The highest levels of transient GUS expression in Gladiolus have been achieved using the GUBQ1 promoter. This promoter should be useful for genetic engineering of disease resistance in Gladiolus, rose, and freesia, where high levels of gene expression are important.

In vivo evaluation of [18F]FEAnGA-Me: a PET tracer for imaging β-glucuronidase (β-GUS) activity in a tumor/inflammation rodent model.

PubMed

Antunes, Inês F; Haisma, Hidde J; Elsinga, Philip H; Sijbesma, Jurgen W A; Waarde, Aren van; Willemsen, Antoon T M; Dierckx, Rudi A; de Vries, Erik F J

2012-08-01

The PET tracer, 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-β-d-glucopyronuronate ([(18)F]FEAnGA), was recently developed for PET imaging of extracellular β-glucuronidase (β-GUS). However, [(18)F]FEAnGA exhibited rapid renal clearance, which resulted in a relatively low tracer uptake in the tumor. To improve the pharmacokinetics of [(18)F]FEAnGA, we developed its more lipophilic methyl ester analog, [(18)F]FEAnGA-Me. [(18)F]FEAnGA-Me was obtained by alkylation of the O-protected glucuronide methyl ester precursor with [(18)F]-fluoroethylamine ([(18)F]FEA), followed by removal of the acetate protecting groups with NaOMe/MeOH. The PET tracer was evaluated by in vitro and in vivo studies. [(18)F]FEAnGA-Me was obtained in 5%-10% overall radiochemical yield. It is 10-fold less hydrophilic than [(18)F]FEAnGA and it is stable in PBS and in the presence of β-GUS for 1 h. However, in the presence of esterase or plasma [(18)F]FEAnGA-Me is converted to [(18)F]FEAnGA, and subsequently converted to [(18)F]FEA by β-GUS. MicroPET studies in Wistar rats bearing a C6 glioma and a sterile inflammation showed similar uptake in tumors after injection of either [(18)F]FEAnGA-Me or [(18)F]FEAnGA. Both tracers had a rapid two-phase clearance of total plasma radioactivity with a half-life of 1 and 8 min. The [(18)F]FEAnGA fraction generated from [(18)F]FEAnGA-Me by in vivo hydrolysis had a circulation half-life of 1 and 11 min in plasma. Similar distribution volume in the viable part of the tumor was found after injection of either [(18)F]FEAnGA-Me or [(18)F]FEAnGA. The imaging properties of [(18)F]FEAnGA-Me were not significantly better than those of [(18)F]FEAnGA. Therefore, other strategies should be applied in order to improve the kinetics of these tracers. Copyright © 2012 Elsevier Inc. All rights reserved.

Cloning and functional analysis of the promoters that upregulate carotenogenic gene expression during flower development in Gentiana lutea.

PubMed

Zhu, Changfu; Yang, Qingjie; Ni, Xiuzhen; Bai, Chao; Sheng, Yanmin; Shi, Lianxuan; Capell, Teresa; Sandmann, Gerhard; Christou, Paul

2014-04-01

Over the last two decades, many carotenogenic genes have been cloned and used to generate metabolically engineered plants producing higher levels of carotenoids. However, comparatively little is known about the regulation of endogenous carotenogenic genes in higher plants, and this restricts our ability to predict how engineered plants will perform in terms of carotenoid content and composition. During petal development in the Great Yellow Gentian (Gentiana lutea), carotenoid accumulation, the formation of chromoplasts and the upregulation of several carotenogenic genes are temporally coordinated. We investigated the regulatory mechanisms responsible for this coordinated expression by isolating five G. lutea carotenogenic gene (GlPDS, GlZDS, GlLYCB, GlBCH and GlLYCE) promoters by inverse polymerase chain reaction (PCR). Each promoter was sufficient for developmentally regulated expression of the gusA reporter gene following transient expression in tomato (Solanum lycopersicum cv. Micro-Tom). Interestingly, the GlLYCB and GlBCH promoters drove high levels of gusA expression in chromoplast-containing mature green fruits, but low levels in chloroplast-containing immature green fruits, indicating a strict correlation between promoter activity, tomato fruit development and chromoplast differentiation. As well as core promoter elements such as TATA and CAAT boxes, all five promoters together with previously characterized GlZEP promoter contained three common cis-regulatory motifs involved in the response to methyl jasmonate (CGTCA) and ethylene (ATCTA), and required for endosperm expression (Skn-1_motif, GTCAT). These shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation. Our data provide insight into the regulatory basis of the coordinated upregulation of carotenogenic gene expression during flower development in G. lutea. © 2013 Scandinavian Plant Physiology Society.

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 mediates environmental stress responses in plants.

PubMed

Hong, Jeum Kyu; Hwang, Byung Kook

2009-01-01

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 was analyzed by an Agrobacterium-mediated transient expression assay in tobacco leaves. Several stress-related cis-acting elements (GT-1, W-box and ABRE) are located within the CaPIMP1 promoter. In tobacco leaf tissues transiently transformed with a CaPIMP1 promoter-beta-glucuronidase (GUS) gene fusion, serially 5'-deleted CaPIMP1 promoters were differentially activated by Pseudomonas syringae pv. tabaci, ethylene, methyl jasmonate, abscisic acid, and nitric oxide. The -1,193 bp region of the CaPIMP1 gene promoter sequence exhibited full promoter activity. The -417- and -593 bp promoter regions were sufficient for GUS gene activation by ethylene and methyl jasmonate treatments, respectively. However, CaPIMP1 promoter sequences longer than -793 bp were required for promoter activation by abscisic acid and sodium nitroprusside treatments. CaPIMP1 expression was activated in pepper leaves by treatment with ethylene, methyl jasmonate, abscisic acid, beta-amino-n-butyric acid, NaCl, mechanical wounding, and low temperature, but not with salicylic acid. Overexpression of CaPIMP1 in Arabidopsis conferred hypersensitivity to mannitol, NaCl, and ABA during seed germination but not during seedling development. In contrast, transgenic plants overexpressing CaPIMP1 exhibited enhanced tolerance to oxidative stress induced by methyl viologen during germination and early seedling stages. These results suggest that CaPIMP1 expression may alter responsiveness to environmental stress, as well as to pathogen infection.

The promoter of an A9 homolog from the conifer Cryptomeria japonica imparts male strobilus-dominant expression in transgenic trees.

PubMed

Kurita, Manabu; Konagaya, Ken-ichi; Watanabe, Atsushi; Kondo, Teiji; Ishii, Katsuaki; Taniguchi, Toru

2013-02-01

KEY MESSAGE : GUS analysis in Cryptomeria japonica revealed that the CjMALE1 promoter is activated in the male strobilus of C. japonica. Toward the development of male sterile technology for Cryptomeria japonica, a male strobilus-dominant promoter of C. japonica was isolated. The CjMALE1 gene was isolated from a male strobilus-specific suppression subtractive hybridization (SSH) library, and the promoter was isolated by the TAIL-PCR method. To characterize the CjMALE1 promoter, β-glucuronidase (GUS)-fused genes were constructed and introduced into C. japonica using Agrobacterium tumefaciens. GUS expression from CjMALE1-2.5 K (2,718 bp fragment)::GUS C. japonica and CjMALE1-1 K (1,029 bp fragment)::GUS C. japonica was detected in the tapetum and microspore mother cells. These promoter fragments were comparably active in the pre-meiotic stage of the male strobilus of C. japonica. Our analysis showed that the 1,029 bp promoter had all the cis-elements necessary for male strobilus-dominant expression of CjMALE1. When CjMALE1-1 K::GUS was introduced into Arabidopsis, GUS expression was detected in the same spatiotemporal pattern as in C. japonica. These results suggest that the CjMALE1 promoter is subject to transcriptional regulatory systems consisting of cis- and trans-elements that have been highly conserved during evolution.

Gene-for-genes interactions between cotton R genes and Xanthomonas campestris pv. malvacearum avr genes.

PubMed

De Feyter, R; Yang, Y; Gabriel, D W

1993-01-01

Six plasmid-borne avirulence (avr) genes were previously cloned from strain XcmH of the cotton pathogen, Xanthomonas campestris pv. malvacearum. We have now localized all six avr genes on the cloned fragments by subcloning and Tn5-gusA insertional mutagenesis. None of these avr genes appeared to exhibit exclusively gene-for-gene patterns of interactions with cotton R genes, and avrB4 was demonstrated to confer avr gene-for-R genes (plural) avirulence to X. c. pv. malvacearum on congenic cotton lines carrying either of two different resistance loci, B1 or B4. Furthermore, the B1 locus appeared to confer R gene-for-avr genes resistance to cotton against isogenic X. c. pv. malvacearum strains carrying any one of three avr genes: avrB4, avrb6, or avrB102. Restriction enzyme, Southern blot hybridization, and DNA sequence analyses showed that the XcmH avr genes are all highly similar to each other, to avrBs3 and avrBsP from the pepper pathogen X. c. pv. vesicatoria, and to the host-specific virulence gene pthA from the citrus pathogen X. citri. The XcmH avr genes differed primarily in the multiplicity of a tandemly repeated 102-base pair motif within the central portions of the genes, repeated from 14 to 23 times in members of this gene family. The complete nucleotide sequence of avrb6 revealed that it is 97% identical in DNA sequence to avrB4, avrBs3, avrBsP, and pthA and that 62-bp inverted terminal repeats mark the boundaries of homology between avrb6 and all members of this Xanthomonas virulence/avirulence gene family sequenced to date. The terminal 38 bp of both inverted repeats are highly similar to the 38-bp consensus terminal sequence of the Tn3 family of transposons. Up to 11 members of the avr gene family appear to be present in North American strains of X. c. pv. malvacearum, including XcmH. The high level of homology observed among these avr genes and their presence in multiple copies may explain the gene-for-genes interactions and also the observed high

Generation of β-glucuronidase reporter-tagged strain to monitor Ustilaginoidea virens infection in rice.

PubMed

Andargie, Mebeaselassie; Yang, Chao; Li, Jianxiong

2016-12-01

An Agrobacterium-mediated genetic transformation system for the rice false smut fungus Ustilaginoidea virens was developed using conidia as recipients. A binary vector, pCAMBIA1301-P gpdA -GUS-T trpC , was constructed. The gpdA promoter (P gpdA ) from Aspergillus nidulans was used to drive the expression of the β-glucuronidase (GUS) gene which enabled GUS activity visualization. The conidia transformation efficiency reached approximately 110 to 250 transformants per 1×10 5 conidia. Based on the analysis made on five successive generations of subcultures and PCR, the pCAMBIA1301-GUS cassette had integrated into the genomes of all transformants and clearly showed mitotic stability. The novel reporter vector constructed will promote the functional characterization of genes and the construction of genetically engineered strains of this important fungus. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of a Non-Conservative Sequence on the Properties of β-glucuronidase from Aspergillus terreus Li-20

PubMed Central

Liu, Yanli; Huangfu, Jie; Qi, Feng; Kaleem, Imdad; E, Wenwen; Li, Chun

2012-01-01

We cloned the β-glucuronidase gene (AtGUS) from Aspergillus terreus Li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Based on sequence alignment, the C-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence AtGUS(-3t) (1–592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS(-3t) were expressed in E. coli BL21, producing AtGUS-E and AtGUS(-3t)-E, respectively. At the similar optimum temperature (55°C) and pH (AtGUS-E, 6.6; AtGUS(-3t)-E, 7.0) conditions, the thermal stability of AtGUS(-3t)-E was enhanced at 65°C, and the metal ions Co2+, Ca2+ and Ni2+ showed opposite effects on AtGUS-E and AtGUS(-3t)-E, respectively. Furthermore, Km of AtGUS(-3t)-E (1.95 mM) was just nearly one-seventh that of AtGUS-E (12.9 mM), whereas the catalytic efficiency of AtGUS(-3t)-E was 3.2 fold higher than that of AtGUS-E (7.16 vs. 2.24 mM s−1), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of AtGUS. Conformational analysis illustrated significant difference in the secondary structure between AtGUS-E and AtGUS(-3t)-E by circular dichroism (CD). The results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme. PMID:22347419

Biolistic transformation of Scoparia dulcis L.

PubMed

Srinivas, Kota; Muralikrishna, Narra; Kumar, Kalva Bharath; Raghu, Ellendula; Mahender, Aileni; Kiranmayee, Kasula; Yashodahara, Velivela; Sadanandam, Abbagani

2016-01-01

Here, we report for the first time, the optimized conditions for microprojectile bombardment-mediated genetic transformation in Vassourinha (Scoparia dulcis L.), a Plantaginaceae medicinal plant species. Transformation was achieved by bombardment of axenic leaf segments with Binary vector pBI121 harbouring β-glucuronidase gene (GUS) as a reporter and neomycin phosphotransferase II gene (npt II) as a selectable marker. The influence of physical parameters viz., acceleration pressure, flight distance, gap width & macroprojectile travel distance of particle gun on frequency of transient GUS and stable (survival of putative transformants) expressions have been investigated. Biolistic delivery of the pBI121 yielded the best (80.0 %) transient expression of GUS gene bombarded at a flight distance of 6 cm and rupture disc pressure/acceleration pressure of 650 psi. Highest stable expression of 52.0 % was noticed in putative transformants on RMBI-K medium. Integration of GUS and npt II genes in the nuclear genome was confirmed through primer specific PCR. DNA blot analysis showed more than one transgene copy in the transformed plantlet genomes. The present study may be used for metabolic engineering and production of biopharmaceuticals by transplastomic technology in this valuable medicinal plant.

Developmental and environmental regulation of the Nicotiana plumbaginifolia cytosolic Cu/Zn-superoxide dismutase promoter in transgenic tobacco.

PubMed Central

Hérouart, D; Van Montagu, M; Inzé, D

1994-01-01

Superoxide dismutases (SODs) play a key role in the cellular defense against reactive oxygen species. To study the transcriptional regulation at the cellular level, the promoter of the Nicotiana plumbaginifolia cytosolic gene encoding Cu/ZnSOD (SODCc) was fused to the beta-glucuronidase (GUS) reporter gene (gusA) and analyzed in transgenic tobacco plants. The promoter was highly active in vascular bundles of leaves and stems, where it is confined to phloem cells. In flowers, GUS activity was detected in ovules and pollen grains, in pigmented tissues of petals, and in vascular tissue of ovaries and anthers. In response to treatment with the superoxide-generating herbicide paraquat, very strong GUS staining was observed in photosynthetically active cells of leaves and in some epidermal root cells of seedlings. The expression of the SODCc-gusA was also induced in seedlings after heat shock and chilling and after treatment with sulfhydryl antioxidants such as reduced glutathione and cysteine. It is postulated that SODCc expression is directly linked to a cell-specific production of excess superoxide radicals in the cytosol. PMID:8165260

Developmental and environmental regulation of the Nicotiana plumbaginifolia cytosolic Cu/Zn-superoxide dismutase promoter in transgenic tobacco.

PubMed

Hérouart, D; Van Montagu, M; Inzé, D

1994-03-01

Superoxide dismutases (SODs) play a key role in the cellular defense against reactive oxygen species. To study the transcriptional regulation at the cellular level, the promoter of the Nicotiana plumbaginifolia cytosolic gene encoding Cu/ZnSOD (SODCc) was fused to the beta-glucuronidase (GUS) reporter gene (gusA) and analyzed in transgenic tobacco plants. The promoter was highly active in vascular bundles of leaves and stems, where it is confined to phloem cells. In flowers, GUS activity was detected in ovules and pollen grains, in pigmented tissues of petals, and in vascular tissue of ovaries and anthers. In response to treatment with the superoxide-generating herbicide paraquat, very strong GUS staining was observed in photosynthetically active cells of leaves and in some epidermal root cells of seedlings. The expression of the SODCc-gusA was also induced in seedlings after heat shock and chilling and after treatment with sulfhydryl antioxidants such as reduced glutathione and cysteine. It is postulated that SODCc expression is directly linked to a cell-specific production of excess superoxide radicals in the cytosol.

A mechanism for negative gene regulation in Autographa californica multinucleocapsid nuclear polyhedrosis virus

USGS Publications Warehouse

Leisy, D.J.; Rasmussen, C.; Owusu, E.O.; Rohrmann, G.F.

1997-01-01

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-1) is thought to play a central role in stimulating early viral transcription. IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulate the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2. Our results indicate that IE-1 negatively regulates the expression of certain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') near their mRNA start sites. The IE-1 binding motif was also found within the palindromic sequences of AcMNPV homologous repeat (hr) regions that have been shown to bind IE-1. The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by introducing mutations in these promoters in which the central 6 bp were replaced with Bg/II sites. GUS reporter constructs containing ie-2 and pe-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containing plasmid (ple-1). Comparisons of GUS expression produced by the mutant and wild-type constructs demonstrated that the IE-1 binding motif mediated a significant decrease in expression from the ie-2 and pe-38 promoters in response to increasing pIe-1 concentrations. Electrophoretic mobility shift assays with pIe-1-transfected cell extracts and supershift assays with IE-1- specific antiserum demonstrated that IE-1 binds to promoter fragments containing the IE-1 binding motif but does not bind to promoter fragments lacking this motif.

The tonoplast intrinsic aquaporin (TIP) subfamily of Eucalyptus grandis: Characterization of EgTIP2, a root-specific and osmotic stress-responsive gene.

PubMed

Rodrigues, Marcela I; Bravo, Juliana P; Sassaki, Flávio T; Severino, Fábio E; Maia, Ivan G

2013-12-01

Aquaporins have important roles in various physiological processes in plants, including growth, development and adaptation to stress. In this study, a gene encoding a root-specific tonoplast intrinsic aquaporin (TIP) from Eucalyptus grandis (named EgTIP2) was investigated. The root-specific expression of EgTIP2 was validated over a panel of five eucalyptus organ/tissues. In eucalyptus roots, EgTIP2 expression was significantly induced by osmotic stress imposed by PEG treatment. Histochemical analysis of transgenic tobacco lines (Nicotiana tabacum SR1) harboring an EgTIP2 promoter:GUS reporter cassette revealed major GUS staining in the vasculature and in root tips. Consistent with its osmotic-stress inducible expression in eucalyptus, EgTIP2 promoter activity was up-regulated by mannitol treatment, but was down-regulated by abscisic acid. Taken together, these results suggest that EgTIP2 might be involved in eucalyptus response to drought. Additional searches in the eucalyptus genome revealed the presence of four additional putative TIP coding genes, which could be individually assigned to the classical TIP1-5 groups. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Expression Patterns Conferred by Tyrosine/Dihydroxyphenylalanine Decarboxylase Promoters from Opium Poppy Are Conserved in Transgenic Tobacco1

PubMed Central

Facchini, Peter J.; Penzes-Yost, Catherine; Samanani, Nailish; Kowalchuk, Brett

1998-01-01

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the β-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species. PMID:9733527

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest

PubMed Central

Ji, Hyeonso; Kim, Sung-Ryul; Kim, Yul-Ho; Suh, Jung-Pil; Park, Hyang-Mi; Sreenivasulu, Nese; Misra, Gopal; Kim, Suk-Man; Hechanova, Sherry Lou; Kim, Hakbum; Lee, Gang-Seob; Yoon, Ung-Han; Kim, Tae-Ho; Lim, Hyemin; Suh, Suk-Chul; Yang, Jungil; An, Gynheung; Jena, Kshirod K.

2016-01-01

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars. PMID:27682162

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest.

PubMed

Ji, Hyeonso; Kim, Sung-Ryul; Kim, Yul-Ho; Suh, Jung-Pil; Park, Hyang-Mi; Sreenivasulu, Nese; Misra, Gopal; Kim, Suk-Man; Hechanova, Sherry Lou; Kim, Hakbum; Lee, Gang-Seob; Yoon, Ung-Han; Kim, Tae-Ho; Lim, Hyemin; Suh, Suk-Chul; Yang, Jungil; An, Gynheung; Jena, Kshirod K

2016-09-29

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars.

Characterization of the Dioscorin Gene Family in Dioscorea alata Reveals a Role in Tuber Development and Environmental Response.

PubMed

Liu, Linya; Huang, Yacheng; Huang, Xiaolong; Yang, Jianghua; Wu, Wenqiang; Xu, Yun; Cong, Ziwen; Xie, Jun; Xia, Wei; Huang, Dongyi

2017-07-20

Dioscorin is one of the major soluble proteins in yam tubers. Unlike other well-known plant storage proteins, such as patatin and sporamin, dioscorin is argued for its function as storage proteins, and the molecular mechanisms underlying its expressional complexity are little understood. In this study, we isolated five dioscorin genes from Dioscorea alata L., comprising three class A ( Da-dio1 , - 3 and - 4 ) and two class B ( Da-dio2 and - 5 ) isoforms. Expressions of all dioscorin genes gradually decreased in mother tubers during yam sprouting and regrowth. On the other hand, all dioscorin genes accumulated transcripts progressively with tuber development in new tubers, with Da-dio5 being the most prominent isoform. In yam leaves, the expressions of Da-dio 5 were up-regulated by the treatments of five phytohormones (gibberellic acid, salicylic acid, indole-3-acetic acid, abscisic acid, and ethylene), and three abiotic stresses (high-temperature, low-temperature and drought). To further elucidate the regulatory mechanisms of Da-dio5 expressions, transgenic Arabidopsis plants harboring the Da-dio5 promoter-β-glucuronidase (GUS) fusion were generated. GUS staining showed that expressions of the Da-dio5 promoter were detected mainly in the shoot apical meristem (SAM) and hypocotyls, and enhanced by the treatments of the five hormones, and the three abiotic stresses mentioned above. These results suggest diverse roles of Da-dio5 in yam sprouting, regrowth, and tuberization, as well as in response to enviromental cues.

Identification of a 467 bp Promoter of Maize Phosphatidylinositol Synthase Gene (ZmPIS) Which Confers High-Level Gene Expression and Salinity or Osmotic Stress Inducibility in Transgenic Tobacco

PubMed Central

Zhang, Hongli; Hou, Jiajia; Jiang, Pingping; Qi, Shoumei; Xu, Changzheng; He, Qiuxia; Ding, Zhaohua; Wang, Zhiwu; Zhang, Kewei; Li, Kunpeng

2016-01-01

Salinity and drought often affect plant growth and crop yields. Cloning and identification of salinity and drought stress inducible promoters is of great significance for their use in the genetic improvement of crop resistance. Previous studies showed that phosphatidylinositol synthase is involved in plant salinity and drought stress responses but its promoter has not been characterized by far. In the study, the promoter (pZmPIS, 1834 bp upstream region of the translation initiation site) was isolated from maize genome. To functionally validate the promoter, eight 5′ deletion fragments of pZmPIS in different lengths were fused to GUS to produce pZmPIS::GUS constructs and transformed into tobacco, namely PZ1–PZ8. The transcription activity and expression pattern obviously changed when the promoter was truncated. Previous studies have demonstrated that NaCl and PEG treatments are usually used to simulate salinity and drought treatments. The results showed that PZ1–PZ7 can respond well upon NaCl and PEG treatments, while PZ8 not. PZ7 (467 bp) displayed the highest transcription activity in all tissues of transgenic tobacco amongst 5′ deleted promoter fragments, which corresponds to about 20 and 50% of CaMV35S under normal and NaCl or PEG treatment, respectively. This implied that PZ7 is the core region of pZmPIS which confers high-level gene expression and NaCl or PEG inducible nature. The 113 bp segment between PZ7 and PZ8 (-467 to -355 bp) was considered as the key sequence for ZmPIS responding to NaCl or PEG treatment. GUS transient assay in tobacco leaves showed that this segment was sufficient for the NaCl or PEG stress response. Bioinformatic analysis revealed that the 113 bp sequence may contain new elements that are crucial for ZmPIS response to NaCl or PEG stress. These results promote our understanding on transcriptional regulation mechanism of ZmPIS and the characterized PZ7 promoter fragment would be an ideal candidate for the overexpression of

Analysis of tomato plasma membrane H(+)-ATPase gene family suggests a mycorrhiza-mediated regulatory mechanism conserved in diverse plant species.

PubMed

Liu, Junli; Liu, Jianjian; Chen, Aiqun; Ji, Minjie; Chen, Jiadong; Yang, Xiaofeng; Gu, Mian; Qu, Hongye; Xu, Guohua

2016-10-01

In plants, the plasma membrane H(+)-ATPase (HA) is considered to play a crucial role in regulating plant growth and respoding to environment stresses. Multiple paralogous genes encoding different isozymes of HA have been identified and characterized in several model plants, while limited information of the HA gene family is available to date for tomato. Here, we describe the molecular and expression features of eight HA-encoding genes (SlHA1-8) from tomato. All these genes are interrupted by multiple introns with conserved positions. SlHA1, 2, and 4 were widely expressed in all tissues, while SlHA5, 6, and 7 were almost only expressed in flowers. SlHA8, the transcripts of which were barely detectable under normal or nutrient-/salt-stress growth conditions, was strongly activated in arbuscular mycorrhizal (AM) fungal-colonized roots. Extreme lack of SlHA8 expression in M161, a mutant defective to AM fungal colonization, provided genetic evidence towards the dependence of its expression on AM symbiosis. A 1521-bp SlHA8 promoter could direct the GUS reporter expression specifically in colonized cells of transgenic tobacco, soybean, and rice mycorrhizal roots. Promoter deletion assay revealed a 223-bp promoter fragment of SlHA8 containing a variant of AM-specific cis-element MYCS (vMYCS) sufficient to confer the AM-induced activity. Targeted deletion of this motif in the corresponding promoter region causes complete abolishment of GUS staining in mycorrhizal roots. Together, these results lend cogent evidence towards the evolutionary conservation of a potential regulatory mechanism mediating the activation of AM-responsive HA genes in diverse mycorrhizal plant species.

Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens.

PubMed

Eklund, D Magnus; Thelander, Mattias; Landberg, Katarina; Ståldal, Veronika; Nilsson, Anders; Johansson, Monika; Valsecchi, Isabel; Pederson, Eric R A; Kowalczyk, Mariusz; Ljung, Karin; Ronne, Hans; Sundberg, Eva

2010-04-01

The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.

Transgenic plants of Petunia hybrida harboring the CYP2E1 gene efficiently remove benzene and toluene pollutants and improve resistance to formaldehyde

PubMed Central

Zhang, Daoxiang; Xiang, Taihe; Li, Peihan; Bao, Lumin

2011-01-01

The CYP2E1 protein belongs to the P450 enzymes family and plays an important role in the metabolism of small molecular and organic pollutants. In this study we generated CYP2E1 transgenic plants of Petunia using Agrobacterium rhizogenes K599. PCR analysis confirmed that the regenerated plants contained the CYP2E1 transgene and the rolB gene of the Ri plasmid. Southern blotting revealed the presence of multiple copies of CYP2E1 in the genome of transgenic plants. Fluorescent quantitative PCR revealed exogenous CYP2E1 gene expression in CYP2E1 transgenic plants at various levels, whereas no like expression was detected in either GUS transgenic plants or wild-types. The absorption of benzene and toluene by transgenic plants was analyzed through quantitative gas chromatography. Transgenic plants with high CYP2E1 expression showed a significant increase in absorption capacity of environmental benzene and toluene, compared to control GUS transgenic and wild type plants. Furthermore, these plants also presented obvious improved resistance to formaldehyde. This study, besides being the first to reveal that the CYP2E1 gene enhances plant resistance to formaldehyde, also furnishes a new method for reducing pollutants, such as benzene, toluene and formaldehyde, by using transgenic flowering horticultural plants. PMID:22215968

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

Bioreactor performance and functional gene analysis of microbial community in a limited-oxygen fed bioreactor for co-reduction of sulfate and nitrate with high organic input.

PubMed

Xu, Xi-jun; Chen, Chuan; Wang, Ai-jie; Yu, Hao; Zhou, Xu; Guo, Hong-liang; Yuan, Ye; Lee, Duu-jong; Zhou, Jizhong; Ren, Nan-qi

2014-08-15

Limited-oxygen mediated synergistic relationships between sulfate-reducing bacteria (SRB), nitrate-reducing bacteria (NRB) and sulfide-oxidizing bacteria (SOB, including nitrate-reducing, sulfide-oxidizing bacteria NR-SOB) were predicted to simultaneously remove contaminants of nitrate, sulfate and high COD, and eliminate sulfide generation. A lab-scale experiment was conducted to examine the impact of limited oxygen on these oxy-anions degradation, sulfide oxidation and associated microbial functional responses. In all scenarios tested, the reduction of both nitrate and sulfate was almost complete. When limited-oxygen was fed into bioreactors, S(0) formation was significantly improved up to ∼ 70%. GeoChip 4.0, a functional gene microarray, was used to determine the microbial gene diversity and functional potential for nitrate and sulfate reduction, and sulfide oxidation. The diversity of the microbial community in bioreactors was increased with the feeding of limited oxygen. Whereas the intensities of the functional genes involved in sulfate reduction did not show a significant difference, the abundance of the detected denitrification genes decreased in limited oxygen samples. More importantly, sulfide-oxidizing bacteria may alter their populations/genes in response to limited oxygen potentially to function more effectively in sulfide oxidation, especially to elemental sulfur. The genes fccA/fccB from nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB), such as Paracoccus denitrificans, Thiobacillus denitrificans, Beggiatoa sp., Thiomicrospira sp., and Thioalkalivibrio sp., were more abundant under limited-oxygen condition. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of a Nicotiana plumbaginifolia plasma membrane H(+)-ATPase gene expressed in the pollen tube.

PubMed

Lefebvre, Benoit; Arango, Miguel; Oufattole, Mohammed; Crouzet, Jérôme; Purnelle, Bénédicte; Boutry, Marc

2005-08-01

In Nicotiana plumbaginifolia, plasma membrane H(+)-ATPases (PMAs) are encoded by a gene family of nine members. Here, we report on the characterization of a new isogene, NpPMA5 (belonging to subfamily IV), and the determination of its expression pattern using the beta-glucuronidase (gusA) reporter gene. pNpPMA5-gusA was expressed in cotyledons, in vascular tissues of the stem (mainly in nodal zones), and in the flower and fruit. In the flower, high expression was found in the pollen tube after in vitro or in vivo germination. Northern blotting analysis confirmed that NpPMA5 was expressed in the pollen tube contrary to NpPMA2 (subfamily I) or NpPMA4 (subfamily II), two genes highly expressed in other tissues. The subcellular localization of PM H(+)-ATPase in the pollen tube was analyzed by immunocytodecoration. As expected, this enzyme was localized to the plasma membrane. However, neither the tip nor the base of the pollen tube was labeled, showing an asymmetrical distribution of this enzyme. This observation supports the hypothesis that the PM H(+)-ATPase is involved in creating the pH gradient that is observed along the pollen tube and is implicated in cell elongation. Compared to other plant PM H(+)-ATPases, the C-terminal region of NpPMA5 is shorter by 26 amino acid residues and is modified in the last 6 residues, due to a sequence rearrangement, which was also found in the orthologous gene of Nicotiana glutinosa, a Nicotiana species distant from N. plumbaginifolia and Petunia hybrida and Lycopersicon esculentum, other Solanacae species. This modification alters part of the PM H(+)-ATPase regulatory domain and raises the question whether this isoform is still regulated.

Molecular cloning and characterization of the light-regulation and circadian-rhythm of the VDE gene promoter from Zingiber officinale.

PubMed

Zhao, Wenchao; Wang, Shaohui; Li, Xin; Huang, Hongyu; Sui, Xiaolei; Zhang, Zhenxian

2012-08-01

Ginger (Zingiber officinale Rosc.) is prone to photoinhibition under intense sunlight. Excessive light can be dissipated by the xanthophyll cycle, where violaxanthin de-epoxidase (VDE) plays a critical role in protecting the photosynthesis apparatus from the damage of excessive light. We isolated ~2.0 kb of ginger VDE (GVDE) gene promoter, which contained the circadian box, I-box, G-box and GT-1 motif. Histochemical staining of Arabidopsis indicated the GVDE promoter was active in almost all organs, especially green tissues. β-glucuronidase (GUS) activity driven by GVDE promoter was repressed rather than activated by high light. GUS activity was altered by hormones, growth regulators and abiotic stresses, which increased with 2,4-dichlorophenoxyacetic acid and decreased with abscisic acid, salicylic acid, zeatin, salt (sodium chloride) and polyethylene glycol. Interestingly, GUS activities with gibberellin or indole-3-acetic acid increased in the short-term (24 h) and decreased in the long-term (48 and 72 h). Analysis of 5' flank deletion found two crucial functional regions residing in -679 to -833 and -63 to -210. Northern blotting analysis found transcription to be regulated by the endogenous circadian clock. Finally, we found a region necessary for regulating the circadian rhythm and another for the basic promoter activity. Key message A novel promoter, named GVDE promoter, was first isolated and analyzed in this study. We have determined one region crucial for promoter activity and another responsible for keeping circadian rhythms.

Tomato Pathogenesis-related Protein Genes are Expressed in Response to Trialeurodes vaporariorum and Bemisia tabaci Biotype B Feeding

PubMed Central

Puthoff, David P.; Holzer, Frances M.; Perring, Thomas M.

2010-01-01

The temporal and spatial expression of tomato wound- and defense-response genes to Bemisia tabaci biotype B (the silverleaf whitefly) and Trialeurodes vaporariorum (the greenhouse whitefly) feeding were characterized. Both species of whiteflies evoked similar changes in tomato gene expression. The levels of RNAs for the methyl jasmonic acid (MeJA)- or ethylene-regulated genes that encode the basic β-1,3-glucanase (GluB), basic chitinase (Chi9), and Pathogenesis-related protein-1 (PR-1) were monitored. GluB and Chi9 RNAs were abundant in infested leaves from the time nymphs initiated feeding (day 5). In addition, GluB RNAs accumulated in apical non-infested leaves. PR-1 RNAs also accumulated after whitefly feeding. In contrast, the ethylene- and salicylic acid (SA)-regulated Chi3 and PR-4 genes had RNAs that accumulated at low levels and GluAC RNAs that were undetectable in whitefly-infested tomato leaves. The changes in Phenylalanine ammonia lyase5 (PAL5) were variable; in some, but not all infestations, PAL5 RNAs increased in response to whitefly feeding. PAL5 RNA levels increased in response to MeJA, ethylene, and abscisic acid, and declined in response to SA. Transcripts from the wound-response genes, leucine aminopeptidase (LapA1) and proteinase inhibitor 2 (pin2), were not detected following whitefly feeding. Furthermore, whitefly infestation of transgenic LapA1:GUS tomato plants showed that whitefly feeding did not activate the LapA1 promoter, although crushing of the leaf lamina increased GUS activity up to 40 fold. These studies indicate that tomato plants perceive B. tabaci and T. vaporariorum in a manner similar to baterical pathogens and distinct from tissue-damaging insects. PMID:20927641

Expression and function of AtMBD4L, the single gene encoding the nuclear DNA glycosylase MBD4L in Arabidopsis.

PubMed

Nota, Florencia; Cambiagno, Damián A; Ribone, Pamela; Alvarez, María E

2015-06-01

DNA glycosylases recognize and excise damaged or incorrect bases from DNA initiating the base excision repair (BER) pathway. Methyl-binding domain protein 4 (MBD4) is a member of the HhH-GPD DNA glycosylase superfamily, which has been well studied in mammals but not in plants. Our knowledge on the plant enzyme is limited to the activity of the Arabidopsis recombinant protein MBD4L in vitro. To start evaluating MBD4L in its biological context, we here characterized the structure, expression and effects of its gene, AtMBD4L. Phylogenetic analysis indicated that AtMBD4L belongs to one of the seven families of HhH-GPD DNA glycosylase genes existing in plants, and is unique on its family. Two AtMBD4L transcripts coding for active enzymes were detected in leaves and flowers. Transgenic plants expressing the AtMBD4L:GUS gene confined GUS activity to perivascular leaf tissues (usually adjacent to hydathodes), flowers (anthers at particular stages of development), and the apex of immature siliques. MBD4L-GFP fusion proteins showed nuclear localization in planta. Interestingly, overexpression of the full length MBD4L, but not a truncated enzyme lacking the DNA glycosylase domain, induced the BER gene LIG1 and enhanced tolerance to oxidative stress. These results suggest that endogenous MBD4L acts on particular tissues, is capable of activating BER, and may contribute to repair DNA damage caused by oxidative stress. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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.

Regulation and function of the pepper pectin methylesterase inhibitor (CaPMEI1) gene promoter in defense and ethylene and methyl jasmonate signaling in plants.

PubMed

An, Soo Hyun; Choi, Hyong Woo; Hong, Jeum Kyu; Hwang, Byung Kook

2009-11-01

Analysis of the promoters of defense-related genes is valuable for determining stress signaling and transcriptional activation during pathogen infection. Here, we have isolated and functionally characterized the promoter region of the pepper (Capsicum annuum) pectin methylesterase inhibitor 1 (CaPMEI1) gene in transiently transformed tobacco plants and stably transformed Arabidopsis plants. Among four 5' deletion constructs analyzed, the -958-bp CaPMEI1 promoter induced a high level of GUS reporter activity in tobacco leaf tissue, driven by pathogen infection as well as by ethylene and methyl jasmonate (MeJA) treatment. The 204-bp region from -958 bp to -754 bp of the CaPMEI1 promoter is responsible for the stress-responsive expression. In addition, the pepper transcription factor CARAV1 activated the CaPMEI1 promoter in tobacco leaves, whereas the transcription factor CAbZIP1 did not. In the transgenic Arabidopsis plants, the -958 bp CaPMEI1 promoter was functionally regulated by developmental cues, bacterial and oomycete pathogen infections, and treatment with ethylene and MeJA. Histochemical GUS staining analyses of Arabidopsis tissues revealed that the CaPMEI1 promoter was mainly activated in leaf veins in response to various biotic and abiotic stimuli. Together, these results suggest that CaPMEI1 promoter activation may be a critical molecular event for host defense response and ethylene- and MeJA-mediated CaPMEI1 gene expression.

Molecular evolution and transcriptional regulation of the oilseed rape proline dehydrogenase genes suggest distinct roles of proline catabolism during development.

PubMed

Faës, Pascal; Deleu, Carole; Aïnouche, Abdelkader; Le Cahérec, Françoise; Montes, Emilie; Clouet, Vanessa; Gouraud, Anne-Marie; Albert, Benjamin; Orsel, Mathilde; Lassalle, Gilles; Leport, Laurent; Bouchereau, Alain; Niogret, Marie-Françoise

2015-02-01

Six BnaProDH1 and two BnaProDH2 genes were identified in Brassica napus genome. The BnaProDH1 genes are mainly expressed in pollen and roots' organs while BnaProDH2 gene expression is associated with leaf vascular tissues at senescence. Proline dehydrogenase (ProDH) catalyzes the first step in the catabolism of proline. The ProDH gene family in oilseed rape (Brassica napus) was characterized and compared to other Brassicaceae ProDH sequences to establish the phylogenetic relationships between genes. Six BnaProDH1 genes and two BnaProDH2 genes were identified in the B. napus genome. Expression of the three paralogous pairs of BnaProDH1 genes and the two homoeologous BnaProDH2 genes was measured by real-time quantitative RT-PCR in plants at vegetative and reproductive stages. The BnaProDH2 genes are specifically expressed in vasculature in an age-dependent manner, while BnaProDH1 genes are strongly expressed in pollen grains and roots. Compared to the abundant expression of BnaProDH1, the overall expression of BnaProDH2 is low except in roots and senescent leaves. The BnaProDH1 paralogs showed different levels of expression with BnaA&C.ProDH1.a the most strongly expressed and BnaA&C.ProDH1.c the least. The promoters of two BnaProDH1 and two BnaProDH2 genes were fused with uidA reporter gene (GUS) to characterize organ and tissue expression profiles in transformed B. napus plants. The transformants with promoters from different genes showed contrasting patterns of GUS activity, which corresponded to the spatial expression of their respective transcripts. ProDHs probably have non-redundant functions in different organs and at different phenological stages. In terms of molecular evolution, all BnaProDH sequences appear to have undergone strong purifying selection and some copies are becoming subfunctionalized. This detailed description of oilseed rape ProDH genes provides new elements to investigate the function of proline metabolism in plant development.

Cloning and function analysis of an alfalfa (Medicago sativa L.) zinc finger protein promoter MsZPP.

PubMed

Li, Yan; Sun, Yan; Yang, Qingchuan; Kang, Junmei; Zhang, Tiejun; Gruber, Margaret Yvonne; Fang, Feng

2012-08-01

A 1272 bp upstream sequence of MsZFN gene was cloned from alfalfa, which was designed as MsZPP (Genbank accession number: FJ 161979.2) using an adaptor-mediated genome walking method. A sole transcription start site was located 69 bp upstream of the translation start site. Its pattern of expression included roots, stem vascular tissues, floral reproductive organs, and leaves, but the promoter did not express in seeds, petals or sepals. Transcription levels can be stimulated by dark, MeJA, and IAA. However, GUS fusion activities had no change by treatments of GA, ABA, drought and high salt for 3 days. Deletion analysis revealed that all sections of the promoter can drive gus gene expression in the root, stem, leaves and floral reproductive organs; however, only fragments longer than the -460 bp promoter can stimulate strong gus gene expression in these organs. In addition, the -460 bp promoter fragment can drive gus expression not only in the vascular tissue, but also in leaf guard cells. The results suggest that the promoter MsZPP plays roles in the regulation of transgene expression, particularly due to its darkness, MeJA, and IAA responsiveness.

β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes ▿

PubMed Central

Myronovskyi, Maksym; Welle, Elisabeth; Fedorenko, Viktor; Luzhetskyy, Andriy

2011-01-01

Here we describe a versatile and sensitive reporter system for actinomycetes that is based on gusA, which encodes the β-glucuronidase enzyme. A series of gusA-containing transcriptional and translational fusion vectors were constructed and utilized to study the regulatory cascade of the phenalinolactone biosynthetic gene cluster. Furthermore, these vectors were used to study the efficiency of translation initiation at the ATG, GTG, TTG, and CTG start codons. Surprisingly, constructs using a TTG start codon showed the best activity, whereas those using ATG or GTG were approximately one-half or one-third as active, respectively. The CTG fusion showed only 5% of the activity of the TTG fusion. A suicide vector, pKGLP2, carrying gusA in its backbone was used to visually detect merodiploid formation and resolution, making gene targeting in actinomycetes much faster and easier. Three regulatory genes, plaR1, plaR2, and plaR3, involved in phenalinolactone biosynthesis were efficiently replaced with an apramycin resistance marker using this system. Finally, we expanded the genetic code of actinomycetes by introducing the nonproteinogenic amino acid N-epsilon-cyclopentyloxycarbonyl-l-lysine with the GusA protein as a reporter. PMID:21685164

Identification of cis-elements for ethylene and circadian regulation of the Solanum melongena gene encoding cysteine proteinase.

PubMed

Rawat, Reetika; Xu, Zeng-Fu; Yao, Kwok-Ming; Chye, Mee-Len

2005-03-01

We have previously shown that the expression of SmCP which encodes Solanum melongena cysteine proteinase is ethylene-inducible and is under circadian control. To understand the regulation of SmCP, a 1.34-kb SmCP 5'-flanking region and its deletion derivatives were analyzed for cis-elements using GUS and luc fusions and by in vitro binding assays. Analysis of transgenic tobacco transformed with SmCP promoter-GUS constructs confirmed that the promoter region -415/+54 containing Ethylene Responsive Element ERE(-355/-348) conferred threefold ethylene-induction of GUS expression, while -827/+54 which also contains ERE(-683/-676), produced fivefold induction. Using gel mobility shift assays, we demonstrated that each ERE binds nuclear proteins from both ethephon-treated and untreated 5-week-old seedlings, suggesting that different transcriptions factors bind each ERE under varying physiological conditions. Binding was also observed in extracts from senescent, but not young, fruits. The variation in binding at the EREs in fruits and seedlings imply that organ-specific factors may participate in binding. Analysis of transgenic tobacco expressing various SmCP promoter-luc constructs containing wild-type or mutant Evening Elements (EEs) confirmed that both conserved EEs at -795/-787 and -785/-777 are important in circadian control. We confirmed the binding of total nuclear proteins to EEs in gel mobility shift assays and in DNase I footprinting. Our results suggest that multiple proteins bind the EEs which are conserved in plants other than Arabidopsis and that functional EEs and EREs are present in the 5'-flanking region of a gene encoding cysteine proteinase.

A negative regulator encoded by a rice WRKY gene represses both abscisic acid and gibberellins signaling in aleurone cells.

PubMed

Zhang, Zhong-Lin; Shin, Margaret; Zou, Xiaolu; Huang, Jianzhi; Ho, Tun-hua David; Shen, Qingxi J

2009-05-01

Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in aleurone cells represses the expression of two reporter constructs: the beta-glucuronidase gene driven by the GA-inducible Amy32b alpha-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and -71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and -71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling.

Isolation of 4-coumarate Co-A ligase gene promoter from loblolly pine (Pinus taeda) and characterization of tissue-specific activity in transgenic tobacco.

PubMed

Osakabe, Yuriko; Osakabe, Keishi; Chiang, Vincent L

2009-01-01

We characterized promoter activity of a phenylpropanoid biosynthetic gene encoding 4-coumarate Co-A ligase (4CL), Pta4Clalpha, from Pinus taeda. Histochemical- and quantitative assays of GUS expression in the vascular tissue were performed using transgenic tobacco plants expressing promoter-GUS reporters. Deletion analysis of the Pta4Clalpha promoter showed that the region -524 to -252, which has two AC elements, controls the high expression levels in ray-parenchyma cells of older tobacco stems. High activity level of the promoter domain of Pta4CLalpha was also detected in the xylem cells under bending stress. DNA-protein complexes were detected in the reactions of the Pta4CLalpha promoter fragments with the nuclear proteins of xylem of P. taeda. The AC elements in the Pta4CLalpha promoter appeared to have individual roles during xylem development that are activated in a coordinated manner in response to stress in transgenic tobacco.

Introduction of a citrus blight-associated gene into Carrizo citrange [Citrus sinensis (L.) Osbc. x Poncirus trifoliata (L.) Raf.] by Agrobacterium-mediated transformation.

PubMed

Kayim, M; Ceccardi, T L; Berretta, M J G; Barthe, G A; Derrick, K S

2004-11-01

The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous beta-glucuronidase+ (GUS+) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS+ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.

Effects of a petunia scaffold/matrix attachment region on copy number dependency and stability of transgene expression in Nicotiana tabacum.

PubMed

Dietz-Pfeilstetter, Antje; Arndt, Nicola; Manske, Ulrike

2016-04-01

Transgenes in genetically modified plants are often not reliably expressed during development or in subsequent generations. Transcriptional gene silencing (TGS) as well as post-transcriptional gene silencing (PTGS) have been shown to occur in transgenic plants depending on integration pattern, copy number and integration site. In an effort to reduce position effects, to prevent read-through transcription and to provide a more accessible chromatin structure, a P35S-ß-glucuronidase (P35S-gus) transgene flanked by a scaffold/matrix attachment region from petunia (Petun-SAR), was introduced in Nicotiana tabacum plants by Agrobacterium tumefaciens mediated transformation. It was found that Petun-SAR mediates enhanced expression and copy number dependency up to 2 gene copies, but did not prevent gene silencing in transformants with multiple and rearranged gene copies. However, in contrast to the non-SAR transformants where silencing was irreversible and proceeded during long-term vegetative propagation and in progeny plants, gus expression in Petun-SAR plants was re-established in the course of development. Gene silencing was not necessarily accompanied by DNA methylation, while the gus transgene could still be expressed despite considerable CG methylation within the coding region.

Transgene Expression Patterns Indicate That Spaceflight Affects Stress Signal Perception and Transduction in Arabidopsis1

PubMed Central

Paul, Anna-Lisa; Daugherty, Christine J.; Bihn, Elizabeth A.; Chapman, David K.; Norwood, Kelly L.L.; Ferl, Robert J.

2001-01-01

The use of plants as integral components of life support systems remains a cornerstone of strategies for long-term human habitation of space and extraterrestrial colonization. Spaceflight experiments over the past few decades have refined the hardware required to grow plants in low-earth orbit and have illuminated fundamental issues regarding spaceflight effects on plant growth and development. Potential incipient hypoxia, resulting from the lack of convection-driven gas movement, has emerged as a possible major impact of microgravity. We developed transgenic Arabidopsis containing the alcohol dehydrogenase (Adh) gene promoter linked to the β-glucuronidase (GUS) reporter gene to address specifically the possibility that spaceflight induces the plant hypoxia response and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. The staining patterns resulting from a 5-d mission on the orbiter Columbia during mission STS-93 indicate that the Adh/GUS reporter gene was activated in roots during the flight. However, the patterns of expression were not identical to terrestrial control inductions. Moreover, although terrestrial hypoxia induces Adh/GUS expression in the shoot apex, no apex staining was observed in the spaceflight plants. This indicates that either the normal hypoxia response signaling is impaired in spaceflight or that spaceflight inappropriately induces Adh/GUS activity for reasons other than hypoxia. PMID:11402191

Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis.

PubMed

Paul, A L; Daugherty, C J; Bihn, E A; Chapman, D K; Norwood, K L; Ferl, R J

2001-06-01

The use of plants as integral components of life support systems remains a cornerstone of strategies for long-term human habitation of space and extraterrestrial colonization. Spaceflight experiments over the past few decades have refined the hardware required to grow plants in low-earth orbit and have illuminated fundamental issues regarding spaceflight effects on plant growth and development. Potential incipient hypoxia, resulting from the lack of convection-driven gas movement, has emerged as a possible major impact of microgravity. We developed transgenic Arabidopsis containing the alcohol dehydrogenase (Adh) gene promoter linked to the beta-glucuronidase (GUS) reporter gene to address specifically the possibility that spaceflight induces the plant hypoxia response and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. The staining patterns resulting from a 5-d mission on the orbiter Columbia during mission STS-93 indicate that the Adh/GUS reporter gene was activated in roots during the flight. However, the patterns of expression were not identical to terrestrial control inductions. Moreover, although terrestrial hypoxia induces Adh/GUS expression in the shoot apex, no apex staining was observed in the spaceflight plants. This indicates that either the normal hypoxia response signaling is impaired in spaceflight or that spaceflight inappropriately induces Adh/GUS activity for reasons other than hypoxia.

Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis

NASA Technical Reports Server (NTRS)

Paul, A. L.; Daugherty, C. J.; Bihn, E. A.; Chapman, D. K.; Norwood, K. L.; Ferl, R. J.

2001-01-01

The use of plants as integral components of life support systems remains a cornerstone of strategies for long-term human habitation of space and extraterrestrial colonization. Spaceflight experiments over the past few decades have refined the hardware required to grow plants in low-earth orbit and have illuminated fundamental issues regarding spaceflight effects on plant growth and development. Potential incipient hypoxia, resulting from the lack of convection-driven gas movement, has emerged as a possible major impact of microgravity. We developed transgenic Arabidopsis containing the alcohol dehydrogenase (Adh) gene promoter linked to the beta-glucuronidase (GUS) reporter gene to address specifically the possibility that spaceflight induces the plant hypoxia response and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. The staining patterns resulting from a 5-d mission on the orbiter Columbia during mission STS-93 indicate that the Adh/GUS reporter gene was activated in roots during the flight. However, the patterns of expression were not identical to terrestrial control inductions. Moreover, although terrestrial hypoxia induces Adh/GUS expression in the shoot apex, no apex staining was observed in the spaceflight plants. This indicates that either the normal hypoxia response signaling is impaired in spaceflight or that spaceflight inappropriately induces Adh/GUS activity for reasons other than hypoxia.

Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment

NASA Technical Reports Server (NTRS)

Chung, H. J.; Ferl, R. J.

1999-01-01

It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.

High Resolution Crystal Structure of Human β-Glucuronidase Reveals Structural Basis of Lysosome Targeting

PubMed Central

Hassan, Md. Imtaiyaz; Waheed, Abdul; Grubb, Jeffery H.; Klei, Herbert E.; Korolev, Sergey; Sly, William S.

2013-01-01

Human β-glucuronidase (GUS) cleaves β-D-glucuronic acid residues from the non-reducing termini of glycosaminoglycan and its deficiency leads to mucopolysaccharidosis type VII (MPSVII). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases. The structure revealed several new details including a new glycan chain at Asn272, in addition to that previously observed at Asn173, and coordination of the glycan chain at Asn173 with Lys197 of the lysosomal targeting motif which is essential for phosphotransferase recognition. Analysis of the high resolution structure not only provided new insights into the structural basis for lysosomal targeting but showed significant differences between human GUS, which is medically important in its own right, and E. coli GUS, which can be selectively inhibited in the human gut to prevent prodrug activation and is also widely used as a reporter gene by plant biologists. Despite these differences, both human and E. coli GUS share a high structure homology in all three domains with most of the glycosyl hydrolases, suggesting that they all evolved from a common ancestral gene. PMID:24260279

N-Glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris.

PubMed

Zou, Shuping; Huang, Shen; Kaleem, Imdad; Li, Chun

2013-03-10

Recombinant β-glucuronidase (GUS) expressed in Pichia pastoris GS115 is an important glycoprotein, encoded by a gene with four potential N-glycosylation sites. To investigate the impact of N-linked carbohydrate moieties on the stability of recombinant GUS, it was deglycosylated by peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic activities of the glycosylated and deglycosylated GUS were compared under various conditions such as temperature, pH, organic solvents, detergents and chaotropic agent. The results demonstrated that the glycosylated GUS retained greater fraction of maximum enzymatic activity against various types of denaturants compared with the deglycosylated. The conformational stabilities of both GUS were analyzed by monitoring the unfolding equilibrium by using the denaturant guanidinium chloride (dn-HCl). The glycosylated GUS displayed a significant increase in its conformational stability than the deglycosylated counterpart. These results affirmed the key role of N-glycosylation on the structural and functional stability of β-glucuronidase and could have potential applications in the functional enhancement of industrial enzymes. Copyright © 2013 Elsevier B.V. All rights reserved.

Transgenic Russian wildrye (Psathyrostachys juncea) plants obtained by biolistic transformation of embryogenic suspension cells.

PubMed

Wang, Z-Y; Bell, J; Lehmann, D

2004-07-01

Russian wildrye (Psathyrostachys juncea (Fisch.) Nevski) is a cool-season forage species well adapted to semi-arid climates. We are interested in developing biotechnological methods to improve this monocot forage species. Single genotype-derived embryogenic suspension cultures were established from the Russian wildrye cultivar Bozoisky-Select, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric beta-glucuronidase (gusA) gene was co-transformed with hph. Resistant calli were obtained from 29% of the bombarded dishes after selection with 200 mg/l hygromycin. Plants were regenerated from 45% of the hygromycin resistant calli. Thirty-six transgenic Russian wildrye plants were recovered after microprojectile bombardment of suspension cells and subsequent hygromycin selection. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. When a second gene (gusA) was co-transformed with hph, a reasonably high co-transformation frequency of 78% was observed. Transgenic expression of gusA was confirmed by GUS staining of shoot and leaf tissues. Fertile transgenic plants were obtained after two winters of vernalization under field conditions. This is the first report on the generation of transgenic plants in Russian wildrye.

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

PubMed

Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

2010-10-01

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

Agrobacterium tumefaciens-mediated transformation of Phellodendron amurense Rupr. using mature-seed explants.

PubMed

Yang, Jingli; Zhao, Bo; Kim, Yeon Bok; Zhou, Chenguang; Li, Chunyan; Chen, Yunlin; Zhang, Haizhen; Li, Cheng Hao

2013-01-01

An efficient transformation protocol was developed for Agrobacterium-mediated transformation of Phellodendron amurense Rupr. for using explants from mature seeds. The binary vector pCAMBIA1303, which contained hygromycin phosphotransferase (hptII) as a selectable marker gene and β-glucuronidase (GUS) as a reporter gene, was used for transformation studies. Different factors that affect survival of transformed buds, namely Agrobacterium infection method, bacterial strain, pre-culture duration, acetosyringone concentration, co-culture duration, and co-culture temperature were examined and optimized for transformation efficiency on the basis of GUS staining of hygromycin-resistant buds. Polymerase chain reaction (PCR), Southern blot and reverse transcription PCR confirmed the presence of the GUS gene. A transformation frequency of 13.1 % was achieved under optimized conditions for transformation (A. tumefaciens strain EHA105, 4 days co-cultivation at 4 °C, and infection of the pre-cultured mature-seed explants for 2 days). This is the first report of a successful genetic transformation protocol for P. amurense.

Low light and low ammonium are key factors for guayule leaf tissue shoot organogenesis and transformation.

PubMed

Dong, Niu; Montanez, Belen; Creelman, Robert A; Cornish, Katrina

2006-02-01

A new method has been developed for guayule tissue culture and transformation. Guayule leaf explants have a poor survival rate when placed on normal MS medium and under normal culture room light conditions. Low light and low ammonium treatment greatly improved shoot organogenesis and transformation from leaf tissues. Using this method, a 35S promoter driven BAR gene and an ubiquitin-3 promoter driven GUS gene (with intron) have been successfully introduced into guayule. These transgenic guayule plants were resistant to the herbicide ammonium-glufosinate and were positive to GUS staining. Molecular analysis showed the expected band and signal in all GUS positive transformants. The transformation efficiency with glufosinate selection ranged from 3 to 6%. Transformation with a pBIN19-based plasmid containing a NPTII gene and then selection with kanamycin also works well using this method. The ratio of kanamycin-resistant calli to total starting explants reached 50% in some experiments.

Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana

PubMed Central

Halder, Vivek; Kombrink, Erich

2015-01-01

The use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred to as “chemical genetics,” has only recently gained momentum. In addition to cultured cells, the well-characterized, small-sized model plant Arabidopsis thaliana is suitable for cultivation in microplates, which allows employing diverse cell- or phenotype-based chemical screens. In such screens, a chemical's bioactivity is typically assessed either through scoring its impact on morphological traits or quantifying molecular attributes such as enzyme or reporter activities. Here, we describe a facile forward chemical screening methodology for intact Arabidopsis seedlings harboring the β-glucuronidase (GUS) reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-β-D-glucuronide (4-MUG) as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. At the same time, the in situ bioassay is very convenient requiring less effort and time for sample handling in comparison to the conventional quantitative in vitro GUS assay using 4-MUG, as validated with several Arabidopsis lines harboring different GUS reporter constructs. To demonstrate that the developed assays is particularly suitable for large-scale screening projects, we performed a pilot screen for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis PR1p::GUS line. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line. PMID:25688251

Brain derived neurotrophic factor gene (BDNF) and personality traits: the modifying effect of season of birth and sex.

PubMed

Kazantseva, A; Gaysina, D; Kutlumbetova, Yu; Kanzafarova, R; Malykh, S; Lobaskova, M; Khusnutdinova, E

2015-01-02

Personality traits are complex phenotypes influenced by interactions of multiple genetic variants of small effect and environmental factors. It has been suggested that the brain derived neurotrophic factor gene (BDNF) is involved in personality traits. Season of birth (SOB) has also been shown to affect personality traits due to its influences on brain development during prenatal and early postnatal periods. The present study aimed to investigate the effects of BDNF on personality traits; and the modifying effects of SOB and sex on associations between BDNF and personality traits. A sample of 1018 young adults (68% women; age range 17-25years) of Caucasian origin from the Russian Federation was assessed on personality traits (Novelty Seeking, Harm Avoidance, Reward Dependence, Persistence, Self-directedness, Cooperativeness, Self-transcendence) with the Temperament and Character Inventory-125 (TCI-125). Associations between personality traits and 12 BDNF SNPs were tested using linear regression models. The present study demonstrated the effect of rs11030102 on Persistence in females only (PFDR=0.043; r(2)=1.3%). There were significant interaction effects between Val66Met (rs6265) and SOB (PFDR=0.048, r(2)=1.4%), and between rs2030323 and SOB (PFDR=0.042, r(2)=1.3%), on Harm Avoidance. Our findings provide evidence for the modifying effect of SOB on the association between BDNF and Harm Avoidance, and for the modifying effect of sex on the association between BDNF and Persistence. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization of OfWRKY3, a transcription factor that positively regulates the carotenoid cleavage dioxygenase gene OfCCD4 in Osmanthus fragrans.

PubMed

Han, Yuanji; Wu, Miao; Cao, Liya; Yuan, Wangjun; Dong, Meifang; Wang, Xiaohui; Chen, Weicai; Shang, Fude

2016-07-01

The sweet osmanthus carotenoid cleavage dioxygenase 4 (OfCCD4) cleaves carotenoids such as β-carotene and zeaxanthin to yield β-ionone. OfCCD4 is a member of the CCD gene family, and its promoter contains a W-box palindrome with two reversely oriented TGAC repeats, which are the proposed binding sites of WRKY transcription factors. We isolated three WRKY cDNAs from the petal of Osmanthus fragrans. One of them, OfWRKY3, encodes a protein containing two WRKY domains and two zinc finger motifs. OfWRKY3 and OfCCD4 had nearly identical expression profile in petals of 'Dangui' and 'Yingui' at different flowering stages and showed similar expression patterns in petals treated by salicylic acid, jasmonic acid and abscisic acid. Activation of OfCCD4pro:GUS by OfWRKY3 was detected in coinfiltrated tobacco leaves and very weak GUS activity was detected in control tissues, indicating that OfWRKY3 can interact with the OfCCD4 promoter. Yeast one-hybrid and electrophoretic mobility shift assay showed that OfWRKY3 was able to bind to the W-box palindrome motif present in the OfCCD4 promoter. These results suggest that OfWRKY3 is a positive regulator of the OfCCD4 gene, and might partly account for the biosynthesis of β-ionone in sweet osmanthus.

Pathogen Phytosensing: Plants to Report Plant Pathogens.

PubMed

Mazarei, Mitra; Teplova, Irina; Hajimorad, M Reza; Stewart, C Neal

2008-04-14

Real-time systems that provide evidence of pathogen contamination in crops can be an important new line of early defense in agricultural centers. Plants possess defense mechanisms to protect against pathogen attack. Inducible plant defense is controlled by signal transduction pathways, inducible promoters and cis-regulatory elements corresponding to key genes involved in defense, and pathogen-specific responses. Identified inducible promoters and cis-acting elements could be utilized in plant sentinels, or 'phytosensors', by fusing these to reporter genes to produce plants with altered phenotypes in response to the presence of pathogens. Here, we have employed cis-acting elements from promoter regions of pathogen inducible genes as well as those responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and ethylene. Synthetic promoters were constructed by combining various regulatory elements supplemented with the enhancer elements from the Cauliflower mosaic virus (CaMV) 35S promoter to increase basal level of the GUS expression. The inducibility of each synthetic promoter was first assessed in transient expression assays using Arabidopsis thaliana protoplasts and then examined for efficacy in stably transgenic Arabidopsis and tobacco plants. Histochemical and fluorometric GUS expression analyses showed that both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohormone treatments with increased GUS expression when compared to untreated plants. Pathogen-inducible phytosensor studies were initiated by analyzing the sensitivity of the synthetic promoters against virus infection. Transgenic tobacco plants infected with Alfalfa mosaic virus showed an increase in GUS expression when compared to mock-inoculated control plants, whereas Tobacco mosaic virus infection caused no changes in GUS expression. Further research, using these transgenic plants against a range of different pathogens with the regulation of detectable

Pathogen Phytosensing: Plants to Report Plant Pathogens

PubMed Central

Mazarei, Mitra; Teplova, Irina; Hajimorad, M. Reza; Stewart, C. Neal

2008-01-01

Real-time systems that provide evidence of pathogen contamination in crops can be an important new line of early defense in agricultural centers. Plants possess defense mechanisms to protect against pathogen attack. Inducible plant defense is controlled by signal transduction pathways, inducible promoters and cis-regulatory elements corresponding to key genes involved in defense, and pathogen-specific responses. Identified inducible promoters and cis-acting elements could be utilized in plant sentinels, or ‘phytosensors’, by fusing these to reporter genes to produce plants with altered phenotypes in response to the presence of pathogens. Here, we have employed cis-acting elements from promoter regions of pathogen inducible genes as well as those responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and ethylene. Synthetic promoters were constructed by combining various regulatory elements supplemented with the enhancer elements from the Cauliflower mosaic virus (CaMV) 35S promoter to increase basal level of the GUS expression. The inducibility of each synthetic promoter was first assessed in transient expression assays using Arabidopsis thaliana protoplasts and then examined for efficacy in stably transgenic Arabidopsis and tobacco plants. Histochemical and fluorometric GUS expression analyses showed that both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohormone treatments with increased GUS expression when compared to untreated plants. Pathogen-inducible phytosensor studies were initiated by analyzing the sensitivity of the synthetic promoters against virus infection. Transgenic tobacco plants infected with Alfalfa mosaic virus showed an increase in GUS expression when compared to mock-inoculated control plants, whereas Tobacco mosaic virus infection caused no changes in GUS expression. Further research, using these transgenic plants against a range of different pathogens with the regulation of detectable

Genetic transformation of Dichanthium annulatum (Forssk)--an apomictic tropical forage grass.

PubMed

Dalton, S J; Bettany, A J E; Bhat, V; Gupta, M G; Bailey, K; Timms, E; Morris, P

2003-06-01

Eleven Dichanthium annulatum (Forssk) plants were regenerated from embryogenic callus co-transformed with two plasmids encoding either the hygromycin phosphotransferase gene (hph) or the beta-glucuronidase (GUS) gene (uidA). Analysis of these putative transformants showed that three plants were transformed with the hph gene, showed the presence of the hph transcript and expressed hygromycin resistance after transfer to soil. Two of these also contained the uidA gene but did not express GUS and were shown to be the same transformation event. All three of the transformants set seed. Hygromycin resistance varied from 68-100% in the progeny of the three transformants. Transgene transmission appeared to have been mainly through apomixis.

Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

PubMed

Khan, Zainul A; Abdin, Malik Z; Khan, Jawaid A

2015-01-01

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

Isolation and characterization of a novel pollen-specific promoter in maize (Zea mays L.).

PubMed

Wang, He; Fan, Mingxia; Wang, Guohong; Zhang, Chunyu; Shi, Lei; Wei, Zhengyi; Ma, Wenjuan; Chang, Jing; Huang, Senxin; Lin, Feng

2017-06-01

ZmSTK2_USP, located on the long arm of chromosome 4, belongs to the serine/threonine kinase gene in maize. The sequence analysis of 2100 bp upstream from the start codon ATG has shown that it contains cis-element motifs and two types of anther/pollen-specific promoter elements (GTGA and AGAAA), suggesting that it is the pollen-specific promoter. To investigate the function of ZmSTK2_USP promoter, the GUS gene fusion system was employed. In proZmSTK2_USP-GUS genetically modified plants, GUS activity was detected in mature pollen grains and pollen tubes but not found in other floral and vegetative tissues. These results show that proZmSTK2_USP is the pollen-specific promoter and drives pollen-specific activity during the middle stage of pollen development until pollen maturation.

Wound-induced expression of horseradish peroxidase.

PubMed

Kawaoka, A; Kawamoto, T; Ohta, H; Sekine, M; Takano, M; Shinmyo, A

1994-01-01

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the β-glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.

β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30.

PubMed

Sakurama, Haruko; Kishino, Shigenobu; Uchibori, Yoshie; Yonejima, Yasunori; Ashida, Hisashi; Kita, Keiko; Takahashi, Satomi; Ogawa, Jun

2014-05-01

Baicalin (baicalein 7-O-β-D-glucuronide) is one of the major flavonoid glucuronides found in traditional herbal medicines. Because its aglycone, baicalein, is absorbed more quickly and shows more effective properties than baicalin, the conversion of baicalin into baicalein by β-glucuronidase (GUS) has drawn the attention of researchers. Recently, we have found that Lactobacillus brevis subsp. coagulans can convert baicalin to baicalein. Therefore, we aimed to identify and characterize the converting enzyme from L. brevis subsp. coagulans. First, we purified this enzyme from the cell-free extracts of L. brevis subsp. coagulans and cloned its gene. Surprisingly, this enzyme was found to be a GUS belonging to glycoside hydrolase (GH) family 30 (designated as LcGUS30), and its amino acid sequence has little similarity with any GUS belonging to GH families 1, 2, and 79 that have been reported so far. We then established a high-level expression and simple purification system of the recombinant LcGUS30 in Escherichia coli. The detailed analysis of the substrate specificity revealed that LcGUS30 has strict specificity toward glycon but not toward aglycones. Interestingly, LcGUS30 prefers baicalin rather than estrone 3-(β-D-glucuronide), one of the human endogenous steroid hormones. These results indicated that L. brevis subsp. coagulans and LcGUS30 should serve as powerful tools for the construction of a safe bioconversion system for baicalin. In addition, we propose that this novel type of GUS forms a new group in subfamily 3 of GH family 30.

Correction of murine mucopolysaccharidosis VII by a human. beta. -glucuronidase transgene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kyle, J.W.; Vogler, C.; Hoffmann, J.W.

1990-05-01

The authors recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of {beta}-glucuronidase. Affected mice, of genotype gus{sup mps}/gus{sup mps}, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarged vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report they describe the consequences of introducing the human {beta}-glucuronidase gene, GUSB, into gus{sup mps}/gus{supmore » mps} mice that produce virtually no murine {beta}-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human {beta}-glucuronidase activity in all tissues examined and were phenotypically normal. Biochemically, both the intralysosomal storage of glycosaminoglycans and the secondary elevation of other acid hydrolases were corrected. These findings demonstrate that the GUSB transgene is expressed in gus{sup mps}/gus{sup mps} mice and that human {beta}-glucuronidase corrects the murine mucopolysaccharidosis storage disease.« less

The characterization of six auxin-induced tomato GH3 genes uncovers a member, SlGH3.4, strongly responsive to arbuscular mycorrhizal symbiosis.

PubMed

Liao, Dehua; Chen, Xiao; Chen, Aiqun; Wang, Huimin; Liu, Jianjian; Liu, Junli; Gu, Mian; Sun, Shubin; Xu, Guohua

2015-04-01

In plants, the GH3 gene family is widely considered to be involved in a broad range of plant physiological processes, through modulation of hormonal homeostasis. Multiple GH3 genes have been functionally characterized in several plant species; however, to date, limited works to study the GH3 genes in tomato have been reported. Here, we characterize the expression and regulatory profiles of six tomato GH3 genes, SlGH3.2, SlGH3.3, SlGH3.4, SlGH3.7, SlGH3.9 and SlGH3.15, in response to different phytohormone applications and arbuscular mycorrhizal (AM) fungal colonization. All six GH3 genes showed inducible responses to external IAA, and three members were significantly up-regulated in response to AM symbiosis. In particular, SlGH3.4, the transcripts of which were barely detectable under normal growth conditions, was strongly activated in the IAA-treated and AM fungal-colonized roots. A comparison of the SlGH3.4 expression in wild-type plants and M161, a mutant with a defect in AM symbiosis, confirmed that SlGH3.4 expression is highly correlated to mycorrhizal colonization. Histochemical staining demonstrated that a 2,258 bp SlGH3.4 promoter fragment could drive β-glucuronidase (GUS) expression strongly in root tips, steles and cortical cells of IAA-treated roots, but predominantly in the fungal-colonized cells of mycorrhizal roots. A truncated 654 bp promoter failed to direct GUS expression in IAA-treated roots, but maintained the symbiosis-induced activity in mycorrhizal roots. In summary, our results suggest that a mycorrhizal signaling pathway that is at least partially independent of the auxin signaling pathway has evolved for the co-regulation of the auxin- and mycorrhiza-activated GH3 genes in plants. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Phytomonitoring and phytoremediation of agrochemicals and related compounds based on recombinant cytochrome P450s and aryl hydrocarbon receptors (AhRs).

PubMed

Shimazu, Sayuri; Inui, Hideyuki; Ohkawa, Hideo

2011-04-13

Molecular mechanisms of metabolism and modes of actions of agrochemicals and related compounds are important for understanding selective toxicity, biodegradability, and monitoring of biological effects on nontarget organisms. It is well-known that in mammals, cytochrome P450 (P450 or CYP) monooxygenases metabolize lipophilic foreign compounds. These P450 species are inducible, and both CYP1A1 and CYP1A2 are induced by aryl hydrocarbon receptor (AhR) combined with a ligand. Gene engineering of P450 and NADPH cytochrome P450 oxidoreductase (P450 reductase) was established for bioconversion. Also, gene modification of AhRs was developed for recombinant AhR-mediated β-glucronidase (GUS) reporter assay of AhR ligands. Recombinant P450 genes were transformed into plants for phytoremediation, and recombinant AhR-mediated GUS reporter gene expression systems were each transformed into plants for phytomonitoring. Transgenic rice plants carrying CYP2B6 metabolized the herbicide metolachlor and remarkably reduced the residues in the plants and soils under paddy field conditions. Transgenic Arabidopsis plants carrying recombinant guinea pig (g) AhR-mediated GUS reporter genes detected PCB126 at the level of 10 ng/g soils in the presence of biosurfactants MEL-B. Both phytomonitoring and phytoremediation plants were each evaluated from the standpoint of practical uses.

Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

PubMed Central

Khan, Zainul A.; Abdin, Malik Z.; Khan, Jawaid A.

2015-01-01

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells. PMID:25799504

Shortening tobacco life cycle accelerates functional gene identification in genomic research.

PubMed

Ning, G; Xiao, X; Lv, H; Li, X; Zuo, Y; Bao, M

2012-11-01

Definitive allocation of function requires the introduction of genetic mutations and analysis of their phenotypic consequences. Novel, rapid and convenient techniques or materials are very important and useful to accelerate gene identification in functional genomics research. Here, over-expression of PmFT (Prunus mume), a novel FT orthologue, and PtFT (Populus tremula) lead to shortening of the tobacco life cycle. A series of novel short life cycle stable tobacco lines (30-50 days) were developed through repeated self-crossing selection breeding. Based on the second transformation via a gusA reporter gene, the promoter from BpFULL1 in silver birch (Betula pendula) and the gene (CPC) from Arabidopsis thaliana were effectively tested using short life cycle tobacco lines. Comparative analysis among wild type, short life cycle tobacco and Arabidopsis transformation system verified that it is optional to accelerate functional gene studies by shortening host plant material life cycle, at least in these short life cycle tobacco lines. The results verified that the novel short life cycle transgenic tobacco lines not only combine the advantages of economic nursery requirements and a simple transformation system, but also provide a robust, effective and stable host system to accelerate gene analysis. Thus, shortening tobacco life cycle strategy is feasible to accelerate heterologous or homologous functional gene identification in genomic research. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

A R2R3-MYB transcription factor gene in common wheat (namely TaMYBsm1) involved in enhancement of drought tolerance in transgenic Arabidopsis.

PubMed

Li, Meng-Jun; Qiao, Yu; Li, Ya-Qing; Shi, Zhan-Liang; Zhang, Nan; Bi, Cai-Li; Guo, Jin-Kao

2016-11-01

We isolated the TaMYBsm1 genes, encoding R2R3-type MYB proteins in common wheat, aimed to uncover the possible molecular mechanisms related to drought response. The TaMYBsm1 genes, TaMYBsm1-A, TaMYBsm1-B and TaMYBsm1-D, were isolated and analyzed from the common wheat cultivar Shimai 15. Their expression patterns under PEG 6000 and mannitol were monitored by semi-quantitative RT-PCR and β-glucuronidase (Gus) assay. The function of TaMYBsm1-D under drought stress in transgenic Arabidopsis plants was investigated, and the germination rate, water loss rate, as well as the proline and malondialdehyde (MDA) content were compared with that in wild type (WT) plants. The expression of three downstream genes (DREB2A, P5CS1 and RD29A) in TaMYBsm1-D transgenic plants was analyzed. The R2R3-MYB domains of the MYBsm1 proteins were highly conserved in plants. In addition, the TaMYBsm1 proteins were targeted to the nucleus and contained transcriptional activation domains (TADs). Gus assay and semi-quantitative RT-PCR analysis demonstrated that the TaMYBsm1 genes were up-regulated when the wheat was treated by PEG and mannitol. Compared with WT plants, the germination rates were much higher, but the water loss rates were much lower in TaMYBsm1-D overexpression plants. TaMYBsm1-D transgenic plants showed distinct higher proline contents but a lower MDA content than the WT plants. The three downstream genes were highly expressed in TaMYBsm1-D transgenic plants. We concluded from these results that TaMYBsm1 genes play an important role in plant drought stress tolerance through up-regulation of DREB2A, P5CS1 and RD29A. The increase of proline content and decrease of MDA content may also be involved in the drought response.

A Preliminary Study on a Specifically Expressed Arabidopsis Promotor in Vascular Bundle

NASA Astrophysics Data System (ADS)

Yun-hong, Gu; Chuan-xiao, Xie; Li-fang, Wu; Zeng-liang, Yu; Guang-yong, Qin; Yu-ping, Huo

2003-04-01

From a population of about 3500 single plants in Arabidopsis promoter trapping bank, one plant whose GUS-gene had been specifically expressed in vascular bundle, was screened by the method of gus tissue staining. The T-DNA flanking sequence was amplified using TAIL-PCR. This band will be purified and connected to TA cloning vector. After sequencing and searching in the genebank, its function will be demonatrated through transformation.

Plant phosphatidylcholine-hydrolyzing phospholipases C NPC3 and NPC4 with roles in root development and brassinolide signaling in Arabidopsis thaliana.

PubMed

Wimalasekera, Rinukshi; Pejchar, Premysl; Holk, André; Martinec, Jan; Scherer, Günther F E

2010-05-01

Phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphocholine and diacylglycerol (DAG). PC-PLC has a long tradition in animal signal transduction to generate DAG as a second messenger besides the classical phosphatidylinositol splitting phospholipase C (PI-PLC). Based on amino acid sequence similarity to bacterial PC-PLC, six putative PC-PLC genes (NPC1 to NPC6) were identified in the Arabidopsis genome. RT-PCR analysis revealed overlapping expression pattern of NPC genes in root, stem, leaf, flower, and silique. In auxin-treated P(NPC3):GUS and P(NPC4):GUS seedlings, strong increase of GUS activity was visible in roots, leaves, and shoots and, to a weaker extent, in brassinolide-treated (BL) seedlings. P(NPC4):GUS seedlings also responded to cytokinin with increased GUS activity in young leaves. Compared to wild-type, T-DNA insertional knockouts npc3 and npc4 showed shorter primary roots and lower lateral root density at low BL concentrations but increased lateral root densities in response to exogenous 0.05-1.0 μM BL. BL-induced expression of TCH4 and LRX2, which are involved in cell expansion, was impaired but not impaired in repression of CPD, a BL biosynthesis gene, in BL-treated npc3 and npc4. These observations suggest NPC3 and NPC4 are important in BL-mediated signaling in root growth. When treated with 0.1 μM BL, DAG accumulation was observed in tobacco BY-2 cell cultures labeled with fluorescent PC as early as 15 min after application. We hypothesize that at least one PC-PLC is a plant signaling enzyme in BL signal transduction and, as shown earlier, in elicitor signal transduction.

Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

PubMed Central

Park, Jong-Won; Beyene, Getu; Buenrostro-Nava, Marco T.; Molina, Joe; Wang, Xiaofeng; Ciomperlik, Jessica J.; Manabayeva, Shuga A.; Alvarado, Veria Y.; Rathore, Keerti S.; Scholthof, Herman B.; Mirkov, T. Erik

2013-01-01

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane. PMID:23799071

Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.).

PubMed

Song, Guo-Qing; Sink, K C

2004-12-01

Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 microM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 microM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

A proximal promoter region of Arabidopsis DREB2C confers tissue-specific expression under heat stress.

PubMed

Chen, Huan; Je, Jihyun; Song, Chieun; Hwang, Jung Eun; Lim, Chae Oh

2012-09-01

The dehydration-responsive element-binding factor 2C (DREB2C) is a member of the CBF/DREB subfamily of proteins, which contains a single APETALA2/Ethylene responsive element-binding factor (AP2/ERF) domain. To identify the expression pattern of the DREB2C gene, which contains multiple transcription cis-regulatory elements in its promoter, an approximately 1.4 kb upstream DREB2C sequence was fused to the β-glucuronidase reporter gene (GUS) and the recombinant p1244 construct was transformed into Arabidopsis thaliana (L.) Heynh. The promoter of the gene directed prominent GUS activity in the vasculature in diverse young dividing tissues. Upon applying heat stress (HS), GUS staining was also enhanced in the vasculature of the growing tissues. Analysis of a series of 5'-deletions of the DREB2C promoter revealed that a proximal upstream sequence sufficient for the tissue-specific spatial and temporal induction of GUS expression by HS is localized in the promoter region between -204 and -34 bps relative to the transcriptional start site. Furthermore, electrophoretic mobility shift assay (EMSA) demonstrated that nuclear protein binding activities specific to a -120 to -32 bp promoter fragment increased after HS. These results indicate that the TATA-proximal region and some latent trans-acting factors may cooperate in HS-induced activation of the Arabidopsis DREB2C promoter. © 2012 Institute of Botany, Chinese Academy of Sciences.

Wheat TaSP gene improves salt tolerance in transgenic Arabidopsis thaliana.

PubMed

Ma, Xiaoli; Cui, Weina; Liang, Wenji; Huang, Zhanjing

2015-12-01

A novel salt-induced gene with unknown functions was cloned through analysis of gene expression profile of a salt-tolerant wheat mutant RH8706-49 under salt stress. The gene was named Triticum aestivum salt-related protein (TaSP) and deposited in GenBank (Accession No. KF307326). Quantitative polymerase chain reaction (qPCR) results showed that TaSP expression was induced under salt, abscisic acid (ABA), and polyethylene glycol (PEG) stresses. Subcellular localization revealed that TaSP was mainly localized in cell membrane. Overexpression of TaSP in Arabidopsis could improve salt tolerance of 35S::TaSP transgenic Arabidopsis. 35S::TaSP transgenic Arabidopsis lines after salt stress presented better physiological indexes than the control group. In the non-invasive micro-test (NMT), an evident Na(+) excretion was observed at the root tip of salt-stressed 35S::TaSP transgenic Arabidopsis. TaSP promoter was cloned, and its beta-glucuronidase (GUS) activities before and after ABA, salt, cold, heat, and salicylic acid (SA) stresses were determined. Full-length TaSP promoter contained ABA and salt response elements. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Agrobacterium tumefaciens-mediated transformation of Narcissus tazzeta var. chinensis.

PubMed

Lu, Gang; Zou, Qingcheng; Guo, Deping; Zhuang, Xiaoying; Yu, Xiaolin; Xiang, Xun; Cao, Jiashu

2007-09-01

Phytoene synthase (PSY), as a key regulatory enzyme for carotene biosynthesis, plays an important role in regulating color formation in many species. In the present study, a protocol was developed for the transformation of Narcissus tazzeta var chinensis using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pCAMBIA1301 plasmid which contained an antisense phytoene synthase gene, a reporter beta-glucuronidase gene and a selectable marker hygromycin phosphotransferase gene. Effects of some factors on efficiency of transformation and regeneration were examined. Preculture of the explants for 6 days before inoculation enhanced the transient GUS expression. The addition of acetosyringone (AS) at 100 micromol l(-1) for inoculation and a period of 3 days co-cultivation yielded efficient transient GUS expression. Transformants were obtained through selection on MS medium containing 5 mg l(-1) 6-benzylaminopurine (BA), 0.1 mg l(-1)alpha-naphthalene acetic acid (NAA) and 40 mg l(-1) hygromycin. The transformation frequency was 1.24% based on PCR analysis of gus gene. One or two copies of transgene were demonstrated in different transformations by Southern blotting analyses. Northern blotting results confirmed that the transcription of the endogenous psy gene in transgenic plants was inhibited or silenced. The method reported here provides new opportunities for improvement of quality traits of Narcissus tazzeta via genetic transformation.

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.

The 5' untranslated region of the VR-ACS1 mRNA acts as a strong translational enhancer in plants.

PubMed

Wever, Willem; McCallum, Emily J; Chakravorty, David; Cazzonelli, Christopher I; Botella, José R

2010-08-01

The structure and function of untranslated mRNA leader sequences and their role in controlling gene expression remains poorly understood. Previous research has suggested that the 5' untranslated region (5'UTR) of the Vigna radiata aminocyclopropane-1-carboxylate synthase synthase (VR-ACS1) gene may function as a translational enhancer in plants. To test such hypothesis we compared the translation enhancing properties of three different 5'UTRs; those from the VR-ACS1, the chlorophyll a/b binding gene from petunia (Cab22L; a known translational enhancer) and the Vigna radiata pectinacetylesterase gene (PAE; used as control). Identical constructs in which the coding region of the beta-glucuronidase (GUS) gene was fused to each of the three 5'UTRs and placed under the control of the cauliflower mosaic virus 35S promoter were prepared. Transient expression assays in tobacco cell cultures and mung bean leaves showed that the VR-ACS1 and Cab22L 5'UTRs directed higher levels of GUS activity than the PAE 5'UTR. Analysis of transgenic Arabidopsis thaliana seedlings, as well as different tissues from mature plants, confirmed that while transcript levels were equivalent for all constructs, the 5'UTRs from the VR-ACS1 and Cab22L genes can increase GUS activity twofold to fivefold compared to the PAE 5'UTR, therefore confirming the translational enhancing properties of the VR-ACS1 5'UTR.

Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) – a class-I KNOX gene in potato

PubMed Central

Mahajan, Ameya S.; Kondhare, Kirtikumar R.; Rajabhoj, Mohit P.; Kumar, Amit; Ghate, Tejashree; Ravindran, Nevedha; Habib, Farhat; Siddappa, Sundaresha; Banerjee, Anjan K.

2016-01-01

Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato. PMID:27217546

Identification of a core set of rhizobial infection genes using data from single cell-types.

PubMed

Chen, Da-Song; Liu, Cheng-Wu; Roy, Sonali; Cousins, Donna; Stacey, Nicola; Murray, Jeremy D

2015-01-01

Genome-wide expression studies on nodulation have varied in their scale from entire root systems to dissected nodules or root sections containing nodule primordia (NP). More recently efforts have focused on developing methods for isolation of root hairs from infected plants and the application of laser-capture microdissection technology to nodules. Here we analyze two published data sets to identify a core set of infection genes that are expressed in the nodule and in root hairs during infection. Among the genes identified were those encoding phenylpropanoid biosynthesis enzymes including Chalcone-O-Methyltransferase which is required for the production of the potent Nod gene inducer 4',4-dihydroxy-2-methoxychalcone. A promoter-GUS analysis in transgenic hairy roots for two genes encoding Chalcone-O-Methyltransferase isoforms revealed their expression in rhizobially infected root hairs and the nodule infection zone but not in the nitrogen fixation zone. We also describe a group of Rhizobially Induced Peroxidases whose expression overlaps with the production of superoxide in rhizobially infected root hairs and in nodules and roots. Finally, we identify a cohort of co-regulated transcription factors as candidate regulators of these processes.

Expression analysis of chitinase upon challenge inoculation to Alternaria wounding and defense inducers in Brassica juncea.

PubMed

Rawat, Sandhya; Ali, Sajad; Mittra, Bhabatosh; Grover, Anita

2017-03-01

Chitinases are the hydrolytic enzymes which belong to the pathogenesis-related (PR) protein family and play an important role not only in plant defense but also in various abiotic stresses. However, only a limited number of chitinase genes have been characterised in B. juncea . In this study, we have characterised B. juncea class IV chitinase gene (accession no EF586206) in response to fungal infection, salicylic acid (SA), jasmonic acid (JA) treatments and wounding. Gene expression studies revealed that the transcript levels of Bjchitinase ( BjChp ) gene increases significantly both in local and distal tissues after Alternaria infection. Bjchitinase gene was also induced by jasmonic acid and wounding but moderately by salicylic acid. A 2.5 kb class IV chitinase promoter of this gene was isolated from B. juncea by Genome walking (accession no KF055403.1). In-silico analysis of this promoter revealed a number of conserved cis -regulatory elements related to defense, wounding and signalling molecules like SA, and JA. For validation, chitinase promoter was fused to the GUS gene, and the resultant construct was then introduced into Arabidopsis plants. Histochemical analysis of T 2 transgenic Arabidopsis plants showed that higher GUS activity in leaves after fungal infection, wounding and JA treatment but weakly by SA. GUS activity was seen in meristematic tissues, young leaves, seeds and siliques. Finally investigation has led to the identification of a pathogen-inducible, developmentally regulated and organ-specific promoter. Present study revealed that Bjchitinase ( BjChp ) promoter is induced during biotic and environmental stress and it can be used in developing finely tuned transgenics.

Gibberellin 20-oxidase gene OsGA20ox3 regulates plant stature and disease development in rice.

PubMed

Qin, Xue; Liu, Jun Hua; Zhao, Wen Sheng; Chen, Xu Jun; Guo, Ze Jian; Peng, You Liang

2013-02-01

Gibberellin (GA) 20-oxidase (GA20ox) catalyses consecutive steps of oxidation in the late part of the GA biosynthetic pathway. A T-DNA insertion mutant (17S-14) in rice, with an elongated phenotype, was isolated. Analysis of the flanking sequences of the T-DNA insertion site revealed that an incomplete T-DNA integration resulted in enhanced constitutively expression of downstream OsGA20ox3 in the mutant. The accumulation of bioactive GA(1) and GA(4) were increased in the mutant in comparison with the wild-type plant. Transgenic plants overexpressing OsGA20ox3 showed phenotypes similar to those of the 17S-14 mutant, and the RNA interference (RNAi) lines that had decreased OsGA20ox3 expression exhibited a semidwarf phenotype. Expression of OsGA20ox3 was detected in the leaves and roots of young seedlings, immature panicles, anthers, and pollens, based on β-glucuronidase (GUS) activity staining in transgenic plants expressing the OsGA20ox3 promoter fused to the GUS gene. The OsGA20ox3 RNAi lines showed enhanced resistance against rice pathogens Magnaporthe oryzae (causing rice blast) and Xanthomonas oryzae pv. oryzae (causing bacterial blight) and increased expression of defense-related genes. Conversely, OsGA20ox3-overexpressing plants were more susceptible to these pathogens comparing with the wild-type plants. The susceptibility of wild-type plants to X. oryzae pv. oryzae was increased by exogenous application of GA(3) and decreased by S-3307 treatment. Together, the results provide direct evidence for a critical role of OsGA20ox3 in regulating not only plant stature but also disease resistance in rice.

Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive

PubMed Central

Narvaez, Isabel; Khayreddine, Titouh; Pliego, Clara; Cerezo, Sergio; Jiménez-Díaz, Rafael M.; Trapero-Casas, José L.; López-Herrera, Carlos; Arjona-Girona, Isabel; Martín, Carmen; Mercado, José A.; Pliego-Alfaro, Fernando

2018-01-01

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. ‘Picual’ were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae. PMID:29875785

Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive.

PubMed

Narvaez, Isabel; Khayreddine, Titouh; Pliego, Clara; Cerezo, Sergio; Jiménez-Díaz, Rafael M; Trapero-Casas, José L; López-Herrera, Carlos; Arjona-Girona, Isabel; Martín, Carmen; Mercado, José A; Pliego-Alfaro, Fernando

2018-01-01

The antifungal protein (AFP) produced by Aspergillus giganteus , encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix , was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. 'Picual' were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae .

Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

PubMed

Huda, Kazi Md Kamrul; Banu, Mst Sufara Akhter; Pathi, Krishna Mohan; Tuteja, Narendra

2013-01-01

Plasma membrane Ca(2+)ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+)) from the cell, hence regulating Ca(2+) level within cells. Though plant Ca(2+)ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. The 1478 bp promoter sequence of rice plasma membrane Ca(2+)ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+)ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. The rice plasma membrane Ca(2+)ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this

Transgenic Bt cotton driven by the green tissue-specific promoter shows strong toxicity to lepidopteran pests and lower Bt toxin accumulation in seeds.

PubMed

Wang, Qing; Zhu, Yi; Sun, Lin; Li, Lebin; Jin, Shuangxia; Zhang, Xianlong

2016-02-01

A promoter of the PNZIP (Pharbitis nil leucine zipper) gene (1.459 kb) was cloned from Pharbitis nil and fused to the GUS (β-glucuronidase) and Bacillus thuringiensis endotoxin (Cry9C) genes. Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation. Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants. In contrast, GUS staining in the reproductive structures such as petals, anther, and immature seeds of PNZIP::GUS cotton was very faint. Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus (CaMV) 35S::Cry9C line were selected for enzyme-linked immunosorbent assay (ELISA) and insect bioassays. Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from 24.6 to 45.5 μg g(-1) fresh weight. In green tissues such as the leaves, boll rinds, and bracts of the PNZIP::Cry9C line, the Cry9C protein accumulated up to 50.2, 39.7, and 48.3 μg g(-1) fresh weight respectively. In contrast, seeds of the PNZIP::Cry9C line (PZ1.3) accumulated only 0.26 μg g(-1) fresh weight of the Cry9C protein, which was 100 times lower than that recorded for the seeds of the CaMV 35S::Cry9C line. The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm. The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds. These features should allay public concerns about the safety of transgenic foods. We propose the future utility of PNZIP as an economical, environmentally friendly promoter in cotton biotechnology.

Reproductive Organ and Vascular Specific Promoter of the Rice Plasma Membrane Ca2+ATPase Mediates Environmental Stress Responses in Plants

PubMed Central

Huda, Kazi Md. Kamrul; Banu, Mst. Sufara Akhter; Pathi, Krishna Mohan; Tuteja, Narendra

2013-01-01

Background Plasma membrane Ca2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca2+) from the cell, hence regulating Ca2+ level within cells. Though plant Ca2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. Results The 1478 bp promoter sequence of rice plasma membrane Ca2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The −1478 to −886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for −1210 and −886 bp flanking region. The −1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The −1210 and −886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the −886 bp and −519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. Conclusions The rice plasma membrane Ca2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue

[Cloning and bioinformatic analysis and expression analysis of beta-glucuronidase in Scutellaria baicalensis].

PubMed

Guo, Shuang-shuang; Cheng, Lin; Yang, Li-min; Han, Mei

2015-11-01

The β-Glucuronidase gene (sbGUS) cDNA firstly from Scutellari abaicalensis leaf was cloned by RT-PCR, with GenBank accession number KR364726. The full length cDNA of sbGUS was 1 584 bp with an open reading frame (ORF), encoding an unstable protein with 527 amino acids. The bioinformatic analysis showed that the sbGUS encoding protein had isoelectric point (pI) of 5.55 and a calculated molecular weight about 58.724 8 kDa, with a transmembrane regions and signal peptide, had conserved domains of glycoside hydrolase super family and unintegrated trans-glycosidase catalytic structure. In the secondary structure, the percentage of alpha helix, extended strand, β-extended and random coil were 25.62%, 28.84%, 13.28% and 32.26%, respectively. The homologous analysis indicated the nucleotide sequence 98.93% similarity and the amino acid sequence 98.29% similarity with S. baicalensis (BAA97804.1), in the nine positions were different. The expression level of sGUS was the highest in root based on a real-time PCR analysis, followed by flower and stem, and the lowest was in stem. The results provide a foundation for exploring the molecular function of sbGUS involved in baicalcin biosynthesis based on synthetic biology approach in S. baicalensis plants.

FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis.

PubMed

Kilby, N J; Davies, G J; Snaith, M R

1995-11-01

FLP site-specific recombinase was expressed in stably transformed tobacco and Arabidopsis. FLP-expressing tobacco lines were crossed with other transformed tobacco lines that contained a stably integrated FLP recognition target construct(s). The target construct consisted of two directly-oriented FLP recognition targets (FRTs), flanking a hygromycin resistance cassette located between a GUS coding region and an upstream 35S CaMV promoter. Excision of the hygromycin resistance cassette by FLP-mediated recombination between FRTs brings the GUS coding region under the transcriptional control of the CaMV 35S promoter. In the absence of FLP-mediated recombination, the GUS gene is transcriptionally silent. GUS activity was observed in the progeny of all crosses made between FLP recombinase-expressing and target-containing tobacco lines, but not in the selfs of parents. The predicted recombination product remaining after excision was confirmed by PCR and Southern analysis. In Arabidopsis, inducible expression of FLP recombinase was achieved from the soybean Gmhsp 17.6L heat-shock promoter. Heat-shock induction of FLP expression in plants containing the target construct led to activation of constitutive GUS expression in a subset of cells, whose progeny, therefore, were GUS-positive. A variety of clonal sectors were produced in plants derived from seed that was heat-shocked during germination. The ability to control the timing of GUS activation was demonstrated by heat-shock of unopened flower heads which produced large sectors. It was concluded that heat-shock-induced expression of FLP recombinase provides a readily controllable method for generating marked clonal sectors in Arabidopsis, the size and distribution of which reflects the timing of applied heat-shock.

Sulfur-oxidizing bacterial populations within cyanobacterial dominated coral disease lesions.

PubMed

Bourne, David G; van der Zee, Marc J J; Botté, Emmanuelle S; Sato, Yui

2013-08-01

This study investigated the diversity and quantitative shifts of sulfur-oxidizing bacteria (SOB) during the onset of black band disease (BBD) in corals using quantitative PCR (qPCR) and cloning approaches targeting the soxB gene, involved in sulfur oxidation. Four Montipora sp. coral colonies identified with lesions previously termed cyanobacterial patches (CP) (comprising microbial communities different from those of BBD lesions), was monitored in situ as CP developed into BBD. The overall abundance of SOB in both CP and BBD lesions were very low and near the detection limit of the qPCR assay, although consistently indicated that SOB populations decreased as the lesions transitioned from CP to BBD. Phylogenetic assessment of retrieved soxB genes showed that SOB in both CP and BBD lesions were dominated by one sequence type, representing > 70% of all soxB gene sequences and affiliated with members of the Rhodobacteraceae within the α-Proteobacteria. This study represents the first assessment targeting SOB within BBD lesions and clearly shows that SOB are not highly diverse or abundant in this complex microbial mat. The lack of oxidation of reduced sulfur compounds by SOB likely aids the accumulation of high levels of sulfide at the base of the BBD mat, a compound contributing to the pathogenicity of BBD lesions. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

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

A WRKY transcription factor recruits the SYG1-like protein SHB1 to activate gene expression and seed cavity enlargement.

PubMed

Kang, Xiaojun; Li, Wei; Zhou, Yun; Ni, Min

2013-01-01

Seed development in Arabidopsis and in many dicots involves an early proliferation of the endosperm to form a large embryo sac or seed cavity close to the size of the mature seed, followed by a second phase during which the embryo grows and replaces the endosperm. Short hypocotyl under BLUE1 (SHB1) is a member of the SYG1 protein family in fungi, Caenorhabditis elegans, flies, and mammals. SHB1 gain-of-function enhances endosperm proliferation, increases seed size, and up-regulates the expression of the WRKY transcription factor gene MINISEED3 (MINI3) and the LRR receptor kinase gene HAIKU2 (IKU2). Mutations in either IKU2 or MINI3 retard endosperm proliferation and reduce seed size. However, the molecular mechanisms underlying the establishment of the seed cavity and hence the seed size remain largely unknown. Here, we show that the expression of MINI3 and IKU2 is repressed before fertilization and after 4 days after pollination (DAP), but is activated by SHB1 from 2 to 4 DAP prior to the formation of the seed cavity. SHB1 associates with their promoters but without a recognizable DNA binding motif, and this association is abolished in mini3 mutant. MINI3 binds to W-boxes in, and recruits SHB1 to, its own and IKU2 promoters. Interestingly, SHB1, but not MINI3, activates transcription of pMINI3::GUS or pIKU2::GUS. We reveal a critical developmental switch through the activation of MINI3 expression by SHB1. The recruitment of SHB1 by MINI3 to its own and IKU2 promoters represents a novel two-step amplification to counter the low expression level of IKU2, which is a trigger for endosperm proliferation and seed cavity enlargement.

A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum.

PubMed

Rud, Ida; Jensen, Peter Ruhdal; Naterstad, Kristine; Axelsson, Lars

2006-04-01

A synthetic promoter library (SPL) for Lactobacillus plantarum has been developed, which generalizes the approach for obtaining synthetic promoters. The consensus sequence, derived from rRNA promoters extracted from the L. plantarum WCFS1 genome, was kept constant, and the non-consensus sequences were randomized. Construction of the SPL was performed in a vector (pSIP409) previously developed for high-level, inducible gene expression in L. plantarum and Lactobacillus sakei. A wide range of promoter strengths was obtained with the approach, covering 3-4 logs of expression levels in small increments of activity. The SPL was evaluated for the ability to drive beta-glucuronidase (GusA) and aminopeptidase N (PepN) expression. Protein production from the synthetic promoters was constitutive, and the most potent promoters gave high protein production with levels comparable to those of native rRNA promoters, and production of PepN protein corresponding to approximately 10-15 % of the total cellular protein. High correlation was obtained between the activities of promoters when tested in L. sakei and L. plantarum, which indicates the potential of the SPL for other Lactobacillus species. The SPL enables fine-tuning of stable gene expression for various applications in L. plantarum.

Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides1[W][OPEN

PubMed Central

Muthukumar, Balasubramaniam; Joyce, Blake L.; Elless, Mark P.; Stewart, C. Neal

2013-01-01

Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid ‘C-fern Express’ (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using β-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns. PMID:23933990

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.

Stable transformation via particle bombardment in two different soybean regeneration systems.

PubMed

Sato, S; Newell, C; Kolacz, K; Tredo, L; Finer, J; Hinchee, M

1993-05-01

The Biolistics(®) particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for β-glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

PubMed

Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

Tobacco PR-2d promoter is induced in transgenic cucumber in response to biotic and abiotic stimuli.

PubMed

Yin, Zhimin; Hennig, Jacek; Szwacka, Maria; Malepszy, Stefan

2004-05-01

The PR-2d promoter/uidA (GUS) gene construct was introduced into the cucumber (Cucumis sativus L.) genome and several transgenic lines were produced. Activation of the PR-2d promoter was investigated in these plants in response to inoculation with fungal pathogens and after salicylic acid (SA) or cold treatments. Treatment with exogenous SA increased GUS activity 2 to 11 fold over that of the control. Endogenous SA and its conjugate salicylic acid glucoside (SAG) rose in parallel after inoculation with the fungal pathogen Pseudoperonospora cubensis, with SAG becoming the predominant form. The free SA levels increased 15 fold above the basal level at 5 dpi and preceded the induction of the PR-2d promoter by five days, which occurred at 10 dpi with a 12 fold increase over the control. Inoculation with another fungal pathogen, Erysiphe polyphage, increased GUS activity 4 to 44 fold over that of the control. During normal development of flowers in the cucumber, the PR-2d/uidA gene expressed in the floral organs was similar to that of the primary host. In addition, we present the first evidence that the PR-2d promoter was induced (624 fold) under cold stress. We demonstrate that in the heterologous state the gene construct was expressed according to the signalling pattern of the native species and was stably transmitted to progeny over four generations.

Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses

PubMed Central

Raikwar, Shailendra; Srivastava, Vineet K.; Gill, Sarvajeet S.; Tuteja, Renu; Tuteja, Narendra

2015-01-01

Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression. PMID:26734018

Cloning and Functional Analysis of Phosphoethanolamine Methyltransferase Promoter from Maize (Zea mays L.)

PubMed Central

Niu, Gai-Li; Gou, Wei; Han, Xiang-Long; Qin, Cheng; Zhang, Li-Xin; Ashraf, Muhammad

2018-01-01

Betaine, a non-toxic osmoprotectant, is believed to accumulate considerably in plants under stress conditions to maintain the osmotic pressure and promote a variety of processes involved in growth and development. Phosphoethanolamine N-methyltransferase (PEAMT), a key enzyme for betaine synthesis, is reported to be regulated by its upstream promoter. In the present investigation, by using the transgenic approach, a 1048 bp long promoter region of ZmPEAMT gene from Zea mays was cloned and functionally characterized in tobacco. Computational analysis affirmed the existence of abiotic stress responsive cis-elements like ABRE, MYC, HST, LST etc., as well as pathogen, wound and phytohormone responsive motifs. For transformation in tobacco, four 5′-deletion constructs of 826 bp (P2), 642 bp (P3), 428 bp (P4) and 245 bp (P5) were constructed from the 1048 bp (P1) promoter fragment. The transgenic plants generated through a single event exhibited a promising expression of GUS reporter protein in the leaf tissues of treated with salt, drought, oxidative and cold stress as well as control plants. The GUS expression level progressively reduced from P1 to P5 in the leaf tissues, whereas a maximal expression was observed with the P3 construct in the leaves of control plants. The expression of GUS was noted to be higher in the leaves of osmotically- or salt-treated transgenic plants than that in the untreated (control) plants. An effective expression of GUS in the transgenic plants manifests that this promoter can be employed for both stress-inducible and constitutive expression of gene(s). Due to this characteristic, this potential promoter can be effectively used for genetic engineering of several crops. PMID:29316727

Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses.

PubMed

Raikwar, Shailendra; Srivastava, Vineet K; Gill, Sarvajeet S; Tuteja, Renu; Tuteja, Narendra

2015-01-01

Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression.

AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression.

PubMed

Klimyuk, V I; Jones, J D

1997-01-01

Based on homologies between the yeast DMC1 and the lily LIM15 meiosis-specific genes, degenerate PCR primers were designed that amplified the Arabidopsis DMC1 gene (AtDMC1). AtDMC1 genomic DNA (8 kb) was sequenced, and the transcript was characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) and by 5' and 3' RACE (rapid amplification of cDNA ends). The AtDMC1 gene contains 15 exons and 14 introns. RNA in situ hybridization analysis showed that expression of the AtDMC1 is restricted to pollen mother cells in anthers and to megaspore mother cells in ovules. The AtDMC1 promoter was fused to the GUS reporter gene, and conferred meiosis-associated expression in both male and female floral lineages. Comparison of AtDMC1 isolated from Landsberg erecta ecotype to its Columbia allele ArLIM15, revealed the presence of a 1874 bp transposon-like element within the promoter region of ArLIM15. RT-PCR analysis showed that the expression levels of AtDMC1 and ArLIM15 are similar. Possible uses for the AtDMC1 promoter are discussed.

Identification of gravitropic response indicator genes in Arabidopsis inflorescence stems

PubMed Central

Taniguchi, Masatoshi; Nakamura, Moritaka; Tasaka, Masao; Morita, Miyo Terao

2014-01-01

Differential organ growth during gravitropic response is caused by differential accumulation of auxin, that is, relative higher auxin concentration in lower flanks than in upper flanks of responding organs. Auxin responsive reporter systems such as DR5::GUS and DR5::GFP have usually been used as indicators of gravitropic response in roots and hypocotyls of Arabidopsis. However, in the inflorescence stems, the reporter systems don’t work well to monitor gravitropic response. Here, we aim to certify appropriate gravitropic response indicators (GRIs) in inflorescence stems. We performed microarray analysis comparing gene expression profiles between upper and lower flanks of Arabidopsis inflorescence stems after gravistimulation. Thirty genes showed > 2-fold differentially increased expression in lower flanks at 30 min, of which 19 were auxin response genes. We focused on IAA5 and IAA2 and verified whether they are appropriate GRIs by real-time qRT-PCR analyses. Transcript levels of IAA5 and IAA2 were remarkably higher in lower flanks than in upper flanks after gravistimulation. The biased IAA5 or IAA2 expression is disappeared in sgr2–1 mutant which is defective in gravity perception, indicating that gravity perception process is essential for formation of the biased gene expression during gravitropism. IAA5 expression was remarkably increased in lower flanks at 30 min after gravistimulation, whereas IAA2 expression was gradually decreased in upper flanks in a time-dependent manner. Therefore, we conclude that IAA5 is a sensitive GRI to monitor asymmetric auxin signaling caused by gravistimulation in Arabidopsis inflorescence stems. PMID:25763694

AtERF38 (At2g35700), an AP2/ERF family transcription factor gene from Arabidopsis thaliana, is expressed in specific cell types of roots, stems and seeds that undergo suberization.

PubMed

Lasserre, Eric; Jobet, Edouard; Llauro, Christel; Delseny, Michel

2008-12-01

An inverse genetic approach was used to gain insight into the role of AP2/ERF-type transcription factors genes during plant development in Arabidopsis thaliana. Here we show that the expression pattern of AtERF38, which is, among the organs tested, more intensively expressed in mature siliques and floral stems, is closely associated with tissues that undergo secondary cell wall modifications. Firstly, public microarray data sets analysis indicates that AtERF38 is coregulated with several genes involved in secondary wall thickening. Secondly, this was experimentally confirmed in different types of cells expressing a Pro(AtERF38)::GUS fusion: histochemical analysis revealed strong and specific GUS activity in outer integument cells of mature seeds, endodermal cells of the roots in the primary developmental stage and some sclerified cells of mature inflorescence stems. All of these cells are known or shown here to be characterized by a reinforced wall. The latter, which have not been well characterized to date in Arabidopsis and may be suberized, could benefit of the use of AtERF38 as a specific marker. We were not able to detect any phenotype in an insertion line in which ectopic expression of AtERF38 is caused by the insertion of a T-DNA in its promoter. Nevertheless, AtERF28 may be considered as a candidate regulator of secondary wall metabolism in particular cell types that are not reinforced by the typical deposition of lignin and cellulose, but that have at least in common accumulation of suberin-like lipid polyesters in their walls.

Evaluation of promoters and visual markers for transformation of eastern white pine

Treesearch

Allan Zipf; Alex M. Diner; Rufina Ward; Suman Bharara; George Brown; R. Nagmani; L.K. Pareek; Govind C. Sharma

2001-01-01

This report serves to evaluate possible promoters for use in the production of trmsgenic eastern white pine (Pinus strobus L.). Embryogenic cultures of eastern white pine were bombarded with gold particles coated separately with a variety of gene constructs containing the Uida B-glucoronidase (GUS) or green flourescent protein (GFP) reporter gene....

Three grape CBF/DREB1 genes respond to low temperature, drought and abscisic acid.

PubMed

Xiao, Huogen; Siddiqua, Mahbuba; Braybrook, Siobhan; Nassuth, Annette

2006-07-01

The C-repeat (CRT)-binding factor/dehydration-responsive element (DRE) binding protein 1 (CBF/ DREB1) transcription factors control an important pathway for increased freezing and drought tolerance in plants. Three CBF/DREB1-like genes, CBF 1-3, were isolated from both freezing-tolerant wild grape (Vitis riparia) and freezing-sensitive cultivated grape (Vitis vinifera). The deduced proteins in V. riparia are 63-70% identical to each other and 96-98% identical to the corresponding proteins in V. vinifera. All Vitis CBF proteins are 42-51% identical to AtCBF1 and contain CBF-specific amino acid motifs, supporting their identification as CBF proteins. Grape CBF sequences are unique in that they contain 20-29 additional amino acids and three serine stretches. Agro-infiltration experiments revealed that VrCBF1b localizes to the nucleus. VrCBF1a, VrCBF1b and VvCBF1 activated a green fluorescent protein (GFP) or glucuronidase (GUS) reporter gene behind CRT-containing promoters. Expression of the endogenous CBF genes was low at ambient temperature and enhanced upon low temperature (4 degrees C) treatment, first for CBF1, followed by CBF2, and about 2 d later by CBF3. No obvious significant difference was observed between V. riparia and V. vinifera genes. The expression levels of all three CBF genes were higher in young tissues than in older tissues. CBF1, 2 and 3 transcripts also accumulated in response to drought and exogenous abscisic acid (ABA) treatment, indicating that grape contains unique CBF genes.

Genomic analysis reveals versatile heterotrophic capacity of a potentially symbiotic sulfur-oxidizing bacterium in sponge.

PubMed

Tian, Ren-Mao; Wang, Yong; Bougouffa, Salim; Gao, Zhao-Ming; Cai, Lin; Bajic, Vladimir; Qian, Pei-Yuan

2014-11-01

Sulfur-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB) play essential roles in marine sponges. However, the detailed characteristics and physiology of the bacteria are largely unknown. Here, we present and analyse the first genome of sponge-associated SOB using a recently developed metagenomic binning strategy. The loss of transposase and virulence-associated genes and the maintenance of the ancient polyphosphate glucokinase gene suggested a stabilized SOB genome that might have coevolved with the ancient host during establishment of their association. Exclusive distribution in sponge, bacterial detoxification for the host (sulfide oxidation) and the enrichment for symbiotic characteristics (genes-encoding ankyrin) in the SOB genome supported the bacterial role as an intercellular symbiont. Despite possessing complete autotrophic sulfur oxidation pathways, the bacterium developed a much more versatile capacity for carbohydrate uptake and metabolism, in comparison with its closest relatives (Thioalkalivibrio) and to other representative autotrophs from the same order (Chromatiales). The ability to perform both autotrophic and heterotrophic metabolism likely results from the unstable supply of reduced sulfur in the sponge and is considered critical for the sponge-SOB consortium. Our study provides insights into SOB of sponge-specific clade with thioautotrophic and versatile heterotrophic metabolism relevant to its roles in the micro-environment of the sponge body. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters.

PubMed

Erpen, L; Tavano, E C R; Harakava, R; Dutt, M; Grosser, J W; Piedade, S M S; Mendes, B M J; Mourão Filho, F A A

2018-05-23

Regulatory sequences from the citrus constitutive genes cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1) were isolated, fused to the uidA gene, and qualitatively and quantitatively evaluated in transgenic sweet orange plants. The 5' upstream region of a gene (the promoter) is the most important component for the initiation and regulation of gene transcription of both native genes and transgenes in plants. The isolation and characterization of gene regulatory sequences are essential to the development of intragenic or cisgenic genetic manipulation strategies, which imply the use of genetic material from the same species or from closely related species. We describe herein the isolation and evaluation of the promoter sequence from three constitutively expressed citrus genes: cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1). The functionality of the promoters was confirmed by a histochemical GUS assay in leaves, stems, and roots of stably transformed citrus plants expressing the promoter-uidA construct. Lower uidA mRNA levels were detected when the transgene was under the control of citrus promoters as compared to the expression under the control of the CaMV35S promoter. The association of the uidA gene with the citrus-derived promoters resulted in mRNA levels of up to 60-41.8% of the value obtained with the construct containing CaMV35S driving the uidA gene. Moreover, a lower inter-individual variability in transgene expression was observed amongst the different transgenic lines, where gene constructs containing citrus-derived promoters were used. In silico analysis of the citrus-derived promoter sequences revealed that their activity may be controlled by several putative cis-regulatory elements. These citrus promoters will expand the availability of regulatory sequences for driving gene expression in citrus gene-modification programs.

A comparative analysis of the avirulence and translational transactivator functions of gene VI of Cauliflower mosaic virus.

PubMed

Palanichelvam, Karuppaiah; Schoelz, James E

2002-02-15

The primary function associated at present with the gene VI product of Cauliflower mosaic virus (CaMV) is that of a translational transactivator (TAV). In this capacity, it alters the host translational machinery to allow reinitiation of translation of other CaMV genes on the polycistronic 35S RNA of CaMV. In addition, the gene VI protein can elicit a specific type of plant defense response called the hypersensitive response (HR) in Nicotiana edwardsonii. In this study, we have adapted the agroinfiltration technique to compare the sequences of CaMV gene VI required for TAV function and elicitation of HR. To measure the activity of the TAV, we coagroinfiltrated gene VI of CaMV strain W260 with a bicistronic GUS reporter plasmid. TAV function could be assayed 4 days postinfiltration, before the onset of HR in N. edwardsonii. Through the use of the TAV and HR assays, we could show that the TAV functions of gene VI of CaMV strains W260 and D4 were equivalent, but only W260 gene VI elicited HR. A mutational analysis of W260 gene VI showed that the structural requirements for elicitation of HR were much more stringent than those for TAV function. Small deletions from either the 5' or 3' end of W260 gene VI abolished its ability to elicit HR, although the TAV function was retained in the mutant. The TAV function could also tolerate a small insertion within gene VI; this insertion abolished the elicitor function. This study provides direct evidence that the TAV function of gene VI is separate from its role as an elicitor of HR.

Nuclear-encoded mitochondrial complex I gene expression is restored to normal levels by inhibition of unedited ATP9 transgene expression in Arabidopsis thaliana.

PubMed

Busi, María V; Gómez-Casati, Diego F; Perales, Mariano; Araya, Alejandro; Zabaleta, Eduardo

2006-01-01

Mitochondria play an important role during sporogenesis in plants. The steady state levels of the nuclear-encoded mitochondrial complex I (nCI), PSST, TYKY and NADHBP transcripts increase in flowers of male-sterile plants with impairment of mitochondrial function generated by the expression of the unedited version of ATP9 (u-ATP9). This suggests a nuclear control of nCI genes in response to the mitochondrial flaw. To evaluate this hypothesis, transgenic plants carrying the GUS reporter gene, under the control of the PSST, TYKY and NADHBP promoters, were constructed. We present evidence that suppression by antisense strategy of the expression of u-ATP9 restores the normal levels of three nCI transcripts, indicating that the increase in PSST, TYKY and NADHBP in plants with a mitochondrial flaw occurs at the transcriptional level. The data presented here support the hypothesis that a mitochondrial dysfunction triggers a retrograde signaling which induce some nuclear-encoded mitochondrial genes. Moreover, these results demonstrate that this is a valuable experimental model for studying nucleus-mitochondria cross-talk events.

Development of A Flexible System for the Simultaneous Conversion of Biomass to Industrial Chemicals and the Production of Industrial Biocatalysts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Johnway; Hooker, Brian S.; Skeen, R S.

2002-01-01

A flexible system was developed for the simultaneous conversion of biomass to industrial chemicals and the production of industrial biocatalysts. In particular, the expression of a bacterial enzyme, beta-glucuronidase (GUS), was investigated using a genetically modified starch-degrading Saccharomyces strain in suspension cultures in starch media. Different sources of starch including corn and waste potato starch were used for yeast biomass accumulation and GUS expression studies under controls of inducible and constitutive promoters. A thermostable bacterial cellulase, Acidothermus cellulolyticus E1 endoglucanase gene was also cloned into an episomal plasmid expression vector and expressed in the starch-degrading Saccharomyces strain.

Genome-wide analyses of late pollen-preferred genes conserved in various rice cultivars and functional identification of a gene involved in the key processes of late pollen development.

PubMed

Moon, Sunok; Oo, Moe Moe; Kim, Backki; Koh, Hee-Jong; Oh, Sung Aeong; Yi, Gihwan; An, Gynheung; Park, Soon Ki; Jung, Ki-Hong

2018-04-23

Understanding late pollen development, including the maturation and pollination process, is a key component in maintaining crop yields. Transcriptome data obtained through microarray or RNA-seq technologies can provide useful insight into those developmental processes. Six series of microarray data from a public transcriptome database, the Gene Expression Omnibus of the National Center for Biotechnology Information, are related to anther and pollen development. We performed a systematic and functional study across the rice genome of genes that are preferentially expressed in the late stages of pollen development, including maturation and germination. By comparing the transcriptomes of sporophytes and male gametes over time, we identified 627 late pollen-preferred genes that are conserved among japonica and indica rice cultivars. Functional classification analysis with a MapMan tool kit revealed a significant association between cell wall organization/metabolism and mature pollen grains. Comparative analysis of rice and Arabidopsis demonstrated that genes involved in cell wall modifications and the metabolism of major carbohydrates are unique to rice. We used the GUS reporter system to monitor the expression of eight of those genes. In addition, we evaluated the significance of our candidate genes, using T-DNA insertional mutant population and the CRISPR/Cas9 system. Mutants from T-DNA insertion and CRISPR/Cas9 systems of a rice gene encoding glycerophosphoryl diester phosphodiesterase are defective in their male gamete transfer. Through the global analyses of the late pollen-preferred genes from rice, we found several biological features of these genes. First, biological process related to cell wall organization and modification is over-represented in these genes to support rapid tube growth. Second, comparative analysis of late pollen preferred genes between rice and Arabidopsis provide a significant insight on the evolutional disparateness in cell wall biogenesis

The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28.

PubMed

Pla, M; Vilardell, J; Guiltinan, M J; Marcotte, W R; Niogret, M F; Quatrano, R S; Pagès, M

1993-01-01

The maize gene rab28 has been identified as ABA-inducible in embryos and vegetative tissues. It is also induced by water stress in young leaves. The proximal promoter region contains the conserved cis-acting element CCACGTGG (ABRE) reported for ABA induction in other plant genes. Transient expression assays in rice protoplasts indicate that a 134 bp fragment (-194 to -60 containing the ABRE) fused to a truncated cauliflower mosaic virus promoter (35S) is sufficient to confer ABA-responsiveness upon the GUS reporter gene. Gel retardation experiments indicate that nuclear proteins from tissues in which the rab28 gene is expressed can interact specifically with this 134 bp DNA fragment. Nuclear protein extracts from embryo and water-stressed leaves generate specific complexes of different electrophoretic mobility which are stable in the presence of detergent and high salt. However, by DMS footprinting the same guanine-specific contacts with the ABRE in both the embryo and leaf binding activities were detected. These results indicate that the rab28 promoter sequence CCACGTGG is a functional ABA-responsive element, and suggest that distinct regulatory factors with apparent similar affinity for the ABRE sequence may be involved in the hormone action during embryo development and in vegetative tissues subjected to osmotic stress.

Seamless editing of the chloroplast genome in plants.

PubMed

Martin Avila, Elena; Gisby, Martin F; Day, Anil

2016-07-29

Gene editing technologies enable the precise insertion of favourable mutations and performance enhancing trait genes into chromosomes whilst excluding all excess DNA from modified genomes. The technology gives rise to a new class of biotech crops which is likely to have widespread applications in agriculture. Despite progress in the nucleus, the seamless insertions of point mutations and non-selectable foreign genes into the organelle genomes of crops have not been described. The chloroplast genome is an attractive target to improve photosynthesis and crop performance. Current chloroplast genome engineering technologies for introducing point mutations into native chloroplast genes leave DNA scars, such as the target sites for recombination enzymes. Seamless editing methods to modify chloroplast genes need to address reversal of site-directed point mutations by template mediated repair with the vast excess of wild type chloroplast genomes that are present early in the transformation process. Using tobacco, we developed an efficient two-step method to edit a chloroplast gene by replacing the wild type sequence with a transient intermediate. This was resolved to the final edited gene by recombination between imperfect direct repeats. Six out of 11 transplastomic plants isolated contained the desired intermediate and at the second step this was resolved to the edited chloroplast gene in five of six plants tested. Maintenance of a single base deletion mutation in an imperfect direct repeat of the native chloroplast rbcL gene showed the limited influence of biased repair back to the wild type sequence. The deletion caused a frameshift, which replaced the five C-terminal amino acids of the Rubisco large subunit with 16 alternative residues resulting in a ~30-fold reduction in its accumulation. We monitored the process in vivo by engineering an overlapping gusA gene downstream of the edited rbcL gene. Translational coupling between the overlapping rbcL and gusA genes

A sensitive synthetic reporter for visualizing cytokinin signaling output in rice.

PubMed

Tao, Jinyuan; Sun, Huwei; Gu, Pengyuan; Liang, Zhihao; Chen, Xinni; Lou, Jiajing; Xu, Guohua; Zhang, Yali

2017-01-01

Cytokinins play many essential roles in plant growth and development, mainly through signal transduction pathways. Although the cytokinin signaling pathway in rice has been clarified, no synthetic reporter for cytokinin signaling output has been reported for rice. The sensitive synthetic reporter two-component signaling sensor ( TCSn ) is used in the model plant Arabidopsis; however, whether the reporter reflects the cytokinin signaling output pattern in rice remains unclear. Early-cytokinin-responsive type-A OsRR-binding element (A/G)GAT(C/T) was more clustered in the 15 type-A OsRRs than in the 13 control genes. Quantitative polymerase chain reaction analysis showed that the relative expression of seven type-A OsRRs in roots and shoots was significantly induced by exogenous cytokinin application, and that of seven OsRRs , mainly in roots, was inhibited by exogenous auxin application. We constructed a transgenic rice plant harboring a beta-glucuronidase (GUS) driven by the synthetic promoter TCSn . TCSn::GUS was expressed in the meristem of germinated rice seed and rice seedlings. Furthermore, TCSn::GUS expression in rice seedlings was induced specifically by exogenous cytokinin application and decreased by exogenous auxin application. Moreover, no obvious reduction in GUS levels was observed after three generations of selfing of transgenic plants, indicating that TCSn::GUS is not subject to transgene silencing. We report here a robust and sensitive synthetic sensor for monitoring the transcriptional output of the cytokinin signaling network in rice.

Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification.

PubMed

Pawar, Prashant Mohan-Anupama; Ratke, Christine; Balasubramanian, Vimal K; Chong, Sun-Li; Gandla, Madhavi Latha; Adriasola, Mathilda; Sparrman, Tobias; Hedenström, Mattias; Szwaj, Klaudia; Derba-Maceluch, Marta; Gaertner, Cyril; Mouille, Gregory; Ezcurra, Ines; Tenkanen, Maija; Jönsson, Leif J; Mellerowicz, Ewa J

2017-06-01

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula × tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Intein-mediated assembly of a functional β-glucuronidase in transgenic plants

PubMed Central

Yang, Jianjun; Fox, George C.; Henry-Smith, Tina V.

2003-01-01

The DnaE intein in Synechocystis sp. strain PCC6803 is the first and only naturally split intein that has been identified so far. It is capable of catalyzing a protein trans-splicing mechanism to assemble a mature protein from two separate precursors. Therefore, it is a powerful tool for protein modification and engineering. Inteins have not been identified, nor have intein-mediated protein splicing reactions been demonstrated, in plant cells. In this paper, we describe the use of the Ssp DnaE split intein in transgenic plants for reconstitution of a protein trans-splicing reaction. We have synthesized artificial genes that encode for N-terminal half (Int-n) and C-terminal half (Int-c) fragments of Ssp DnaE split intein and divided β-glucuronidase (GUS) gene to encode GUS-n and GUS-c parts of the enzyme as reporter. The in-frame fusions of GUSn/Intn and Intc/GUSc were constructed and transfected into Arabidopsis. We have observed in vivo reassembly of functional β-glucuronidase when both GUSn/Intn and Intc/GUSc constructs were introduced into the same Arabidopsis genome either by cotransformation or through genetic crossing, hereby signifying an intein-mediated protein trans-splicing mechanism reconstituted in plant cells. PMID:12629210

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.

GUS expression in Gladiolus plant controlled by two Gladiolus ubinquitin promotors

USDA-ARS?s Scientific Manuscript database

Ubiquitin represents a conserved family of genes that is involved in many metabolic processes. The most commonly used promoter for genetic engineering of cereal monocots is the maize ubiquitin promoter because it directs high levels of expression in most of the plant’s tissues, but this promoter re...

A Rosa canina WUSCHEL-related homeobox gene, RcWOX1, is involved in auxin-induced rhizoid formation.

PubMed

Gao, Bin; Wen, Chao; Fan, Lusheng; Kou, Yaping; Ma, Nan; Zhao, Liangjun

2014-12-01

Homeobox (HB) proteins are important transcription factors that regulate the developmental decisions of eukaryotes. WUSCHEL-related homeobox (WOX) transcription factors, known as a plant-specific HB family, play a key role in plant developmental processes. Our previous work has indicated that rhizoids are induced by auxin in rose (Rosa spp.), which acts as critical part of an efficient plant regeneration system. However, the function of WOX genes in auxin-induced rhizoid formation remains unclear. Here, we isolated and characterized a WUSCHEL-related homeobox gene from Rosa canina, RcWOX1, containing a typical homeodomain with 65 amino acid residues. Real-time reverse transcription PCR (qRT-PCR) analysis revealed that RcWOX1 was expressed in the whole process of callus formation and in the early stage of rhizoid formation. Moreover, its expression was induced by auxin treatment. In Arabidopsis transgenic lines expressing the RcWOX1pro::GUS and 35S::GFP-RcWOX1, RcWOX1 was specifically expressed in roots and localized to the nucleus. Overexpression of RcWOX1 in Arabidopsis increased lateral root density and induced upregulation of PIN1 and PIN7 genes. Therefore, we postulated that RcWOX1 is a functional transcription factor that plays an essential role in auxin-induced rhizoid formation.

Identification of a cluster IV pleiotropic drug resistance transporter gene expressed in the style of Nicotiana plumbaginifolia.

PubMed

Trombik, Tomasz; Jasinski, Michal; Crouzet, Jérome; Boutry, Marc

2008-01-01

ATP-binding cassette transporters of the pleiotropic drug resistance (PDR) subfamily are composed of five clusters. We have cloned a gene, NpPDR2, belonging to the still uncharacterized cluster IV from Nicotiana plumbaginifolia. NpPDR2 transcripts were found in the roots and mature flowers. In the latter, NpPDR2 expression was restricted to the style and only after pollination. A 1.5-kb genomic sequence containing the putative NpPDR2 transcription promoter was fused to the beta-glucuronidase reporter gene. The GUS expression pattern confirmed the RT-PCR results that NpPDR2 was expressed in roots and the flower style and showed that it was localized around the conductive tissues. Unlike other PDR genes, NpPDR2 expression was not induced in leaf tissues by none of the hormones typically involved in biotic and abiotic stress response. Moreover, unlike NpPDR1 known to be involved in biotic stress response, NpPDR2 expression was not induced in the style upon Botrytis cinerea infection. In N. plumbaginifolia plants in which NpPDR2 expression was prevented by RNA interference, no unusual phenotype was observed, including at the flowering stage, which suggests that NpPDR2 is not essential in the reproductive process under the tested conditions.

An ABI3-interactor of conifers responds to multiple hormones.

PubMed

Zeng, Ying; Zhao, Tiehan; Kermode, Allison

2013-11-01

CnAIP2 (Callitropsis nootkatensis ABI3-Interacting Protein 2) was previously identified as a protein that interacts with the yellow-cedar ABI3 protein. CnAIP2 plays important roles during several key transitions of the plant lifecycle and acts as a global regulator with functions opposite to those of ABI3 proteins. Here we report that the CnAIP2 gene promoter is strongly upregulated by all of the major plant hormones. Young Arabidopsis seedlings expressing a chimeric CnAIP2pro-GUS construct were subjected to exogenously applied hormones; the maximum fold-enhancement of GUS activity was as high as 47-fold, and each hormone showed a distinctive cell/tissue-specific pattern of GUS induction. By far the greatest response was elicited by the synthetic auxin 2,4-D (47-fold induction); the other hormones tested stimulated GUS activities by 8- to 21-fold. The CnAIP2 promoter also responded to glucose and salt (NaCl), albeit to a lesser extent (2- to 3-fold induction). As well as acting in an antagonistic way to the global regulator ABI3, CnAIP2 appears to participate in multiple hormonal crosstalk pathways to carry out its functions.

Heat shock protein Hsp90-2 expression in the Arabidopsis thaliana seedlings under clinorotation

NASA Astrophysics Data System (ADS)

Kozeko, Liudmyla

Heat shock proteins 90 kDa (Hsp90) are abundant under normal conditions and induced by stress. This family is distinguished from other chaperones in that most of its substrates are signal transduction proteins. Previously, we determined some time-dependent increase in the Hsp90 level in pea seedlings in response to simulated microgravity that indicated a stress-reaction. However, expression of the individual members of the Hsp90 family have specific pattern. The purpose of this study was to investigate possible alterations in the gene expression pattern of cytosolic Hsp90-2 in Arabidopsis thaliana seedlings under 2D-clinorotation. To obtain detailed expression pattern of the HSP90-2 genes we used seeds that provides a resource of loss-of-function mutations gene expression patterns via translational fusions with the reporter gene, GUS (a line N 166718, NASC). There were two variants of the experiment: 1) seedlings grew under clinorotation for 10, 12, 14 d; 2) seedlings grew in the stationary conditions for 10 d followed by clinorotation for 3 h -at 22o C and 16h light cycle. The seedlings grown in the stationary conditions were used as a control. GUS staining showed that HSP90-2 expression was regulated during seedling development and affected by clinorotation in the heterozygous mutant plants. In the homozygous for the mutation plants, HSP90-2 expression was stable during seedling development and not affected by clinorotation. GUS staining was observed in cotyledons, leaves and hypocotyls of the seedlings (especially intense in vascular bundles), indicating intensive cellular processes with participation of this chaperone. Possible pathways of influence of clinorotation on HSP90-2 expression are discussed.

Analysis of gene-disruption mutants of a sucrose phosphate synthase gene in rice, OsSPS1, shows the importance of sucrose synthesis in pollen germination.

PubMed

Hirose, Tatsuro; Hashida, Yoichi; Aoki, Naohiro; Okamura, Masaki; Yonekura, Madoka; Ohto, Chikara; Terao, Tomio; Ohsugi, Ryu

2014-08-01

The molecular function of an isoform of sucrose phosphate synthase (SPS) in rice, OsSPS1, was investigated using gene-disruption mutant lines generated by retrotransposon insertion. The progeny of the heterozygote of disrupted OsSPS1 (SPS1(+/-)) segregated into SPS1(+/+), SPS1(+/-), and SPS1(-/-) at a ratio of 1:1:0. This distorted segregation ratio, together with the expression of OsSPS1 in the developing pollen revealed by quantitative RT-PCR analysis and promoter-beta-glucuronidase (GUS) fusion assay, suggested that the disruption of OsSPS1 results in sterile pollen. This hypothesis was reinforced by reciprocal crosses of SPS1(+/-) plants with wild-type plants in which the disrupted OsSPS1 was not paternally transmitted to the progeny. While the pollen grains of SPS(+/-) plants normally accumulated starch during their development, pollen germination on the artificial media was reduced to half of that observed in the wild-type control. Overall, our data suggests that sucrose synthesis via OsSPS1 is essential in pollen germination in rice. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Differential distribution of proteins expressed in companion cells in the sieve element-companion cell complex of rice plants.

PubMed

Fukuda, Akari; Fujimaki, Syu; Mori, Tomoko; Suzui, Nobuo; Ishiyama, Keiki; Hayakawa, Toshihiko; Yamaya, Tomoyuki; Fujiwara, Toru; Yoneyama, Tadakatsu; Hayashi, Hiroaki

2005-11-01

Sieve tubes are comprised of sieve elements, enucleated cells that are incapable of RNA and protein synthesis. The proteins in sieve elements are supplied from the neighboring companion cells through plasmodesmata. In rice plants, it was unclear whether or not all proteins produced in companion cells had the same distribution pattern in the sieve element-companion cell complex. In this study, the distribution pattern of four proteins, beta-glucuronidase (GUS), green fluorescent protein (GFP), thioredoxin h (TRXh) and glutathione S-transferase (GST) were analyzed. The foreign proteins GUS and GFP were expressed in transgenic rice plants under the control of the TRXh gene promoter (PTRXh), a companion cell-specific promoter. Analysis of leaf cross-sections of PTRXh-GUS and PTRXh-GFP plants indicated high accumulation of GUS and GFP, respectively, in companion cells rather than in sieve elements. GUS and GFP were also detected in phloem sap collected from leaf sheaths of the transgenic rice plants, suggesting these proteins could enter sieve elements. Relative amounts of GFP and endogenous phloem proteins, TRXh and GST, in phloem sap and total leaf extracts were compared. Compared to TRXh and GST, GFP content was higher in total leaf extracts, but lower in phloem sap, suggesting that GFP accumulated mainly in companion cells rather than in sieve elements. On the other hand, TRXh and GST appeared to accumulate in sieve elements rather than in companion cells. These results indicate the evidence for differential distribution of proteins between sieve elements and companion cells in rice plants.

Cell-Type Specificity of the Expression of Os BOR1, a Rice Efflux Boron Transporter Gene, Is Regulated in Response to Boron Availability for Efficient Boron Uptake and Xylem Loading

PubMed Central

Nakagawa, Yuko; Hanaoka, Hideki; Kobayashi, Masaharu; Miyoshi, Kazumaru; Miwa, Kyoko; Fujiwara, Toru

2007-01-01

We describe a boron (B) transporter, Os BOR1, in rice (Oryza sativa). Os BOR1 is a plasma membrane–localized efflux transporter of B and is required for normal growth of rice plants under conditions of limited B supply (referred to as -B). Disruption of Os BOR1 reduced B uptake and xylem loading of B. The accumulation of Os BOR1 transcripts was higher in roots than that in shoots and was not affected by B deprivation; however, Os BOR1 was detected in the roots of wild-type plants under -B conditions, but not under normal conditions, suggesting regulation of protein accumulation in response to B nutrition. Interestingly, tissue specificity of Os BOR1 expression is affected by B treatment. Transgenic rice plants containing an Os BOR1 promoter–β-glucuronidase (GUS) fusion construct grown with a normal B supply showed the strongest GUS activity in the steles, whereas after 3 d of -B treatment, GUS activity was elevated in the exodermis. After 6 d of -B treatment, GUS activity was again strong in the stele. Our results demonstrate that Os BOR1 is required both for efficient B uptake and for xylem loading of B. Possible roles of the temporal changes in tissue-specific patterns of Os BOR1 expression in response to B condition are discussed. PMID:17675406

An efficient and reproducible protocol for the production of salt tolerant transgenic wheat plants expressing the Arabidopsis AtNHX1 gene.

PubMed

Moghaieb, Reda E A; Sharaf, Ahmed N; Soliman, Mohamed H; El-Arabi, Nagwa I; Momtaz, Osama A

2014-01-01

We present an efficient method for the production of transgenic salt tolerant hexaploid wheat plants expressing the Arabidopsis AtNHX1 gene. Wheat mature zygotic embryos were isolated from two hexaploid bread wheat (Triticum aestivum) cultivars (namely: Gemmeiza 9 and Gemmeiza 10) and were transformed with the A. tumefaciens LBA4404 harboring the pBI-121 vector containing the AtNHX1 gene. Transgenic wheat lines that express the gus intron was obtained and used as control. The results confirmed that npt-II gene could be transmitted and expressed in the T2 following 3:1 Mendelian segregation while the control plant couldn't. The data indicate that, the AtNHX1 gene was integrated in a stable manner into the wheat genome and the corresponding transcripts were expressed. The transformation efficiency was 5.7 and 7.5% for cultivars Gemmeiza 10 and Gemmeiza 9, respectively. A greenhouse experiment was conducted to investigate the effect of AtNHX1 gene in wheat salt tolerance. The transgenic wheat lines could maintain high growth rate under salt stress condition (350 mM NaCl) while the control plant couldn't. The results confirmed that Na(+)/H(+) antiporter gene AtNHX1 increased salt tolerance by increasing Na(+) accumulation and keeping K+/Na(+) balance. Thus, transgenic plants showed high tolerance to salt stress and can be considered as a new genetic resource in breeding programs.

Treatment of lysosomal storage disease in MPS VII mice using a recombinant adeno-associated virus.

PubMed

Watson, G L; Sayles, J N; Chen, C; Elliger, S S; Elliger, C A; Raju, N R; Kurtzman, G J; Podsakoff, G M

1998-12-01

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by a genetic deficiency of beta-glucuronidase (GUS). We used a recombinant adeno-associated virus vector (AAV-GUS) to deliver GUS cDNA to MPS VII mice. The route of vector administration had a dramatic effect on the extent and distribution of GUS activity. Intramuscular injection of AAV-GUS resulted in high, localized production of GUS, while intravenous administration produced low GUS activity in several tissues. This latter treatment of MPS VII mice reduced glycosaminoglycan levels in the liver to normal and reduced storage granules dramatically. We show that a single administration of AAV-GUS can provide sustained expression of GUS in a variety of cell types and is sufficient to reverse the disease phenotype at least in the liver.

Flower-predominant expression of a gene encoding a novel class I chitinase in rice (Oryza sativa L.).

PubMed

Takakura, Y; Ito, T; Saito, H; Inoue, T; Komari, T; Kuwata, S

2000-04-01

A flower-predominant cDNA for a gene, termed OsChia 1;175, was isolated from a cDNA library of rice pistils. Northern blot and RT-PCR analyses revealed that the OsChia 1;175 gene is highly expressed in floral organs (pistils, stamens and lodicules at the heading stage) but not or at an extremely low level in vegetative organs. OsChia 1;175 encodes a protein that consists of 340 amino acid residues, and the putative mature protein shows 52% to 63% amino acid identity to class I chitinases of rice or other plants. The phylogenetic tree shows that the OsChia 1;175 protein is a new type of plant class I chitinase in rice. The expression of OsChia 1;175 in vegetative organs is not induced by several chemicals, UV, and wounding. The soluble putative mature OsChia 1;175 protein expressed in Escherichia coli exhibited chitinase activity in the assay with colloidal chitin as a substrate. Genomic Southern analysis revealed that the OsChia 1;175 gene was organized as a low-copy gene family. The rice genomic library was screened and a genome clone corresponding to OsChia 1;175 was isolated. The transcription start sites of the OsChia 1;175 gene were mapped by primer extension analysis. The 1.2 kb putative promoter region of the OsChia 1;175 gene was fused to the GUS (beta-glucuronidase) gene, and this chimeric gene was introduced to rice by Agrobacterium-mediated transformation. The flower-predominant gene expression was identified also in the transgenic rice plants. The high promoter activity was detected in the stigmas, styles, stamens and lodicules in transgenic plants. The possible functions of OsChia 1;175 are discussed.

Efficient and stable transformation of hop (Humulus lupulus L.) var. Eroica by particle bombardment.

PubMed

Batista, Dora; Fonseca, Sandra; Serrazina, Susana; Figueiredo, Andreia; Pais, Maria Salomé

2008-07-01

To the best of our knowledge, this is the first accurate and reliable protocol for hop (Humulus lupulus L.) genetic transformation using particle bombardment. Based on the highly productive regeneration system previously developed by us for hop var. Eroica, two efficient transformation protocols were established using petioles and green organogenic nodular clusters (GONCs) bombarded with gusA reporter and hpt selectable genes. A total of 36 hygromycin B-resistant (hyg(r)) plants obtained upon continuous selection were successfully transferred to the greenhouse, and a first generation group of transplanted plants was followed after spending a complete vegetative cycle. PCR analysis showed the presence of one of both transgenes in 25 plants, corresponding to an integration frequency of 69.4% and an overall transformation efficiency of 7.5%. Although all final transformants were GUS negative, the integration frequency of gusA gene was higher than that of hpt gene. Petiole-derived transgenic plants showed a higher co-integration rate of 76.9%. Real-time PCR analysis confirmed co-integration in 86% of the plants tested and its stability until the first generation, and identified positive plants amongst those previously assessed as hpt (+) only by conventional PCR. Our results suggest that the integration frequencies presented here, as well as those of others, may have been underestimated, and that PCR results should be taken with precaution not only for false positives, but also for false negatives. The protocols here described could be very useful for future introduction of metabolic or resistance traits in hop cultivars even if slight modifications for other genotypes are needed.

In planta transformation method for T-DNA transfer in orchids

NASA Astrophysics Data System (ADS)

Semiarti, Endang; Purwantoro, Aziz; Mercuriani, Ixora S.; Anggriasari, Anida M.; Jang, Seonghoe; Suhandono, Sony; Machida, Yasunori; Machida, Chiyoko

2014-03-01

Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro∷PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1α2 pro∷GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromicin containing NP (New Phalaenopsis) medium. The existance of transgene in putative transformant protocorm (developing orchid embryo) genome was confirmed using PCR with specific primers of either PaFT or GUS genes. Histochemical GUS assay was also performed to the putative transformants. The result showed that transformation frequencies were 2.1 % in P. amabilis, and 0,53% in S. plicata. These results indicates that in planta transformation method could be used for Agrobacterium-mediated genetic transformation, with advantage easier and more secure work from contaminants than that of the in vitro method.

Evaluation of pollen dispersal and cross pollination using transgenic grapevine plants.

PubMed

Harst, Margit; Cobanov, Beatrix-Axinja; Hausmann, Ludger; Eibach, Rudolf; Töpfer, Reinhard

2009-01-01

Public debate about the possible risk of genetically modified plants often concerns putative effects of pollen dispersal and out-crossing into conventional fields in the neighborhood of transgenic plants. Though Vitis vinifera (grapevine) is generally considered to be self-pollinating, it cannot be excluded that vertical gene transfer might occur. For monitoring pollen flow and out-crossing events, transgenic plants of Vitis vinifera cv. 'Dornfelder' harboring the gus-int gene were planted in the center of a field experiment in Southwest Germany in 1999. The rate of pollen dispersal was determined by pollen traps placed at radial distances of 5-150 m from the pollen-donor plants, at 1.00 and 1.80 m above ground. Transgenic pollen was evaluated by GUS staining, and could clearly be distinguished from pollen originating from non-transgenic grapevine plants. Transgenic pollen was observed up to 150 m from the pollen donors. The rate of out-crossing was determined by sampling seeds of selected grapevines at a distance of 10 m to the pollen source, and of a sector at 20 m distance, respectively, followed by GUS analysis of seedlings. The average cross-pollination rate during the experiment (2002-2004) was 2.7% at a distance of 20 m. The results of this first pilot study present a good base for further assessment under the conditions of normal viticulture practice.

The GMD1 and GMD2 genes of Arabidopsis encode isoforms of GDP-D-mannose 4,6-dehydratase with cell type-specific expression patterns.

PubMed

Bonin, Christopher P; Freshour, Glenn; Hahn, Michael G; Vanzin, Gary F; Reiter, Wolf-Dieter

2003-06-01

l-Fucose (l-Fuc) is a monosaccharide constituent of plant cell wall polysaccharides and glycoproteins. The committing step in the de novo synthesis of l-Fuc is catalyzed by GDP-d-mannose 4,6-dehydratase, which, in Arabidopsis, is encoded by the GMD1 and GMD2 (MUR1) genes. To determine the functional significance of this genetic redundancy, the expression patterns of both genes were investigated via promoter-beta-glucuronidase fusions and immunolocalization of a Fuc-containing epitope. GMD2 is expressed in most cell types of the root, with the notable exception of the root tip where strong expression of GMD1 is observed. Within shoot organs, GMD1::GUS expression is confined to stipules and pollen grains leading to fucosylation of the walls of these cell types in the mur1 mutant. These results suggest that GMD2 represents the major housekeeping gene for the de novo synthesis of GDP-l-Fuc, whereas GMD1 expression is limited to a number of specialized cell types. We conclude that the synthesis of GDP-l-Fuc is controlled in a cell-autonomous manner by differential expression of two isoforms of the same enzyme.

A low-temperature-responsive element involved in the regulation of the Arabidopsis thaliana At1g71850/At1g71860 divergent gene pair.

PubMed

Liu, Shijuan; Chen, Huiqing; Li, Xiulan; Zhang, Wei

2016-08-01

The bidirectional promoter of the Arabidopsis thaliana gene pair At1g71850/At1g71860 harbors low-temperature-responsive elements, which participate in anti-correlated transcription regulation of the driving genes in response to environmental low temperature. A divergent gene pair is defined as two adjacent genes organized head to head in opposite orientation, sharing a common promoter region. Divergent gene pairs are mainly coexpressed, but some display opposite regulation. The mechanistic basis of such anti-correlated regulation is not well understood. Here, the regulation of the Arabidopsis thaliana gene pair At1g71850/At1g71860 was investigated. Semi-quantitative RT-PCR and Genevestigator analyses showed that while one of the pair was upregulated by exposure to low temperature, the same treatment downregulated the other. Promoter::GUS fusion transgenes were used to show that this behavior was driven by a bidirectional promoter, which harbored an as-1 motif, associated with the low-temperature response; mutation of this sequence produced a significant decrease in cold-responsive expression. With regard to the as-1 motif in the native orientation repressing the promoter's low-temperature responsiveness, the same as-1 motif introduced in the reverse direction showed a slight enhancement in the promoter's responsiveness to low-temperature exposure, indicating that the orientation of the motif was important for the promoter's activity. These findings provide new insights into the complex transcriptional regulation of bidirectional gene pairs as well as plant stress response.

Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane.

PubMed

Joyce, Priya; Kuwahata, Melissa; Turner, Nicole; Lakshmanan, Prakash

2010-02-01

A reproducible method for transformation of sugarcane using various strains of Agrobacterium tumefaciens (A. tumefaciens) (AGL0, AGL1, EHA105 and LBA4404) has been developed. The selection system and co-cultivation medium were the most important factors determining the success of transformation and transgenic plant regeneration. Plant regeneration at a frequency of 0.8-4.8% occurred only when callus was transformed with A. tumefaciens carrying a newly constructed superbinary plasmid containing neomycin phosphotransferase (nptII) and beta-glucuronidase (gusA) genes, both driven by the maize ubiquitin (ubi-1) promoter. Regeneration was successful in plants carrying the nptII gene but not the hygromycin phosphotransferase (hph) gene. NptII gene selection was imposed at a concentration of 150 mg/l paromomycin sulphate and applied either immediately or 4 days after the co-cultivation period. Co-cultivation on Murashige and Skoog (MS)-based medium for a period of 4 days produced the highest number of transgenic plants. Over 200 independent transgenic lines were created using this protocol. Regenerated plants appeared phenotypically normal and contained both gusA and nptII genes. Southern blot analysis revealed 1-3 transgene insertion events that were randomly integrated in the majority of the plants produced.

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

Transcriptional activation of a 37 kDa ethylene responsive cysteine protease gene, RbCP1, is associated with protein degradation during petal abscission in rose

PubMed Central

Tripathi, Siddharth Kaushal; Singh, Amar Pal; Sane, Aniruddha P.; Nath, Pravendra

2009-01-01

Cysteine proteases play an important role in several developmental processes in plants, particularly those related to senescence and cell death. A cysteine protease gene, RbCP1, has been identified that encodes a putative protein of 357 amino acids and is expressed in the abscission zone (AZ) of petals in rose. The gene was responsive to ethylene in petals, petal abscission zones, leaves, and thalamus. The expression of RbCP1 increased during both ethylene-induced as well as natural abscission and was inhibited by 1-MCP. Transcript accumulation of RbCP1 was accompanied by the appearance of a 37 kDa cysteine protease, a concomitant increase in protease activity and a substantial decrease in total protein content in the AZ of petals. Agro-injection of rose petals with a 2.0 kb region upstream of the RbCP1 gene could drive GUS expression in an abscission zone-specific manner and was blocked by 1-MCP. It is concluded that petal abscission is associated with a decrease in total protein content resulting from rapid transcription of RbCP1 and the expression of a 37 kDa protease. PMID:19346241

Long Circulating Enzyme Replacement Therapy Rescues Bone Pathology in Mucopolysaccharidosis VII Murine Model

PubMed Central

Rowan, Daniel J.; Tomatsu, Shunji; Grubb, Jeffrey H.; Haupt, Bisong; Montaño, Adriana M.; Oikawa, Hirotaka; Sosa, Catalina; Chen, Anping; Sly, William S.

2012-01-01

Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by deficiency of the lysosomal enzyme β-glucuronidase (GUS), leading to accumulation of glycosaminoglycans (GAGs). Enzyme replacement therapy (ERT) effectively clears GAG storage in the viscera. Recent studies showed that a chemically modified form of GUS (PerT-GUS), which escaped clearance by mannose 6-phosphate and mannose receptors and showed prolonged circulation, reduced CNS storage more effectively than native GUS. Clearance of storage in bone has been limited due to the avascularity of the growth plate. To evaluate the effectiveness of long-circulating PerT-GUS in reducing the skeletal pathology, we treated MPS VII mice for 12 weeks beginning at 5 weeks of age with PerT-GUS or native GUS and used micro-CT, radiographs, and quantitative histopathological analysis for assessment of bones. Micro-CT findings showed PerT-GUS treated mice had a significantly lower BMD. Histopathological analysis also showed reduced storage material and a more organized growth plate in PerT-GUS treated mice compared with native GUS treated mice. Long term treatment with PerT-GUS from birth up to 57 weeks also significantly improved bone lesions demonstrated by micro-CT, radiographs and quantitative histopathological assay. In conclusion, long-circulating PerT-GUS provides a significant impact to rescue of bone lesions and CNS involvement. PMID:22902520

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.

Spatial and temporal activity of the foxtail millet (Setaria italica) seed-specific promoter pF128.

PubMed

Pan, Yanlin; Ma, Xin; Liang, Hanwen; Zhao, Qian; Zhu, Dengyun; Yu, Jingjuan

2015-01-01

pF128 drives GUS specifically expressed in transgenic seeds of foxtail millet and Zea mays with higher activity than the constitutive CaMV35S promoter and the maize seed-specific 19Z promoter. Foxtail millet (Setaria italica), a member of the Poaceae family, is an important food and fodder crop in arid regions. Foxtail millet is an excellent C4 crop model owing to its small genome (~490 Mb), self-pollination and availability of a complete genome sequence. F128 was isolated from a cDNA library of foxtail millet immature seeds. Real-time PCR analysis revealed that F128 mRNA was specifically expressed in immature and mature seeds. The highest F128 mRNA level was observed 5 days after pollination and gradually decreased as the seed matured. Sequence analysis suggested that the protein encoded by F128 is likely a protease inhibitor/seed storage protein/lipid-transfer protein. The 1,053 bp 5' flanking sequence of F128 (pF128) was isolated and fused to the GUS reporter gene. The corresponding vector was then transformed into Arabidopsis thaliana, foxtail millet and Zea mays. GUS analysis revealed that pF128 drove GUS expression efficiently and specifically in the seeds of transgenic Arabidopsis, foxtail millet and Zea mays. GUS activity was also detected in Arabidopsis cotyledons. Activity of pF128 was higher than that observed for the constitutive CaMV35S promoter and the maize seed-specific 19 Zein (19Z) promoter. These results indicate that pF128 is a seed-specific promoter. Its application is expected to be of considerable value in plant genetic engineering.

Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis.

PubMed

Kim, Mi Jung; Baek, Kon; Park, Chung-Mo

2009-08-01

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The beta-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85-90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.

Controlled Aggregation and Increased Stability of β-Glucuronidase by Cellulose Binding Domain Fusion

PubMed Central

Kim, Moonjung; Kwon, Kil Koang; Fu, Yaoyao; Kim, Haseong; Lee, Hyewon; Lee, Dae-Hee; Jung, Heungchae; Lee, Seung-Goo

2017-01-01

Cellulose-binding domains (CBDs) are protein domains with cellulose-binding activity, and some act as leaders in the localization of cellulosomal scaffoldin proteins to the hydrophobic surface of crystalline cellulose. In this study, we found that a CBD fusion enhanced and improved soluble β-glucuronidase (GusA) enzyme properties through the formation of an artificially oligomeric state. First, a soluble CBD fused to the C-terminus of GusA (GusA-CBD) was obtained and characterized. Interestingly, the soluble GusA-CBD showed maximum activity at higher temperatures (65°C) and more acidic pH values (pH 6.0) than free GusA did (60°C and pH 7.5). Moreover, the GusA-CBD enzyme showed higher thermal and pH stabilities than the free GusA enzyme did. Additionally, GusA-CBD showed higher enzymatic activity in the presence of methanol than free GusA did. Evaluation of the protease accessibility of both enzymes revealed that GusA-CBD retained 100% of its activity after 1 h incubation in 0.5 mg/ml protease K, while free GusA completely lost its activity. Simple fusion of CBD as a single domain may be useful for tunable enzyme states to improve enzyme stability in industrial applications. PMID:28099480

Controlled Aggregation and Increased Stability of β-Glucuronidase by Cellulose Binding Domain Fusion.

PubMed

Yeom, Soo-Jin; Han, Gui Hwan; Kim, Moonjung; Kwon, Kil Koang; Fu, Yaoyao; Kim, Haseong; Lee, Hyewon; Lee, Dae-Hee; Jung, Heungchae; Lee, Seung-Goo

2017-01-01

Cellulose-binding domains (CBDs) are protein domains with cellulose-binding activity, and some act as leaders in the localization of cellulosomal scaffoldin proteins to the hydrophobic surface of crystalline cellulose. In this study, we found that a CBD fusion enhanced and improved soluble β-glucuronidase (GusA) enzyme properties through the formation of an artificially oligomeric state. First, a soluble CBD fused to the C-terminus of GusA (GusA-CBD) was obtained and characterized. Interestingly, the soluble GusA-CBD showed maximum activity at higher temperatures (65°C) and more acidic pH values (pH 6.0) than free GusA did (60°C and pH 7.5). Moreover, the GusA-CBD enzyme showed higher thermal and pH stabilities than the free GusA enzyme did. Additionally, GusA-CBD showed higher enzymatic activity in the presence of methanol than free GusA did. Evaluation of the protease accessibility of both enzymes revealed that GusA-CBD retained 100% of its activity after 1 h incubation in 0.5 mg/ml protease K, while free GusA completely lost its activity. Simple fusion of CBD as a single domain may be useful for tunable enzyme states to improve enzyme stability in industrial applications.

The AAP gene family for amino acid permeases contributes to development of the cyst nematode Heterodera schachtii in roots of Arabidopsis.

PubMed

Elashry, Abdelnaser; Okumoto, Sakiko; Siddique, Shahid; Koch, Wolfgang; Kreil, David P; Bohlmann, Holger

2013-09-01

The beet cyst nematode Heterodera schachtii is able to infect Arabidopsis plants and induce feeding sites in the root. These syncytia are the only source of nutrients for the nematodes throughout their life and are a nutrient sink for the host plant. We have studied here the role of amino acid transporters for nematode development. Arabidopsis contains a large number of different amino acid transporters in several gene families but those of the AAP family were found to be especially expressed in syncytia. Arabidopsis contains 8 AAP genes and they were all strongly expressed in syncytia with the exception of AAP5 and AAP7, which were slightly downregulated. We used promoter::GUS lines and in situ RT-PCR to confirm the expression of several AAP genes and LHT1, a lysine- and histidine-specific amino acid transporter, in syncytia. The strong expression of AAP genes in syncytia indicated that these transporters are important for the transport of amino acids into syncytia and we used T-DNA mutants for several AAP genes to test for their influence on nematode development. We found that mutants of AAP1, AAP2, and AAP8 significantly reduced the number of female nematodes developing on these plants. Our study showed that amino acid transport into syncytia is important for the development of the nematodes. Copyright © 2013 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses.

PubMed

Narusaka, Yoshihiro; Nakashima, Kazuo; Shinwari, Zabta K; Sakuma, Yoh; Furihata, Takashi; Abe, Hiroshi; Narusaka, Mari; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2003-04-01

Many abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (-174 to -55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis.

The context of transcription start site regions is crucial for transcription of a plant tRNA(Lys)(UUU) gene group both in vitro and in vivo.

PubMed

Yukawa, Yasushi; Akama, Kazuhito; Noguchi, Kanta; Komiya, Masaaki; Sugiura, Masahiro

2013-01-10

Nuclear tRNA genes are transcribed by RNA polymerase III. The A- and B-boxes located within the transcribed regions are essential promoter elements for nuclear tRNA gene transcription. The Arabidopsis genome contains ten annotated genes encoding identical tRNA(Lys)(UUU) molecules, which are scattered on the five chromosomes. In this study, we prepared ten tDNA constructs including each of the tRNA(Lys)(UUU) coding sequences with their individual 5' and 3' flanking sequences, and assayed tRNA expression using an in vitro RNA polymerase III-dependent transcription system. Transcription levels differed significantly among the ten genes and two of the tRNA genes were transcribed at a very low level, despite possessing A- and B-boxes identical to those of the other tRNA genes. To examine whether the in vitro results were reproducible in vivo, the 5' flanking sequence of an amber suppressor tRNA gene was then replaced with those of the ten tRNA(Lys) genes. An in vivo experiment based on an amber suppressor tRNA that mediates suppression of a premature amber codon in a β-glucuronidase (GUS) reporter gene in plant tissues generated nearly identical results to those obtained in vitro. Analysis of mutated versions of the amber suppressor tRNA gene, which contained base substitutions around the transcription start site (TSS), showed that the context around the transcription start sites is a crucial determinant for transcription of plant tRNA(Lys)(UUU) both in vitro and in vivo. The above transcription regulation by context around TSS differed between tRNA genes and other Pol III-dependent genes. Copyright © 2012 Elsevier B.V. All rights reserved.

A Gossypium hirsutum GDSL lipase/hydrolase gene (GhGLIP) appears to be involved in promoting seed growth in Arabidopsis.

PubMed

Ma, Rendi; Yuan, Hali; An, Jing; Hao, Xiaoyun; Li, Hongbin

2018-01-01

GDSL lipase (GLIP) plays a pivotal role in plant cell growth as a multifunctional hydrolytic enzyme. Herein, a cotton (Gossypium hirsutum L. cv Xuzhou 142) GDSL lipase gene (GhGLIP) was obtained from developing ovules and fibers. The GhGLIP cDNA contained an open reading frame (ORF) of 1,143 base pairs (bp) and encodes a putative polypeptide of 380 amino acid residues. Sequence alignment indicated that GhGLIP includes four enzyme catalytic amino acid residue sites of Ser (S), Gly (G), Asn (N) and His (H), located in four conserved blocks. Phylogenetic tree analysis showed that GhGLIP belongs to the typical class IV lipase family with potential functions in plant secondary metabolism. Subcellular distribution analysis demonstrated that GhGLIP localized to the nucleus, cytoplasm and plasma membrane. GhGLIP was expressed predominantly at 5-15 day post anthesis (dpa) in developing ovules and elongating fibers, measured as mRNA levels and enzyme activity. Ectopic overexpression of GhGLIP in Arabidopsis plants resulted in enhanced seed development, including length and fresh weight. Meanwhile, there was increased soluble sugar and protein storage in transgenic Arabidopsis plants, coupled with the promotion of lipase activity. Moreover, the expression of cotton GhGLIP is induced by ethylene (ETH) treatment in vitro. A 1,954-bp GhGLIP promoter was isolated and expressed high activity in driving green fluorescence protein (GFP) expression in tobacco leaves. Cis-acting element analysis of the GhGLIP promoter (pGhGLIP) indicated the presence of an ethylene-responsive element (ERE), and transgenic tobacco leaves with ectopic expression of pGhGLIP::GFP-GUS showed increased GUS activity after ETH treatment. In summary, these results suggest that GhGLIP is a functional enzyme involved in ovule and fiber development and performs significant roles in seed development.

A Gossypium hirsutum GDSL lipase/hydrolase gene (GhGLIP) appears to be involved in promoting seed growth in Arabidopsis

PubMed Central

An, Jing; Hao, Xiaoyun

2018-01-01

GDSL lipase (GLIP) plays a pivotal role in plant cell growth as a multifunctional hydrolytic enzyme. Herein, a cotton (Gossypium hirsutum L. cv Xuzhou 142) GDSL lipase gene (GhGLIP) was obtained from developing ovules and fibers. The GhGLIP cDNA contained an open reading frame (ORF) of 1,143 base pairs (bp) and encodes a putative polypeptide of 380 amino acid residues. Sequence alignment indicated that GhGLIP includes four enzyme catalytic amino acid residue sites of Ser (S), Gly (G), Asn (N) and His (H), located in four conserved blocks. Phylogenetic tree analysis showed that GhGLIP belongs to the typical class IV lipase family with potential functions in plant secondary metabolism. Subcellular distribution analysis demonstrated that GhGLIP localized to the nucleus, cytoplasm and plasma membrane. GhGLIP was expressed predominantly at 5–15 day post anthesis (dpa) in developing ovules and elongating fibers, measured as mRNA levels and enzyme activity. Ectopic overexpression of GhGLIP in Arabidopsis plants resulted in enhanced seed development, including length and fresh weight. Meanwhile, there was increased soluble sugar and protein storage in transgenic Arabidopsis plants, coupled with the promotion of lipase activity. Moreover, the expression of cotton GhGLIP is induced by ethylene (ETH) treatment in vitro. A 1,954-bp GhGLIP promoter was isolated and expressed high activity in driving green fluorescence protein (GFP) expression in tobacco leaves. Cis-acting element analysis of the GhGLIP promoter (pGhGLIP) indicated the presence of an ethylene-responsive element (ERE), and transgenic tobacco leaves with ectopic expression of pGhGLIP::GFP-GUS showed increased GUS activity after ETH treatment. In summary, these results suggest that GhGLIP is a functional enzyme involved in ovule and fiber development and performs significant roles in seed development. PMID:29621331

Structural requirements of oleosin domains for subcellular targeting to the oil body.

PubMed Central

van Rooijen, G J; Moloney, M M

1995-01-01

We have investigated the protein domains responsible for the correct subcellular targeting of plant seed oleosins. We have attempted to study this targeting in vivo using "tagged" oleosins in transgenic plants. Different constructs were prepared lacking gene sequences encoding one of three structural domains of natural oleosins. Each was fused in frame to the Escherichia coli uid A gene encoding beta-glucuronidase (GUS). These constructs were introduced into Brassica napus using Agrobacterium-mediated transformation. GUS activity was measured in washed oil bodies and in the soluble protein fraction of the transgenic seeds. It was found that complete Arabidopsis oleosin-GUS fusions undergo correct subcellular targeting in transgenic Brassica seeds. Removal of the C-terminal domain of the Arabidopsis oleosin comprising the last 48 amino acids had no effect on overall subcellular targeting. In contrast, loss of the first 47 amino acids (N terminus) or amino acids 48 to 113 (which make up a lipophilic core) resulted in impaired targeting of the fusion protein to the oil bodies and greatly reduced accumulation of the fusion protein. Northern blotting revealed that this reduction is not due to differences in mRNA accumulation. Results from these measurements indicated that both the N-terminal and central oleosin domain are important for targeting to the oil body and show that there is a direct correlation between the inability to target to the oil body and protein stability. PMID:8539295

Molecular characterization of the sweet potato peroxidase SWPA4 promoter which responds to abiotic stresses and pathogen infection.

PubMed

Ryu, Sun-Hwa; Kim, Yun-Hee; Kim, Cha Young; Park, Soo-Young; Kwon, Suk-Yoon; Lee, Haeng-Soon; Kwak, Sang-Soo

2009-04-01

Previously, the swpa4 peroxidase gene has been shown to be inducible by a variety of abiotic stresses and pathogenic infections in sweet potato (Ipomoea batatas). To elucidate its regulatory mechanism at the transcriptional level under various stress conditions, we isolated and characterized the promoter region (2374 bp) of swpa4 (referred to as SWPA4). We performed a transient expression assay in tobacco protoplasts with deletions from the 5'-end of SWPA4 promoter fused to the beta-glucuronidase (GUS) reporter gene. The -1408 and -374 bp deletions relative to the transcription start site (+1) showed 8 and 4.5 times higher GUS expression than the cauliflower mosaic virus 35S promoter, respectively. In addition, transgenic tobacco plants expressing GUS under the control of -2374, -1408 or -374 bp region of SWPA4 promoter were generated and studied in various tissues under abiotic stresses and pathogen infection. Gel mobility shift assays revealed that nuclear proteins from sweet potato cultured cells specifically interacted with 60-bp fragment (-178/-118) in -374 bp promoter region. In silico analysis indicated that four kinds of cis-acting regulatory sequences, reactive oxygen species-related element activator protein 1 (AP1), CCAAT/enhancer-binding protein alpha element, ethylene-responsive element (ERE) and heat-shock element, are present in the -60 bp region (-178/-118), suggesting that the -60 bp region might be associated with stress inducibility of the SWPA4 promoter.

Promoter regions of potato vacuolar invertase gene in response to sugars and hormones.

PubMed

Ou, Yongbin; Song, Botao; Liu, Xun; Xie, Conghua; Li, Meng; Lin, Yuan; Zhang, Huiling; Liu, Jun

2013-08-01

Potato vacuolar acid invertase (StvacINV1) (β-fructofuranosidase; EC 3.2.1.26) has been confirmed to play an important role in cold-induced sweetening of potato tubers. However, the transcriptional regulation mechanisms of StvacINV1 are largely unknown. In this study, the 5'-flanking sequence of StvacINV1 was cloned and the cis-acting elements were predicted. Histochemical assay showed that the StvacINV1 promoter governed β-glucuronidase (GUS) expression in potato leaves, stems, roots and tubers. Quantitative analysis of GUS expression suggested that the activity of StvacINV1 promoter was suppressed by sucrose, glucose, fructose, and cold, while enhanced by indole-3-acetic acid (IAA), and gibberellic acid (GA3). Further deletion analysis clarified that the promoter regions from -118 to -551, -551 to -1021, and -1021 to -1521 were required for responding to sucrose/glucose, GA3, and IAA, respectively. These findings provide essential information regarding transcriptional regulation mechanisms of StvacINV1. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz).

PubMed

Beltrán, J; Prías, M; Al-Babili, S; Ladino, Y; López, D; Beyer, P; Chavarriaga, P; Tohme, J

2010-05-01

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.

A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence.

PubMed

Balazadeh, Salma; Siddiqui, Hamad; Allu, Annapurna D; Matallana-Ramirez, Lilian P; Caldana, Camila; Mehrnia, Mohammad; Zanor, Maria-Inés; Köhler, Barbara; Mueller-Roeber, Bernd

2010-04-01

The onset and progression of senescence are under genetic and environmental control. The Arabidopsis thaliana NAC transcription factor ANAC092 (also called AtNAC2 and ORE1) has recently been shown to control age-dependent senescence, but its mode of action has not been analysed yet. To explore the regulatory network administered by ANAC092 we performed microarray-based expression profiling using estradiol-inducible ANAC092 overexpression lines. Approximately 46% of the 170 genes up-regulated upon ANAC092 induction are known senescence-associated genes, suggesting that the NAC factor exerts its role in senescence through a regulatory network that includes many of the genes previously reported to be senescence regulated. We selected 39 candidate genes and confirmed their time-dependent response to enhanced ANAC092 expression by quantitative RT-PCR. We also found that the majority of them (24 genes) are up-regulated by salt stress, a major promoter of plant senescence, in a manner similar to that of ANAC092, which itself is salt responsive. Furthermore, 24 genes like ANAC092 turned out to be stage-dependently expressed during seed growth with low expression at early and elevated expression at late stages of seed development. Disruption of ANAC092 increased the rate of seed germination under saline conditions, whereas the opposite occurred in respective overexpression plants. We also detected a delay of salinity-induced chlorophyll loss in detached anac092-1 mutant leaves. Promoter-reporter (GUS) studies revealed transcriptional control of ANAC092 expression during leaf and flower ageing and in response to salt stress. We conclude that ANAC092 exerts its functions during senescence and seed germination through partly overlapping target gene sets.

A petal-specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops.

PubMed

Azuma, Mirai; Morimoto, Reina; Hirose, Mana; Morita, Yasumasa; Hoshino, Atsushi; Iida, Shigeru; Oshima, Yoshimi; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Shiratake, Katsuhiro

2016-01-01

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal-specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal-specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal-specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β-glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal-specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA-like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal-specific promoter in molecular breeding of floricultural crops. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer.

PubMed

Cheng, X; Sardana, R; Kaplan, H; Altosaar, I

1998-03-17

Over 2,600 transgenic rice plants in nine strains were regenerated from >500 independently selected hygromycin-resistant calli after Agrobacterium-mediated transformation. The plants were transformed with fully modified (plant codon optimized) versions of two synthetic cryIA(b) and cryIA(c) coding sequences from Bacillus thuringiensis as well as the hph and gus genes, coding for hygromycin phosphotransferase and beta-glucuronidase, respectively. These sequences were placed under control of the maize ubiquitin promoter, the CaMV35S promoter, and the Brassica Bp10 gene promoter to achieve high and tissue-specific expression of the lepidopteran-specific delta-endotoxins. The integration, expression, and inheritance of these genes were demonstrated in R0 and R1 generations by Southern, Northern, and Western analyses and by other techniques. Accumulation of high levels (up to 3% of soluble proteins) of CryIA(b) and CryIA(c) proteins was detected in R0 plants. Bioassays with R1 transgenic plants indicated that the transgenic plants were highly toxic to two major rice insect pests, striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas), with mortalities of 97-100% within 5 days after infestation, thus offering a potential for effective insect resistance in transgenic rice plants.

Arabidopsis miR171-Targeted Scarecrow-Like Proteins Bind to GT cis-Elements and Mediate Gibberellin-Regulated Chlorophyll Biosynthesis under Light Conditions

PubMed Central

Ma, Zhaoxue; Hu, Xupeng; Cai, Wenjuan; Huang, Weihua; Zhou, Xin; Luo, Qian; Yang, Hongquan; Wang, Jiawei; Huang, Jirong

2014-01-01

An extraordinarily precise regulation of chlorophyll biosynthesis is essential for plant growth and development. However, our knowledge on the complex regulatory mechanisms of chlorophyll biosynthesis is very limited. Previous studies have demonstrated that miR171-targeted scarecrow-like proteins (SCL6/22/27) negatively regulate chlorophyll biosynthesis via an unknown mechanism. Here we showed that SCLs inhibit the expression of the key gene encoding protochlorophyllide oxidoreductase (POR) in light-grown plants, but have no significant effect on protochlorophyllide biosynthesis in etiolated seedlings. Histochemical analysis of β-glucuronidase (GUS) activity in transgenic plants expressing pSCL27::rSCL27-GUS revealed that SCL27-GUS accumulates at high levels and suppresses chlorophyll biosynthesis at the leaf basal proliferation region during leaf development. Transient gene expression assays showed that the promoter activity of PORC is indeed regulated by SCL27. Consistently, chromatin immunoprecipitation and quantitative PCR assays showed that SCL27 binds to the promoter region of PORC in vivo. An electrophoretic mobility shift assay revealed that SCL27 is directly interacted with G(A/G)(A/T)AA(A/T)GT cis-elements of the PORC promoter. Furthermore, genetic analysis showed that gibberellin (GA)-regulated chlorophyll biosynthesis is mediated, at least in part, by SCLs. We demonstrated that SCL27 interacts with DELLA proteins in vitro and in vivo by yeast-two-hybrid and coimmunoprecipitation analysis and found that their interaction reduces the binding activity of SCL27 to the PORC promoter. Additionally, we showed that SCL27 activates MIR171 gene expression, forming a feedback regulatory loop. Taken together, our data suggest that the miR171-SCL module is critical for mediating GA-DELLA signaling in the coordinate regulation of chlorophyll biosynthesis and leaf growth in light. PMID:25101599

A Genetic Transformation Method for Cadmium Hyperaccumulator Sedum plumbizincicola and Non-hyperaccumulating Ecotype of Sedum alfredii

PubMed Central

Liu, Huan; Zhao, Haixia; Wu, Longhua; Xu, Wenzhong

2017-01-01

The present study demonstrates the development of an Agrobacterium-mediated genetic transformation method for species of the Sedum genus, which includes the Cd/Zn hyperaccumulator Sedum plumbizincicola and the non-hyperaccumulating ecotype of S. alfredii. Multiple shoots were induced from stem nodes of two Sedum plants using Murashige and Skoog (MS) medium containing 0.1 mg/L cytokinin 6-benzyladenine (6-BA) and 1.0 mg/L auxin 1-naphthaleneacetic acid (NAA). The shoot primordia were used as direct targets for Agrobacterium infection. Selection on hygromycin was highly effective in generating Agrobacterium-transformed explants. This callus-free procedure allowed us to obtain transgenic plantlets after rooting hygromycin-resistant shoots on phytohormone-free MS medium containing the antibiotic. The presence and expression of the reporter genes gusA and GFP in transgenic plants were confirmed by a real-time polymerase chain reaction, histochemical GUS assays, and confocal microscopy. This reliable method for genetic transformation of Sedum plants will help us to understand gene functions and the molecular mechanisms underlying Cd hypertolerance and hyperaccumulation in these species. PMID:28670322

Isolation and characterization of Acidithiobacillus caldus from a sulfur-oxidizing bacterial biosensor and its role in detection of toxic chemicals.

PubMed

Hassan, Sedky H A; Van Ginkel, Steven W; Kim, Sung-Min; Yoon, Sung-Hwan; Joo, Jin-Ho; Shin, Beom-Soo; Jeon, Byong-Hun; Bae, Wookeun; Oh, Sang-Eun

2010-08-01

A novel toxicity detection methodology based on sulfur-oxidizing bacteria (SOB) has been developed for the rapid and reliable detection of toxic chemicals in water. The methodology exploits the ability of SOB to oxidize sulfur particles in the presence of oxygen to produce sulfuric acid. The reaction results in an increase in electrical conductivity (EC) and a decrease in pH. The assay is based on the inhibition of SOB in the presence of toxic chemicals by measuring changes in EC and pH. We found that SOB biosensor can detect toxic chemicals, such as heavy metals and CN-, in the 5-2000ppb range. One bacterium was isolated from an SOB biosensor and the 16S rRNA gene of the bacterial strain has 99% and 96% sequence similarity to Acidithiobacillus sp. ORCS6 and Acidithiobacillus caldus DSM 8584, respectively. The isolate was identified as A. caldus SMK. The SOB biosensor is ideally suited for monitoring toxic chemicals in water having the advantages of high sensitivity and quick detection.

Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana.

PubMed

Schliep, Martin; Ebert, Berit; Simon-Rosin, Ulrike; Zoeller, Daniela; Fisahn, Joachim

2010-05-01

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes.

Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana

PubMed Central

Schliep, Martin; Ebert, Berit; Simon-Rosin, Ulrike; Zoeller, Daniela

2010-01-01

Gene expression levels of several transcription factors from Arabidopsis thaliana that were described previously to be involved in leaf development and trichome formation were analysed in trichome, basal and pavement cells of mature leaves. Single cell samples of these three cells types were collected by glass micro-capillaries. Real-time reverse transcription (RT)-PCR was used to analyse expression patterns of the following transcription factors: MYB23, MYB55, AtHB1, FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1), TRIPTYCHON (TRY) and CAPRICE (CPC). A difference in the expression patterns of TRY and CPC was revealed. Contrary to the CPC expression pattern, no transcripts of TRY could be detected in pavement cells. FIL/YAB1 was exclusively expressed in trichome cells. AtHB1 was highly expressed throughout all three cell types. MYB55 was higher expressed in basal cells than in trichome and pavement cells. MYB23 showed a pattern of low expression in pavement cells, medium in basal cells and high expression in trichomes. Expression patterns obtained by single cell sampling and real-time RT-PCR were compared to promoter GUS fusions of the selected transcription factors. Therefore, we regenerated two transgenic Arabidopsis lines that expressed the GUS reporter gene under control of the promoters of MYB55 and YAB1. In conclusion, despite their function in leaf morphogenesis, all six transcription factors were detected in mature leaves. Furthermore, single cell sampling and promoter GUS staining patterns demonstrated the predominant presence of MYB55 in basal cells as compared to pavement cells and trichomes. PMID:20101514

Process and economic evaluation of the extraction and purification of recombinant beta-glucuronidase from transgenic corn

PubMed

Evangelista; Kusnadi; Howard; Nikolov

1998-07-01

A process model for the recovery and purification of recombinant beta-glucuronidase (rGUS) from transgenic corn was developed, and the process economics were estimated. The base-case bioprocessing plant operates 7500 h/year processing 1.74 million (MM) kg of transgenic corn containing 0.015% (db) rGUS. The process consists of milling the corn into flour, extraction of protein by using 50 mM sodium phosphate buffer, and rGUS purification by ion exchange and hydrophobic interaction chromatography. About 137 kg of rGUS of 83% (db) purity can be produced annually. The production cost amounted to $43 000/kg of rGUS. The cost of milling, protein extraction, and rGUS purification accounted for 6, 40, and 48% of annual operating cost, respectively. The cost of transgenic corn was 31% of the raw material costs or 6% of the annual operating cost. About 78% of the cost of buffer and water were incurred in the protein extraction section, while 88% of other consumables were from the purification section. The sensitivity analysis indicated that rGUS can be produced profitably from corn even at the 0.015% (db) expression level, assuming a selling price of $100 000/kg GUS. An increase in rGUS expression levels up to 0.08% significantly improves the process economics.

Structure-activity relationships of flavonoids as natural inhibitors against E. coli β-glucuronidase.

PubMed

Weng, Zi-Miao; Wang, Ping; Ge, Guang-Bo; Dai, Zi-Ru; Wu, Da-Chang; Zou, Li-Wei; Dou, Tong-Yi; Zhang, Tong-Yan; Yang, Ling; Hou, Jie

2017-11-01

Bacterial β-glucuronidases play key roles in the deconjugation of a variety of endogenous and drug glucuronides, thus have been recognized as important targets to modulate the enterohepatic circulation of various glucuronides. In this study, more than 30 natural flavonoids were collected and their inhibitory effects against E. coli β-glucuronidase (EcGUS) were assayed. The results demonstrated that some flavonoids including scutellarein, luteolin, baicalein, quercetin and scutellarin displayed strong to moderate inhibitory effects against EcGUS, with the IC 50 values ranging from 5.76 μM to 29.64 μM, while isoflavones and dihydroflavones displayed weak inhibitory effects against EcGUS. Further investigation on inhibition kinetics revealed that scutellarein and luteolin functioned as potent competitive inhibitors against EcGUS-mediated PNPG hydrolysis, with the K i values less than 3.0 μM. Molecular docking simulations demonstrated that scutellarein and luteolin could be well-docked into the catalytic site of EcGUS, while the binding areas of these two natural inhibitors on EcGUS were highly overlapped with that of PNPG on EcGUS. Additionally, the structure-inhibition relationships of natural flavonoids against EcGUS are also summarized, which will be very helpful for the medicinal chemists to design and develop more potent flavonoid-type inhibitors against EcGUS. Copyright © 2017 Elsevier Ltd. All rights reserved.

The expression pattern of the Picea glauca Defensin 1 promoter is maintained in Arabidopsis thaliana, indicating the conservation of signalling pathways between angiosperms and gymnosperms.

PubMed

Germain, Hugo; Lachance, Denis; Pelletier, Gervais; Fossdal, Carl Gunnar; Solheim, Halvor; Séguin, Armand

2012-01-01

A 1149 bp genomic fragment corresponding to the 5' non-coding region of the PgD1 (Picea glauca Defensin 1) gene was cloned, characterized, and compared with all Arabidopsis thaliana defensin promoters. The cloned fragment was found to contain several motifs specific to defence or hormonal response, including a motif involved in the methyl jasmonate reponse, a fungal elicitor responsive element, and TC-rich repeat cis-acting element involved in defence and stress responsiveness. A functional analysis of the PgD1 promoter was performed using the uidA (GUS) reporter system in stably transformed Arabidopsis and white spruce plants. The PgD1 promoter was responsive to jasmonic acid (JA), to infection by fungus and to wounding. In transgenic spruce embryos, GUS staining was clearly restricted to the shoot apical meristem. In Arabidopsis, faint GUS coloration was observed in leaves and flowers and a strong blue colour was observed in guard cells and trichomes. Transgenic Arabidopsis plants expressing the PgD1::GUS construct were also infiltrated with the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. It caused a suppression of defensin expression probably resulting from the antagonistic relationship between the pathogen-stimulated salicylic acid pathway and the jasmonic acid pathway. It is therefore concluded that the PgD1 promoter fragment cloned appears to contain most if not all the elements for proper PgD1 expression and that these elements are also recognized in Arabidopsis despite the phylogenetic and evolutionary differences that separates them.

Analysis of CFB, a cytokinin-responsive gene of Arabidopsis thaliana encoding a novel F-box protein regulating sterol biosynthesis.

PubMed

Brenner, Wolfram G; Leuendorf, Jan Erik; Cortleven, Anne; Martin, Laetitia B B; Schaller, Hubert; Schmülling, Thomas

2017-05-17

Protein degradation by the ubiquitin-26S proteasome pathway is important for the regulation of cellular processes, but the function of most F-box proteins relevant to substrate recognition is unknown. We describe the analysis of the gene Cytokinin-induced F-box encoding (CFB, AT3G44326), identified in a meta-analysis of cytokinin-related transcriptome studies as one of the most robust cytokinin response genes. F-box domain-dependent interaction with the E3 ubiquitin ligase complex component ASK1 classifies CFB as a functional F-box protein. Apart from F-box and transmembrane domains, CFB contains no known functional domains. CFB is expressed in all plant tissues, predominantly in root tissue. A ProCFB:GFP-GUS fusion gene showed strongest expression in the lateral root cap and during lateral root formation. CFB-GFP fusion proteins were mainly localized in the nucleus and the cytosol but also at the plasma membrane. cfb mutants had no discernible phenotype, but CFB overexpressing plants showed several defects, such as a white upper inflorescence stem, similar to the hypomorphic cycloartenol synthase mutant cas1-1. Both CFB overexpressing plants and cas1-1 mutants accumulated the CAS1 substrate 2,3-oxidosqualene in the white stem tissue, the latter even more after cytokinin treatment, indicating impairment of CAS1 function. This suggests that CFB may link cytokinin and the sterol biosynthesis pathway. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Characterization of promoter of EgPAL1, a novel PAL gene from the oil palm Elaeis guineensis Jacq.

PubMed

Yusuf, Chong Yu Lok; Abdullah, Janna Ong; Shaharuddin, Noor Azmi; Abu Seman, Idris; Abdullah, Mohd Puad

2018-02-01

The oil palm EgPAL1 gene promoter and its regulatory region were functional as a promoter in the heterologous system of Arabidopsis according to the cis-acting elements present in that region. The promoter was developmentally regulated, vascular tissue specific and responsive to water stress agents. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) is the key enzyme of the phenylpropanoid pathway which plays important roles in plant development and adaptation. To date, there is no report on the study of PAL from oil palm (Elaeis guineensis), an economically important oil crop. In this study, the 5' regulatory sequence of a highly divergent oil palm PAL gene (EgPAL1) was isolated and fused with GUS in Arabidopsis to create two transgenic plants carrying the minimal promoter with (2302 bp) and without its regulatory elements (139 bp). The regulatory sequence contained cis-acting elements known to be important for plant development and stress response including the AC-II element for lignin biosynthesis and several stress responsive elements. The promoter and its regulatory region were fully functional in Arabidopsis. Its activities were characterised by two common fundamental features of PAL which are responsive to plant internal developmental programme and external factors. The promoter was developmentally regulated in certain organs; highly active in young organs but less active or inactive in mature organs. The presence of the AC elements and global activity of the EgPAL1 promoter in all organs resembled the property of lignin-related genes. The existence of the MBS element and enhancement of the promoter activity by PEG reflected the behaviour of drought-responsive genes. Our findings provide a platform for evaluating oil palm gene promoters in the heterologous system of Arabidopsis and give insights into the activities of EgPAL1 promoter in oil palm.

AGO1 controls arabidopsis inflorescence architecture possibly by regulating TFL1 expression.

PubMed

Fernández-Nohales, P; Domenech, M J; Martínez de Alba, A E; Micol, J L; Ponce, M R; Madueño, F

2014-11-01

The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators. Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (β-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype. A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants. The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression. © The Author 2014. Published by Oxford

Spatial Control of Gene Expression by miR319-Regulated TCP Transcription Factors in Leaf Development.

PubMed

Bresso, Edgardo G; Chorostecki, Uciel; Rodriguez, Ramiro E; Palatnik, Javier F; Schommer, Carla

2018-02-01

The characteristic leaf shapes we see in all plants are in good part the outcome of the combined action of several transcription factor networks that translate into cell division activity during the early development of the organ. We show here that wild-type leaves have distinct transcriptomic profiles in center and marginal regions. Certain transcripts are enriched in margins, including those of CINCINNATA -like TCPs ( TEOSINTE BRANCHED, CYCLOIDEA and PCF1/2 ) and members of the NGATHA and STYLISH gene families. We study in detail the contribution of microRNA319 (miR319)-regulated TCP transcription factors to the development of the center and marginal regions of Arabidopsis ( Arabidopsis thaliana ) leaves. We compare in molecular analyses the wild type, the tcp2 tcp4 mutant that has enlarged flat leaves, and the tcp2 tcp3 tcp4 tcp10 mutant with strongly crinkled leaves. The different leaf domains of the tcp mutants show changed expression patterns for many photosynthesis-related genes, indicating delayed differentiation, especially in the marginal parts of the organ. At the same time, we found an up-regulation of cyclin genes and other genes that are known to participate in cell division, specifically in the marginal regions of tcp2 tcp3 tcp4 tcp10 Using GUS reporter constructs, we confirmed extended mitotic activity in the tcp2 tcp3 tcp4 tcp10 leaf, which persisted in small defined foci in the margins when the mitotic activity had already ceased in wild-type leaves. Our results describe the role of miR319-regulated TCP transcription factors in the coordination of activities in different leaf domains during organ development. © 2018 American Society of Plant Biologists. All Rights Reserved.

Genetic transformation of carnation (Dianthus caryophylus L.).

PubMed

Nontaswatsri, Chalermsri; Fukai, Seiichi

2010-01-01

This chapter describes a rapid and efficient protocol for explant preparation and genetic transformation of carnation. Node explants from greenhouse-grown plants and leaf explants from in vitro plants are infected with Agrobacterium tumefaciens AGL0 harboring pKT3 plasmid, consisting of GUS and NPTII genes. Explant preparation is an important factor to obtain the transformed plants. The GUS-staining area was located only on the cut end of explants and only explants with a cut end close to the connecting area between node and leaf, produced transformed shoots. The cocultivation medium is also an important factor for the successful genetic transformation of carnation node and leaf explants. High genetic transformation efficiency of node and leaf explants cocultured with Agrobacterium tumefaciens was achieved when the explants were cocultivated on a filter paper soaked with water or water and acetosyringone mixture (AS).

Functional dissection of a napin gene promoter: identification of promoter elements required for embryo and endosperm-specific transcription.

PubMed

Ellerström, M; Stålberg, K; Ezcurra, I; Rask, L

1996-12-01

The promoter region (-309 to +44) of the Brassica napus storage protein gene napA was studied in transgenic tobacco by successive 5' as well as internal deletions fused to the reporter gene GUS (beta-glucuronidase). The expression in the two main tissues of the seed, the endosperm and the embryo, was shown to be differentially regulated. This tissue-specific regulation within the seed was found to affect the developmental expression during seed development. The region between -309 to -152, which has a large effect on quantitative expression, was shown to harbour four elements regulating embryo and one regulating endosperm expression. This region also displayed enhancer activity. Deletion of eight bp from position -152 to position -144 totally abolished the activity of the napA promoter. This deletion disrupted a cis element with similarity to an ABA-responsive element (ABRE) overlapping with an E-box, demonstrating its crucial importance for quantitative expression. An internal deletion of the region -133 to -120, resulted in increased activity in both leaves and endosperm and a decreased activity in the embryo. Within this region, a cis element similar to the (CA)n element, found in other storage protein promoters, was identified. This suggest that the (CA)n element is important for conferring seed specificity by serving both as an activator and a repressor element.

Identifying the location and population served by domestic wells in California

USGS Publications Warehouse

Johnson, Tyler D.; Belitz, Kenneth

2015-01-01

Aggregating the results indicates that three hydrogeologic provinces contain nearly 80% of all domestic wells and also have the highest density of domestic well users: Central Valley (31.6%), Sierra Nevada (31.5%), and Northern Coast Ranges (16.6%). Results were also aggregated into groundwater basins and highland areas, collectively called Groundwater Units (GUs). Twenty-eight of the 938 GUs contain more than 50% of the total population served by domestic wells, 70 GUs contain more than 75%, and 150 GUs contain 90%. The 28 GUs are mostly located in the eastern and southern San Joaquin Valley (11), the Sacramento Valley (7), and the western foothills of the Sierra Nevada province (5). Using the information presented in this research along with other information about domestic-well use, the US Geological Survey has begun sampling high-use GUs for the Shallow Aquifer Assessment component of the Groundwater Ambient Assessment (GAMA) program.

A new mutant of Arabidopsis disturbed in its roots, right-handed slanting, and gravitropism defines a gene that encodes a heat-shock factor.

PubMed

Fortunati, A; Piconese, S; Tassone, P; Ferrari, S; Migliaccio, F

2008-01-01

A new mutant of Arabidopsis named rha1 is characterized and the gene involved cloned. In roots, the mutant shows minimal right-handed slanting, reduced gravitropic response, notable resistance to 2,4-D, but scarce resistance to IAA and NAA. The roots also show a clear resistance to the auxin transport inhibitors TIBA and NPA, and to ethylene. Other characteristics are a reduced number of lateral roots and reduced size of shoot and root in the seedlings. The gene, cloned through TAIL-PCR, was found to be a heat-shock factor that maps on chromosome 5, close to and above the RFLP marker m61. The rha1 structure, mRNA, and translation product are reported. Since, so far, no other gravitropic mutant has been described as mutated in a heat-shock factor, rha1 belongs to a new group of mutants disturbed in slanting, gravitropism, and auxin physiology. As shown through the RT-PCR analyses of its expression, the gene retains the function connected with heat shock. If the characteristics connected with auxin physiology are considered, however, it is also likely that the gene, as a transcription factor, could be involved in root circumnutation, gravitropic response, and hormonal control of differentiation. Since GUS staining under the gene promoter was localized mainly in the mature tissues, rha1 does not seem to be involved in the first steps of gravitropism, but is rather related to the general response to auxin. The alterations in slanting (mainly due to reduced chiral circumnutation) and gravitropism lead to the supposition that the two processes may have, at least in part, common origins.

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens.

PubMed

Norzagaray-Valenzuela, Claudia D; Germán-Báez, Lourdes J; Valdez-Flores, Marco A; Hernández-Verdugo, Sergio; Shelton, Luke M; Valdez-Ortiz, Angel

2018-05-16

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD 600  = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications. Copyright © 2018 Elsevier B.V. All rights reserved.

A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana.

PubMed

Tripathi, Jaindra N; Oduor, Richard O; Tripathi, Leena

2015-01-01

Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties "Cavendish Williams" and "Gros Michel" were developed using multiple buds, whereas ECS of "Sukali Ndiizi" was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000-50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20-70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa.

Wheat Chloroplast Targeted sHSP26 Promoter Confers Heat and Abiotic Stress Inducible Expression in Transgenic Arabidopsis Plants

PubMed Central

Khurana, Neetika; Chauhan, Harsh; Khurana, Paramjit

2013-01-01

The small heat shock proteins (sHSPs) have been found to play a critical role in physiological stress conditions in protecting proteins from irreversible aggregation. To characterize the hloroplast targeted sHSP26 promoter in detail, deletion analysis of the promoter is carried out and analysed via transgenics in Arabidopsis. In the present study, complete assessment of the importance of CCAAT-box elements along with Heat shock elements (HSEs) in the promoter of sHSP26 was performed. Moreover, the importance of 5′ untranslated region (UTR) has also been established in the promoter via Arabidopsis transgenics. An intense GUS expression was observed after heat stress in the transgenics harbouring a full-length promoter, confirming the heat-stress inducibility of the promoter. Transgenic plants without UTR showed reduced GUS expression when compared to transgenic plants with UTR as was confirmed at the RNA and protein levels by qRT-PCR and GUS histochemical assays, thus suggesting the possible involvement of some regulatory elements present in the UTR in heat-stress inducibility of the promoter. Promoter activity was also checked under different abiotic stresses and revealed differential expression in different deletion constructs. Promoter analysis based on histochemical assay, real-time qPCR and fluorimetric analysis revealed that HSEs alone could not transcribe GUS gene significantly in sHSP26 promoter and CCAAT box elements contribute synergistically to the transcription. Our results also provide insight into the importance of 5`UTR of sHsp26 promoter thus emphasizing the probable role of imperfect CCAAT-box element or some novel cis-element with respect to heat stress. PMID:23349883

Characterization of a type-A response regulator differentially expressed during adventitious caulogenesis in Pinus pinaster.

PubMed

Alvarez, José M; Cortizo, Millán; Ordás, Ricardo J

2012-12-15

The molecular cloning and characterization of PipsRR1, a type-A response regulator in Pinus pinaster, is reported here. Type-A response regulators mediate downstream responses to cytokinin and act as negative feedback regulators of the signal transduction pathway. Some type-A response regulators in Arabidopsis have been related to de novo meristem formation. However, little information exists in Pinus spp. The PipsRR1 gene contains 5 exons, as do all type-A response regulators in Arabidopsis, and the deduced protein contains a receiver domain with the conserved DDK residues and a short C terminal extension. Expression analysis showed that the PipsRR1 gene is differentially expressed during the first phases of adventitious caulogenesis induced by benzyladenine in P. pinaster cotyledons, suggesting that PipsRR1 plays a role in caulogenesis in conifers. Additionally, a binary vector carrying the PipsRR1 promoter driving GFP:GUS expression was constructed to analyze the promoter activity in P. pinaster somatic embryos. The results of genetic transformation showed GUS activity during somatic embryo mass proliferation and embryo maturation. Copyright © 2012 Elsevier GmbH. All rights reserved.

Induction of hypoxic root metabolism results from physical limitations in O 2 bioavailability in microgravity

NASA Astrophysics Data System (ADS)

Liao, J.; Liu, G.; Monje, O.; Stutte, G. W.; Porterfield, D. M.

2004-01-01

Numerous spaceflight experiments have noted changes in the roots that are consistent with hypoxia in the rootzone. These observations include general ultrastructure analysis and biochemical measurements to direct measurements of stress specific enzymes. In experiments that have monitored alcohol dehydrogenase (ADH), the data shows this hypoxically responsive gene is induced and is associated with increased ADH activity in microgravity. These changes in ADH could be induced either by spaceflight hypoxia resulting from inhibition of gravity mediated O 2 transport, or by a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in a series of two experiments. The objective of the first experiment was to determine if physical changes in gravity-mediated O 2 transport can be directly measured, while the second series of experiments tested whether disruption of gravisensing can induce a non-specific ADH response. To directly measure O 2 bioavailability as a function of gravity, we designed a sensor that mimics metabolic oxygen consumption in the rhizosphere. Because of these criteria, the sensor is sensitive to any changes in root O 2 bioavailability that may occur in microgravity. In a KC-135 experiment, the sensor was implanted in a moist granular clay media and exposed to microgravity during parabolic flight. The resulting data indicated that root O 2 bioavailability decreased in phase with gravity. In experiments that tested for non-specific induction of ADH, we compared the response of transgenic Arabidopsis plants (ADH promoted GUS marker gene) exposed to clinostat, control, and waterlogged conditions. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH

Production of herbicide-resistant transgenic Panax ginseng through the introduction of the phosphinothricin acetyl transferase gene and successful soil transfer.

PubMed

Choi, Y E; Jeong, J H; In, J K; Yang, D C

2003-02-01

Herbicide-resistant transgenic Panax ginseng plants were produced by introducing the phosphinothricin acetyl transferase (PAT) gene that confers resistance to the herbicide Basta (bialaphos) through Agrobacterium tumefaciens co-cultivation. Embryogenic callus gathered from cotyledon explants of P. ginseng were pre-treated with 0.5 M sucrose or 0.05 M MgSO(4 )before Agrobacterium infection. This pre-treatment process markedly enhanced the transient expression of the beta-glucuronidase (GUS) gene. Embryogenic callus was initially cultured on MS medium supplemented with 400 mg/l cefotaxime for 3 weeks and subsequently subcultured five times to a medium containing 25 mg/l kanamycin and 300 mg/l cefotaxime. Somatic embryos formed on the surfaces of kanamycin-resistant callus. Upon development into the cotyledonary stage, these somatic embryos were transferred to a medium containing 50 mg/l kanamycin and 5 mg/l gibberellic acid to induce germination and strong selection. Integration of the transgene into the plants was confirmed by polymerase chain reaction and Southern analyses. Transfer of the transgenic ginseng plantlets to soil was successfully accomplished via acclimatization in autoclaved perlite. Not all of the plantlets survived in soil that had not been autoclaved because of fungal infection, particularly in the region between the roots and leaves. Transgenic plants growing in soil were observed to be strongly resistant to Basta application.

Jatropha (Jatropha curcas L.).

PubMed

Maravi, Devendra Kumar; Mazumdar, Purabi; Alam, Shamsher; Goud, Vaibhav V; Sahoo, Lingaraj

2015-01-01

The seed oil of Jatropha (Jatropha curcas L.) as a source of biodiesel fuel is gaining worldwide importance. Commercial-scale exploration of Jatropha has not succeeded due to low and unstable seed yield in semiarid lands unsuitable for the food production and infestation to diseases. Genetic engineering is promising to improve various agronomic traits in Jatropha and to understand the molecular functions of key Jatropha genes for molecular breeding. We describe a protocol routinely followed in our laboratory for stable and efficient Agrobacterium tumefaciens-mediated transformation of Jatropha using cotyledonary leaf as explants. The 4-day-old explants are infected with Agrobacterium tumefaciens strain EHA105 harboring pBI121 plant binary vector, which contains nptII as plant selectable marker and gus as reporter. The putative transformed plants are selected on kanamycin, and stable integration of transgene(s) is confirmed by histochemical GUS assay, polymerase chain reaction, and Southern hybridization.

Development of Useful Recombinant Promoter and Its Expression Analysis in Different Plant Cells Using Confocal Laser Scanning Microscopy

PubMed Central

Kumar, Deepak; Sahoo, Dipak K.; Maiti, Indu B.; Dey, Nrisingha

2011-01-01

Background Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s). Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. Methodology/Principal Findings We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS) of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27) and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, −271 to +31). Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt) showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN) and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. Conclusion and Significance We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in a wide variety

Overexpression of OsRAA1 Causes Pleiotropic Phenotypes in Transgenic Rice Plants, including Altered Leaf, Flower, and Root Development and Root Response to Gravity1

PubMed Central

Ge, Lei; Chen, Hui; Jiang, Jia-Fu; Zhao, Yuan; Xu, Ming-Li; Xu, Yun-Yuan; Tan, Ke-hui; Xu, Zhi-Hong; Chong, Kang

2004-01-01

There are very few root genes that have been described in rice as a monocotyledonous model plant so far. Here, the OsRAA1 (Oryza sativa Root Architecture Associated 1) gene has been characterized molecularly. OsRAA1 encodes a 12.0-kD protein that has 58% homology to the AtFPF1 (Flowering Promoting Factor 1) in Arabidopsis, which has not been reported as modulating root development yet. Data of in situ hybridization and OsRAA1∷GUS transgenic plant showed that OsRAA1 expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive expression of OsRAA1 under the control of maize (Zea mays) ubiquitin promoter resulted in phenotypes of reduced growth of primary root, increased number of adventitious roots and helix primary root, and delayed gravitropic response of roots in seedlings of rice (Oryza sativa), which are similar to the phenotypes of the wild-type plant treated with auxin. With overexpression of OsRAA1, initiation and growth of adventitious root were more sensitive to treatment of auxin than those of the control plants, while their responses to 9-hydroxyfluorene-9-carboxylic acid in both transgenic line and wild type showed similar results. OsRAA1 constitutive expression also caused longer leaves and sterile florets at the last stage of plant development. Analysis of northern blot and GUS activity staining of OsRAA1∷GUS transgenic plants demonstrated that the OsRAA1 expression was induced by auxin. At the same time, overexpression of OsRAA1 also caused endogenous indole-3-acetic acid to increase. These data suggested that OsRAA1 as a new gene functions in the development of rice root systems, which are mediated by auxin. A positive feedback regulation mechanism of OsRAA1 to indole-3-acetic acid metabolism may be involved in rice root development in nature. PMID:15247372

Functional Characterization of the Poplar R2R3-MYB Transcription Factor PtoMYB216 Involved in the Regulation of Lignin Biosynthesis during Wood Formation

PubMed Central

Lu, Wanxiang; Yang, Li; Jiang, Yuanzhong; Luo, Keming

2013-01-01

Because of the importance of wood in many industrial applications, tremendous studies have been performed on wood formation, especially in lignin biosynthesis. MYB transcription factors (TFs), which consist of a large family of plant TFs, have been reported to directly regulate lignin biosynthetic genes in a number of plants. In this study, we describe the cloning and functional characterization of PtoMYB216, a cDNA isolated from Chinese white poplar (Populus tomentosa Carr.). PtoMYB216 encodes a protein belonging to the R2R3-MYB family and displays significant similarity with other MYB factors shown to regulate lignin synthesis in Arabidopsis. Gene expression profiling studies showed that PtoMYB216 mRNA is specifically expressed during secondary wall formation in wood. The 1.8-kb promoter sequence of PtoMYB216 was fused to the GUS coding sequence and introduced into wild-type A. thaliana. GUS expression was shown to be restricted to tissues undergoing secondary cell wall formation. Overexpression of PtoMYB216 specifically activated the expression of the upstream genes in the lignin biosynthetic pathway and resulted in ectopic deposition of lignin in cells that are normally unligninified. These results suggest that PtoMYB216 is specific transcriptional activators of lignin biosynthesis and involved in the regulation of wood formation in poplar. PMID:24204619

Gibberellin modulates anther development in rice via the transcriptional regulation of GAMYB.

PubMed

Aya, Koichiro; Ueguchi-Tanaka, Miyako; Kondo, Maki; Hamada, Kazuki; Yano, Kentaro; Nishimura, Mikio; Matsuoka, Makoto

2009-05-01

Gibberellins (GAs) play important roles in regulating reproductive development, especially anther development. Our previous studies revealed that the MYB transcriptional factor GAMYB, an important component of GA signaling in cereal aleurone cells, is also important for anther development. Here, we examined the physiological functions of GA during anther development through phenotypic analyses of rice (Oryza sativa) GA-deficient, GA-insensitive, and gamyb mutants. The mutants exhibited common defects in programmed cell death (PCD) of tapetal cells and formation of exine and Ubisch bodies. Microarray analysis using anther RNAs of these mutants revealed that rice GAMYB is involved in almost all instances of GA-regulated gene expression in anthers. Among the GA-regulated genes, we focused on two lipid metabolic genes, a cytochrome P450 hydroxylase CYP703A3 and beta-ketoacyl reductase, both of which might be involved in providing a substrate for exine and Ubisch body. GAMYB specifically interacted with GAMYB binding motifs in the promoter regions in vitro, and mutation of these motifs in promoter-beta-glucuronidase (GUS) transformants caused reduced GUS expression in anthers. Furthermore, a knockout mutant for CYP703A3 showed gamyb-like defects in exine and Ubisch body formation. Together, these results suggest that GA regulates exine formation and the PCD of tapetal cells and that direct activation of CYP703A3 by GAMYB is key to exine formation.

Expression of ORF2 partial gene of hepatitis E virus in tomatoes and immunoactivity of expression products.

PubMed

Ma, Ying; Lin, Shun-Quan; Gao, Yi; Li, Mei; Luo, Wen-Xin; Zhang, Jun; Xia, Ning-Shao

2003-10-01

To transfer hepatitis E virus (HEV) ORF2 partial gene to tomato plants, to investigate its expression in transformants and the immunoactivity of expression products, and to explore the feasibility of developing a new type of plant-derived HEV oral vaccine. Plant binary expression vector p1301E2, carrying a fragment of HEV open reading frame-2 (named HEV-E2), was constructed by linking the fragment to a constitutive CaMV35s promoter and nos terminator, then directly introduced into Agrobacterium tumefaciens EHA105. With leaf-disc method, tomato plants medicated by EHA105 were transformed and hygromycin-resistant plantlets were obtained in selective medium containing hygromycin. The presence and integration of foreign DNA in transgenic tomato genome were confirmed by Gus gene expression, PCR amplification and Southern dot blotting. The immunoactivity of recombinant protein extracted from transformed plants was examined by enzyme-linked immunosorbant assay (ELISA) using a monoclonal antibody specifically against HEV. ELISA was also used to estimate the recombinant protein content in leaves and fruits of the transformants. Seven positive lines of HEV-E2-transgenic tomato plants confirmed by PCR and Southern blotting were obtained and the immunoactivity of recombinant protein could be detected in extracts of transformants. The expression levels of recombinant protein were 61.22 ng/g fresh weight in fruits and 6.37-47.9 ng/g fresh weight in leaves of the transformants. HEV-E2 gene was correctly expressed in transgenic tomatoes and the recombinant antigen derived from them has normal immunoactivity. Transgenic tomatoes may hold a good promise for producing a new type of low-cost oral vaccine for hepatitis E virus.

Defining the Pathway for Tat-mediated Delivery of β-Glucuronidase in Cultured Cells and MPS VII Mice

PubMed Central

Orii, Koji O.; Grubb, Jeffrey H.; Vogler, Carole; Levy, Beth; Tan, Yun; Markova, Kamelia; Davidson, Beverly L.; Mao, Q.; Orii, Tadao; Kondo, Naomi; Sly, William S.

2008-01-01

We used recombinant forms of human β-glucuronidase (GUS) purified from secretions from stably transfected CHO cells to compare the native enzyme to a GUS-Tat C-terminal fusion protein containing the 11-amino-acid HIV Tat protein transduction domain for: (1) susceptibility to endocytosis by cultured cells, (2) rate of clearance following intravenous infusion, and (3) tissue distribution and effectiveness in clearing lysosomal storage following infusion in the MPS VII mouse. We found: (1) Native GUS was more efficiently taken up by cultured human fibroblasts and its endocytosis was exclusively mediated by the M6P receptor. The GUS-Tat fusion protein showed only 30-50% as much M6P-receptor-mediated uptake, but also was taken up by adsorptive endocytosis through binding of the positively charged Tat peptide to cell surface proteoglycans. (2) GUS-Tat was less rapidly cleared from the circulation in the rat (t1/2 = 13 min vs 7 min). (3) Delivery to most tissues of the MPS VII mouse was similar, but GUS-Tat was more efficiently delivered to kidney. Histology showed that GUS-Tat more efficiently reduced storage in renal tubules, retina, and bone. These studies demonstrate that Tat modification can extend the range of tissues corrected by infused enzyme. PMID:16043103

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

Growing Up Strong: A Mental Wellness and Life Skills Development Program for Fourth Graders.

ERIC Educational Resources Information Center

Hammer, Judith McGowan; O'Bar, Angelina Merenda

Growing Up Strong (GUS) is a curriculum designed to develop strong mental and physical health in kindergarten through sixth grade students, with the objective of preventing subsequent substance abuse. This document contains a teacher's guide for using GUS in fourth grade classrooms (GUS 4) and duplication masters of materials that can be sent home…

Growing Up Strong: A Mental Wellness and Life Skills Development Program for Fifth and Sixth Graders.

ERIC Educational Resources Information Center

Hammer, Judith McGowan; O'Bar, Angelina Merenda

Growing Up Strong (GUS) is a curriculum designed to develop strong mental and physical health in kindergarten through sixth grade students, with the objective of preventing subsequent substance abuse. This document contains a teacher's guide for using GUS in fifth and sixth grade classrooms (GUS 5-6) and duplication masters of materials that can…

Divergent expression of cytokinin biosynthesis, signaling and catabolism genes underlying differences in feeding sites induced by cyst and root-knot nematodes.

PubMed

Dowd, Carola D; Chronis, Demosthenis; Radakovic, Zoran S; Siddique, Shahid; Schmülling, Thomas; Werner, Tomáš; Kakimoto, Tatsuo; Grundler, Florian M W; Mitchum, Melissa G

2017-10-01

Cyst and root-knot nematodes are obligate parasites of economic importance with a remarkable ability to reprogram root cells into unique metabolically active feeding sites. Previous studies have suggested a role for cytokinin in feeding site formation induced by these two types of nematodes, but the mechanistic details have not yet been described. Using Arabidopsis as a host plant species, we conducted a comparative analysis of cytokinin genes in response to the beet cyst nematode (BCN), Heterodera schachtii, and the root-knot nematode (RKN), Meloidogyne incognita. We identified distinct differences in the expression of cytokinin biosynthesis, catabolism and signaling genes in response to infection by BCN and RKN, suggesting differential manipulation of the cytokinin pathway by these two nematode species. Furthermore, we evaluated Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 in response to RKN infection. Similar to our previous studies with BCN, these lines were significantly less susceptible to RKN without compromising nematode penetration, suggesting a requirement of cytokinin signaling in RKN feeding site formation. Moreover, an analysis of ahk double mutants using CycB1;1:GUS/ahk introgressed lines revealed contrasting differences in the cytokinin receptors mediating cell cycle activation in feeding sites induced by BCN and RKN. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies

PubMed Central

2011-01-01

Background Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. Methods Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. Results Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) renal excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. Conclusion GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells. PMID:21989091

Ammonium-induced loss of root gravitropism is related to auxin distribution and TRH1 function, and is uncoupled from the inhibition of root elongation in Arabidopsis.

PubMed

Zou, Na; Li, Baohai; Dong, Gangqiang; Kronzucker, Herbert J; Shi, Weiming

2012-06-01

Root gravitropism is affected by many environmental stresses, including salinity, drought, and nutrient deficiency. One significant environmental stress, excess ammonium (NH(4)(+)), is well documented to inhibit root elongation and lateral root formation, yet little is known about its effects on the direction of root growth. We show here that inhibition of root elongation upon elevation of external NH(4)(+) is accompanied by a loss in root gravitropism (agravitropism) in Arabidopsis. Addition of potassium (K(+)) to the treatment medium partially rescued the inhibition of root elongation by high NH(4)(+) but did not improve gravitropic root curvature. Expression analysis of the auxin-responsive reporter gene DR5::GUS revealed that NH(4)(+) treatment delayed the development of gravity-induced auxin gradients across the root cap but extended their duration once initiated. Moreover, the β-glucuronidase (GUS) signal intensity in root tip cells was significantly reduced under high NH(4)(+) treatment over time. The potassium carrier mutant trh1 displayed different patterns of root gravitropism and DR5::GUS signal intensity in root apex cells compared with the wild type in response to NH(4)(+). Together, the results demonstrate that the effects of NH(4)(+) on root gravitropism are related to delayed lateral auxin redistribution and the TRH1 pathway, and are largely independent of inhibitory effects on root elongation.

Proteomic identification of an embryo-specific 1Cys-Prx promoter and analysis of its activity in transgenic rice.

PubMed

Kim, Je Hein; Jung, In Jung; Kim, Dool Yi; Fanata, Wahyu Indra; Son, Bo Hwa; Yoo, Jae Yong; Harmoko, Rikno; Ko, Ki Seong; Moon, Jeong Chan; Jang, Ho Hee; Kim, Woe Yeon; Kim, Jae-Yean; Lim, Chae Oh; Lee, Sang Yeol; Lee, Kyun Oh

2011-04-29

Proteomic analysis of a rice callus led to the identification of 10 abscisic acid (ABA)-induced proteins as putative products of the embryo-specific promoter candidates. 5'-flanking sequence of 1 Cys-Prx, a highly-induced protein gene, was cloned and analyzed. The transcription initiation site of 1 Cys-Prx maps 96 nucleotides upstream of the translation initiation codon and a TATA-box and putative seed-specific cis-acting elements, RYE and ABRE, are located 26, 115 and 124 bp upstream of the transcription site, respectively. β-glucuronidase (GUS) expression driven by the 1 Cys-Prx promoters was strong in the embryo and aleurone layer and the activity reached up to 24.9 ± 3.3 and 40.5 ± 2.1 pmol (4 MU/min/μg protein) in transgenic rice seeds and calluses, respectively. The activity of the 1 Cys-Prx promoters is much higher than that of the previously-identified embryo-specific promoters, and comparable to that of strong endosperm-specific promoters in rice. GUS expression driven by the 1 Cys-Prx promoters has been increased by ABA treatment and rapidly induced by wounding in callus and at the leaf of the transgenic plants, respectively. Furthermore, ectopic expression of the GUS construct in Arabidopsis suggested that the 1 Cys-Prx promoter also has strong activity in seeds of dicot plants. Copyright © 2011 Elsevier Inc. All rights reserved.

The rose (Rosa hybrida) NAC transcription factor 3 gene, RhNAC3, involved in ABA signaling pathway both in rose and Arabidopsis.

PubMed

Jiang, Guimei; Jiang, Xinqiang; Lü, Peitao; Liu, Jitao; Gao, Junping; Zhang, Changqing

2014-01-01

Plant transcription factors involved in stress responses are generally classified by their involvement in either the abscisic acid (ABA)-dependent or the ABA-independent regulatory pathways. A stress-associated NAC gene from rose (Rosa hybrida), RhNAC3, was previously found to increase dehydration tolerance in both rose and Arabidopsis. However, the regulatory mechanism involved in RhNAC3 action is still not fully understood. In this study, we isolated and analyzed the upstream regulatory sequence of RhNAC3 and found many stress-related cis-elements to be present in the promoter, with five ABA-responsive element (ABRE) motifs being of particular interest. Characterization of Arabidopsis thaliana plants transformed with the putative RhNAC3 promoter sequence fused to the β-glucuronidase (GUS) reporter gene revealed that RhNAC3 is expressed at high basal levels in leaf guard cells and in vascular tissues. Moreover, the ABRE motifs in the RhNAC3 promoter were observed to have a cumulative effect on the transcriptional activity of this gene both in the presence and absence of exogenous ABA. Overexpression of RhNAC3 in A. thaliana resulted in ABA hypersensitivity during seed germination and promoted leaf closure after ABA or drought treatments. Additionally, the expression of 11 ABA-responsive genes was induced to a greater degree by dehydration in the transgenic plants overexpressing RhNAC3 than control lines transformed with the vector alone. Further analysis revealed that all these genes contain NAC binding cis-elements in their promoter regions, and RhNAC3 was found to partially bind to these putative NAC recognition sites. We further found that of 219 A. thaliana genes previously shown by microarray analysis to be regulated by heterologous overexpression RhNAC3, 85 are responsive to ABA. In rose, the expression of genes downstream of the ABA-signaling pathways was also repressed in RhNAC3-silenced petals. Taken together, we propose that the rose RhNAC3 protein

The Rose (Rosa hybrida) NAC Transcription Factor 3 Gene, RhNAC3, Involved in ABA Signaling Pathway Both in Rose and Arabidopsis

PubMed Central

Lü, Peitao; Liu, Jitao; Gao, Junping; Zhang, Changqing

2014-01-01

Plant transcription factors involved in stress responses are generally classified by their involvement in either the abscisic acid (ABA)-dependent or the ABA-independent regulatory pathways. A stress-associated NAC gene from rose (Rosa hybrida), RhNAC3, was previously found to increase dehydration tolerance in both rose and Arabidopsis. However, the regulatory mechanism involved in RhNAC3 action is still not fully understood. In this study, we isolated and analyzed the upstream regulatory sequence of RhNAC3 and found many stress-related cis-elements to be present in the promoter, with five ABA-responsive element (ABRE) motifs being of particular interest. Characterization of Arabidopsis thaliana plants transformed with the putative RhNAC3 promoter sequence fused to the β-glucuronidase (GUS) reporter gene revealed that RhNAC3 is expressed at high basal levels in leaf guard cells and in vascular tissues. Moreover, the ABRE motifs in the RhNAC3 promoter were observed to have a cumulative effect on the transcriptional activity of this gene both in the presence and absence of exogenous ABA. Overexpression of RhNAC3 in A. thaliana resulted in ABA hypersensitivity during seed germination and promoted leaf closure after ABA or drought treatments. Additionally, the expression of 11 ABA-responsive genes was induced to a greater degree by dehydration in the transgenic plants overexpressing RhNAC3 than control lines transformed with the vector alone. Further analysis revealed that all these genes contain NAC binding cis-elements in their promoter regions, and RhNAC3 was found to partially bind to these putative NAC recognition sites. We further found that of 219 A. thaliana genes previously shown by microarray analysis to be regulated by heterologous overexpression RhNAC3, 85 are responsive to ABA. In rose, the expression of genes downstream of the ABA-signaling pathways was also repressed in RhNAC3-silenced petals. Taken together, we propose that the rose RhNAC3 protein

The Engineering Design of Engine/Airframe Integration for the SAENGER Fully Reusable Space Transportation System

DTIC Science & Technology

2010-09-01

1995 Dasa-DA Hot Metallic Structure Dasa-LM Raufoss A/S (N) NBL Partner Plansee (A) *) HTP, initiated 1988 and sponsored by the German Federal Ministry...Dornier NBL Partner TsAGI (Gus) VOLVO (S) CIAM (GUS) NBL Partner VOLVO (S) AEDC (US) VOLVO (S) Dasa-RI Dasa-LM TU Stuttgart RWTH Aachen DLR TsAGI (GUS

Genome-wide analysis of the GH3 family in apple (Malus × domestica)

PubMed Central

2013-01-01

Background Auxin plays important roles in hormone crosstalk and the plant’s stress response. The auxin-responsive Gretchen Hagen3 (GH3) gene family maintains hormonal homeostasis by conjugating excess indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acids (JAs) to amino acids during hormone- and stress-related signaling pathways. With the sequencing of the apple (Malus × domestica) genome completed, it is possible to carry out genomic studies on GH3 genes to indentify candidates with roles in abiotic/biotic stress responses. Results Malus sieversii Roem., an apple rootstock with strong drought tolerance and the ancestral species of cultivated apple species, was used as the experimental material. Following genome-wide computational and experimental identification of MdGH3 genes, we showed that MdGH3s were differentially expressed in the leaves and roots of M. sieversii and that some of these genes were significantly induced after various phytohormone and abiotic stress treatments. Given the role of GH3 in the negative feedback regulation of free IAA concentration, we examined whether phytohormones and abiotic stresses could alter the endogenous auxin level. By analyzing the GUS activity of DR5::GUS-transformed Arabidopsis seedlings, we showed that ABA, SA, salt, and cold treatments suppressed the auxin response. These findings suggest that other phytohormones and abiotic stress factors might alter endogenous auxin levels. Conclusion Previous studies showed that GH3 genes regulate hormonal homeostasis. Our study indicated that some GH3 genes were significantly induced in M. sieversii after various phytohormone and abiotic stress treatments, and that ABA, SA, salt, and cold treatments reduce the endogenous level of axuin. Taken together, this study provides evidence that GH3 genes play important roles in the crosstalk between auxin, other phytohormones, and the abiotic stress response by maintaining auxin homeostasis. PMID:23638690

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

2017-05-01

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 F 1 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 T 0 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 T 1 positive plants, but the gus gene was never found to be silenced in T 1 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. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Genome-wide analysis of the GH3 family in apple (Malus × domestica).

PubMed

Yuan, Huazhao; Zhao, Kai; Lei, Hengjiu; Shen, Xinjie; Liu, Yun; Liao, Xiong; Li, Tianhong

2013-05-02

Auxin plays important roles in hormone crosstalk and the plant's stress response. The auxin-responsive Gretchen Hagen3 (GH3) gene family maintains hormonal homeostasis by conjugating excess indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acids (JAs) to amino acids during hormone- and stress-related signaling pathways. With the sequencing of the apple (Malus × domestica) genome completed, it is possible to carry out genomic studies on GH3 genes to indentify candidates with roles in abiotic/biotic stress responses. Malus sieversii Roem., an apple rootstock with strong drought tolerance and the ancestral species of cultivated apple species, was used as the experimental material. Following genome-wide computational and experimental identification of MdGH3 genes, we showed that MdGH3s were differentially expressed in the leaves and roots of M. sieversii and that some of these genes were significantly induced after various phytohormone and abiotic stress treatments. Given the role of GH3 in the negative feedback regulation of free IAA concentration, we examined whether phytohormones and abiotic stresses could alter the endogenous auxin level. By analyzing the GUS activity of DR5::GUS-transformed Arabidopsis seedlings, we showed that ABA, SA, salt, and cold treatments suppressed the auxin response. These findings suggest that other phytohormones and abiotic stress factors might alter endogenous auxin levels. Previous studies showed that GH3 genes regulate hormonal homeostasis. Our study indicated that some GH3 genes were significantly induced in M. sieversii after various phytohormone and abiotic stress treatments, and that ABA, SA, salt, and cold treatments reduce the endogenous level of axuin. Taken together, this study provides evidence that GH3 genes play important roles in the crosstalk between auxin, other phytohormones, and the abiotic stress response by maintaining auxin homeostasis.

An Intergenic Region Shared by At4g35985 and At4g35987 in Arabidopsis thaliana Is a Tissue Specific and Stress Inducible Bidirectional Promoter Analyzed in Transgenic Arabidopsis and Tobacco Plants

PubMed Central

Banerjee, Joydeep; Sahoo, Dipak Kumar; Dey, Nrisingha; Houtz, Robert L.; Maiti, Indu Bhushan

2013-01-01

On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85) showed stronger expression (about 3.5 fold) compared to the At4g35987 promoter (P87). The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold) under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications. PMID:24260266

Two tobacco AP1-like gene promoters with highly specific, tightly regulated and uniquely expressed activity during floral transition, initiation and development

USDA-ARS?s Scientific Manuscript database

Biotech engineering of agronomic traits requires an array of highly specific and tightly regulated promoters in flower or other tissues. In this study, we isolated and characterized two tobacco AP1-like promoters (termed NtAP1La and NtAP1Lb1) in transgenic plants using GUS reporter and tissue-speci...

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter.

PubMed

Li, Wanying; Yu, Dan; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

2018-03-12

ZmbZIP25 ( Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5' RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from -2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from -2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5'-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5'-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5'-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5'-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from -1117 to -957 that were responsible for the specificity of the ZmbZIP25 5'-flanking sequence.

Functional Analysis of Maize Silk-Specific ZmbZIP25 Promoter

PubMed Central

Li, Wanying; Yu, Dan; Yu, Jingjuan; Zhu, Dengyun; Zhao, Qian

2018-01-01

ZmbZIP25 (Zea mays bZIP (basic leucine zipper) transcription factor 25) is a function-unknown protein that belongs to the D group of the bZIP transcription factor family. RNA-seq data showed that the expression of ZmbZIP25 was tissue-specific in maize silks, and this specificity was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). In situ RNA hybridization showed that ZmbZIP25 was expressed exclusively in the xylem of maize silks. A 5′ RACE (rapid amplification of cDNA ends) assay identified an adenine residue as the transcription start site of the ZmbZIP25 gene. To characterize this silk-specific promoter, we isolated and analyzed a 2450 bp (from −2083 to +367) and a 2600 bp sequence of ZmbZIP25 (from −2083 to +517, the transcription start site was denoted +1). Stable expression assays in Arabidopsis showed that the expression of the reporter gene GUS driven by the 2450 bp ZmbZIP25 5′-flanking fragment occurred exclusively in the papillae of Arabidopsis stigmas. Furthermore, transient expression assays in maize indicated that GUS and GFP expression driven by the 2450 bp ZmbZIP25 5′-flanking sequences occurred only in maize silks and not in other tissues. However, no GUS or GFP expression was driven by the 2600 bp ZmbZIP25 5′-flanking sequences in either stable or transient expression assays. A series of deletion analyses of the 2450 bp ZmbZIP25 5′-flanking sequence was performed in transgenic Arabidopsis plants, and probable elements prediction analysis revealed the possible presence of negative regulatory elements within the 161 bp region from −1117 to −957 that were responsible for the specificity of the ZmbZIP25 5′-flanking sequence. PMID:29534529

Studies on the Expression of Sesquiterpene Synthases Using Promoter-β-Glucuronidase Fusions in Transgenic Artemisia annua L

PubMed Central

Wang, Hongzhen; Han, Junli; Kanagarajan, Selvaraju; Lundgren, Anneli; Brodelius, Peter E.

2013-01-01

In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production. PMID:24278301

A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana

PubMed Central

Tripathi, Jaindra N.; Oduor, Richard O.; Tripathi, Leena

2015-01-01

Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties “Cavendish Williams” and “Gros Michel” were developed using multiple buds, whereas ECS of “Sukali Ndiizi” was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000–50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20–70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa. PMID:26635849

Compositions and Abundances of Sulfate-Reducing and Sulfur-Oxidizing Microorganisms in Water-Flooded Petroleum Reservoirs with Different Temperatures in China

PubMed Central

Tian, Huimei; Gao, Peike; Chen, Zhaohui; Li, Yanshu; Li, Yan; Wang, Yansen; Zhou, Jiefang; Li, Guoqiang; Ma, Ting

2017-01-01

Sulfate-reducing bacteria (SRB) have been studied extensively in the petroleum industry due to their role in corrosion, but very little is known about sulfur-oxidizing bacteria (SOB), which drive the oxidization of sulfur-compounds produced by the activity of SRB in petroleum reservoirs. Here, we surveyed the community structure, diversity and abundance of SRB and SOB simultaneously based on 16S rRNA, dsrB and soxB gene sequencing, and quantitative PCR analyses, respectively in petroleum reservoirs with different physicochemical properties. Similar to SRB, SOB were found widely inhabiting the analyzed reservoirs with high diversity and different structures. The dominant SRB belonged to the classes Deltaproteobacteria and Clostridia, and included the Desulfotignum, Desulfotomaculum, Desulfovibrio, Desulfobulbus, and Desulfomicrobium genera. The most frequently detected potential SOB were Sulfurimonas, Thiobacillus, Thioclava, Thiohalomonas and Dechloromonas, and belonged to Betaproteobacteria, Alphaproteobacteria, and Epsilonproteobacteria. Among them, Desulfovibrio, Desulfomicrobium, Thioclava, and Sulfurimonas were highly abundant in the low-temperature reservoirs, while Desulfotomaculum, Desulfotignum, Thiobacillus, and Dechloromonas were more often present in high-temperature reservoirs. The relative abundances of SRB and SOB varied and were present at higher proportions in the relatively high-temperature reservoirs. Canonical correspondence analysis also revealed that the SRB and SOB communities in reservoirs displayed high niche specificity and were closely related to reservoir temperature, pH of the formation brine, and sulfate concentration. In conclusion, this study extends our knowledge about the distribution of SRB and SOB communities in petroleum reservoirs. PMID:28210252

Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars.

PubMed

Arun, Muthukrishnan; Subramanyam, Kondeti; Theboral, Jeevaraj; Sivanandhan, Ganeshan; Rajesh, Manoharan; Kapil Dev, Gnanajothi; Jaganath, Balusamy; Manickavasagam, Markandan; Girija, Shanmugam; Ganapathi, Andy

2014-02-01

Soybean oil contains high levels of tocopherols which are an important source of vitamin E in human diet. The conversion of γ- to α-tocopherol catalyzed by γ-tocopherol methyltransferase (γ-TMT) is found to be the rate limiting factor in soybean which influences the tocopherol composition. Using Agrobacterium-mediated transformation, we overexpressed the γ-TMT gene of Perilla frutescens under the control of the seed-specific promoter vicillin in cultivar Pusa 16. Transgene integration and expression was confirmed in five independently transformed GUS positive soybean plants by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase-PCR (RT-PCR). High-performance liquid chromatography (HPLC) analysis showed that overexpression of Pf-γ-TMT resulted in efficient conversion of γ-tocopherol to α-tocopherol and concomitant increase in seed α-tocopherol content in RT-PCR positive plants. The protocol was successfully applied to three more cultivars PK 416, Gujarat soybean 1, and VL soya 1 in which seeds of transformed plants showed elevated level of α-tocopherol than wild-type seeds.

A receptor-like kinase gene (GbRLK) from Gossypium barbadense enhances salinity and drought-stress tolerance in Arabidopsis

PubMed Central

2013-01-01

Background Cotton (Gossypium spp.) is widely cultivated due to the important economic value of its fiber. However, extreme environmental degradation impedes cotton growth and production. Receptor-like kinase (RLK) proteins play important roles in signal transduction and participate in a diverse range of processes in response to plant hormones and environmental cues. Here, we introduced an RLK gene (GbRLK) from cotton into Arabidopsis and investigated its role in imparting abiotic stress tolerance. Results GbRLK transcription was induced by exogenously supplied abscisic acid (ABA), salicylic acid, methyl jasmonate, mock drought conditions and high salinity. We cloned the promoter sequence of this gene via self-formed adaptor PCR. Sequence analysis revealed that the promoter region contains many cis-acting stress-responsive elements such as ABRE, W-Box, MYB-core, W-Box core, TCA-element and others. We constructed a vector containing a 1,890-bp sequence in the 5′ region upstream of the initiation codon of this promoter and transformed it into Arabidopsis thaliana. GUS histochemical staining analysis showed that GbRLK was expressed mainly in leaf veins, petioles and roots of transgenic Arabidopsis, but not in the cotyledons or root hairs. GbRLK promoter activity was induced by ABA, PEG, NaCl and Verticillium dahliae. Transgenic Arabidopsis with constitutive overexpression of GbRLK exhibited a reduced rate of water loss in leaves in vitro, along with improved salinity and drought tolerance and increased sensitivity to ABA compared with non-transgenic Col-0 Arabidopsis. Expression analysis of stress-responsive genes in GbRLK Arabidopsis revealed that there was increased expression of genes involved in the ABA-dependent signaling pathway (AtRD20, AtRD22 and AtRD26) and antioxidant genes (AtCAT1, AtCCS, AtCSD2 and AtCSD1) but not ion transporter genes (AtNHX1, AtSOS1). Conclusions GbRLK is involved in the drought and high salinity stresses pathway by activating or

A receptor-like kinase gene (GbRLK) from Gossypium barbadense enhances salinity and drought-stress tolerance in Arabidopsis.

PubMed

Zhao, Jun; Gao, Yulong; Zhang, Zhiyuan; Chen, Tianzi; Guo, Wangzhen; Zhang, Tianzhen

2013-08-06

Cotton (Gossypium spp.) is widely cultivated due to the important economic value of its fiber. However, extreme environmental degradation impedes cotton growth and production. Receptor-like kinase (RLK) proteins play important roles in signal transduction and participate in a diverse range of processes in response to plant hormones and environmental cues. Here, we introduced an RLK gene (GbRLK) from cotton into Arabidopsis and investigated its role in imparting abiotic stress tolerance. GbRLK transcription was induced by exogenously supplied abscisic acid (ABA), salicylic acid, methyl jasmonate, mock drought conditions and high salinity. We cloned the promoter sequence of this gene via self-formed adaptor PCR. Sequence analysis revealed that the promoter region contains many cis-acting stress-responsive elements such as ABRE, W-Box, MYB-core, W-Box core, TCA-element and others. We constructed a vector containing a 1,890-bp sequence in the 5' region upstream of the initiation codon of this promoter and transformed it into Arabidopsis thaliana. GUS histochemical staining analysis showed that GbRLK was expressed mainly in leaf veins, petioles and roots of transgenic Arabidopsis, but not in the cotyledons or root hairs. GbRLK promoter activity was induced by ABA, PEG, NaCl and Verticillium dahliae. Transgenic Arabidopsis with constitutive overexpression of GbRLK exhibited a reduced rate of water loss in leaves in vitro, along with improved salinity and drought tolerance and increased sensitivity to ABA compared with non-transgenic Col-0 Arabidopsis. Expression analysis of stress-responsive genes in GbRLK Arabidopsis revealed that there was increased expression of genes involved in the ABA-dependent signaling pathway (AtRD20, AtRD22 and AtRD26) and antioxidant genes (AtCAT1, AtCCS, AtCSD2 and AtCSD1) but not ion transporter genes (AtNHX1, AtSOS1). GbRLK is involved in the drought and high salinity stresses pathway by activating or participating in the ABA signaling

An engineered promoter driving expression of a microbial avirulence gene confers recognition of TAL effectors and reduces growth of diverse Xanthomonas strains in citrus.

PubMed

Shantharaj, Deepak; Römer, Patrick; Figueiredo, Jose F L; Minsavage, Gerald V; Krönauer, Christina; Stall, Robert E; Moore, Gloria A; Fisher, Latanya C; Hu, Yang; Horvath, Diana M; Lahaye, Thomas; Jones, Jeffrey B

2017-09-01

Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs3 1EBE ), and fused this engineered promoter with multiple EBEs (ProBs3 14EBE ) to either the β-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs3 1EBE and ProBs3 14EBE were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs3 14EBE by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs3 14EBE . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants. © 2016 BSPP AND JOHN WILEY & SONS LTD.

NTL8 Regulates Trichome Formation in Arabidopsis by Directly Activating R3 MYB Genes TRY and TCL11[OPEN

PubMed Central

Tian, Hainan; Wang, Xianling; Guo, Hongyan; Cheng, Yuxin; Hou, Chunjiang

2017-01-01

The NAM, ATAF1/2, and CUC (NAC) are plant-specific transcription factors that regulate multiple aspects of plant growth and development and plant response to environmental stimuli. We report here the identification of NTM1-LIKE8 (NTL8), a membrane-associated NAC transcription factor, as a novel regulator of trichome formation in Arabidopsis (Arabidopsis thaliana). From an activation-tagged Arabidopsis population, we identified a dominant, gain-of-function mutant with glabrous inflorescence stem. By using plasmid rescue and RT-PCR analyses, we found that NTL8 was tagged; thus, the mutant was named ntl8-1 Dominant (ntl8-1D). Recapitulation experiment further confirmed that the phenotype observed in the ntl8-1D mutant was caused by elevated expression of NTL8. Quantitative RT-PCR results showed that the expression level of the single-repeat R3 MYB genes TRIPTYCHON (TRY) and TRICHOMELESS1 (TCL1) was elevated in the ntl8-1D mutant. Genetic analyses demonstrated that NTL8 acts upstream of TRY and TCL1 in the regulation of trichome formation. When recruited to the promoter region of the reporter gene Gal4:GUS by a fused GAL4 DNA-binding domain, NTL8 activated the expression of the reporter gene. Chromatin immunoprecipitation results indicated that TRY and TCL1 are direct targets of NTL8. However, NTL8 did not interact with SQUAMOSA PROMOTER BINDING PROTEIN LIKE9, another transcription factor that regulates the expression of TRY and TCL1, in yeast and plant cells. Taken together, our results suggest that NTL8 negatively regulates trichome formation in Arabidopsis by directly activating the expression of TRY and TCL1. PMID:28649093

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-07-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-11-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium.

PubMed

Chin, Dong Poh; Mishiba, Kei-ichiro; Mii, Masahiro

2007-06-01

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L(-1) gellan gum-solidified NDM containing 10 g L(-1) sucrose, 20 mg L(-1) hygromycin and 40 mg L(-1) meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 muM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.

A single-repeat R3-MYB transcription factor MYBC1 negatively regulates freezing tolerance in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhai, Hong, E-mail: Zhai.h@hotmail.com; Bai, Xi, E-mail: baixi@neau.edu.cn; Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn

2010-04-16

We had previously identified the MYBC1 gene, which encodes a single-repeat R3-MYB protein, as a putative osmotic responding gene; however, no R3-MYB transcription factor has been reported to regulate osmotic stress tolerance. Thus, we sought to elucidate the function of MYBC1 in response to osmotic stresses. Real-time RT-PCR analysis indicated that MYBC1 expression responded to cold, dehydration, salinity and exogenous ABA at the transcript level. mybc1 mutants exhibited an increased tolerance to freezing stress, whereas 35S::MYBC1 transgenic plants exhibited decreased cold tolerance. Transcript levels of some cold-responsive genes, including CBF/DREB genes, KIN1, ADC1, ADC2 and ZAT12, though, were not alteredmore » in the mybc1 mutants or the 35S::MYBC1 transgenic plants in response to cold stress, as compared to the wild type. Microarray analysis results that are publically available were investigated and found transcript level of MYBC1 was not altered by overexpression of CBF1, CBF2, and CBF3, suggesting that MYBC1 is not down regulated by these CBF family members. Together, these results suggested that MYBC1is capable of negatively regulating the freezing tolerance of Arabidopsis in the CBF-independent pathway. In transgenic Arabidopsis carrying an MYBC1 promoter driven {beta}-glucuronidase (GUS) construct, GUS activity was observed in all tissues and was relatively stronger in the vascular tissues. Fused MYBC1 and GFP protein revealed that MYBC1 was localized exclusively in the nuclear compartment.« less

Overexpression of MusaMYB31, a R2R3 type MYB transcription factor gene indicate its role as a negative regulator of lignin biosynthesis in banana

PubMed Central

Ganapathi, T. R.

2017-01-01

Lignin and polyphenols are important cellular components biosynthesized through phenylpropanoid pathway. Phenylpropanoid pathway in plants is regulated by some important transcription factors including R2R3 MYB transcription factors. In this study, we report the cloning and functional characterization of a banana R2R3-MYB transcription factor (MusaMYB31) by overexpression in transgenic banana plants and evaluated its potential role in regulating biosynthesis of lignin and polyphenols. Sequence analysis of MusaMYB31 indicated its clustering with members of subgroup 4 (Sg4) of R2R3MYB family which are well known for their role as repressors of lignin biosynthesis. Expression analysis indicated higher expression of MusaMYB31 in corm and root tissue, known for presence of highly lignified tissue than other organs of banana. Overexpression of MusaMYB31 in banana cultivar Rasthali was carried out and four transgenic lines were confirmed by GUS histochemical staining, PCR analysis and Southern blot. Histological and biochemical analysis suggested reduction of cell wall lignin in vascular elements of banana. Transgenic lines showed alteration in transcript levels of general phenylpropanoid pathway genes including lignin biosynthesis pathway genes. Reduction of total polyphenols content in transgenic lines was in line with the observation related to repression of general phenylpropanoid pathway genes. This study suggested the potential role of MusaMYB31 as repressor of lignin and polyphenols biosynthesis in banana. PMID:28234982

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment.

PubMed

Tanaka, T; Nishihara, M; Seki, M; Sakamoto, A; Tanaka, K; Irifune, K; Morikawa, H

1995-05-01

Gold particles coated with beta-glucuronidase (GUS) mRNA with a 5' cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.

Genetic Transformation of Hordeum vulgare ssp. spontaneum for the Development of a Transposon-Based Insertional Mutagenesis System.

PubMed

Cardinal, Marie-Josée; Kaur, Rajvinder; Singh, Jaswinder

2016-10-01

Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5 mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100 % in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.

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

High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode.

PubMed

Cho, H J; Farrand, S K; Noel, G R; Widholm, J M

2000-01-01

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and beta-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54-95% of the cotyledon explants on MXB selective medium containing 200 microg ml(-1) kanamycin and 500 microg ml(-1) carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4-5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode.

phiC31 Integrase-Mediated Site-Specific Recombination in Barley

PubMed Central

Rubtsova, Myroslava; Kumlehn, Jochen; Gils, Mario

2012-01-01

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity. PMID:23024817

A single molecule perspective on the functional diversity of in vitro evolved β-glucuronidase.

PubMed

Liebherr, Raphaela B; Renner, Max; Gorris, Hans H

2014-04-23

The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62,500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.

FIN219/JAR1 and cryptochrome1 antagonize each other to modulate photomorphogenesis under blue light in Arabidopsis

PubMed Central

2018-01-01

Plant development is affected by the integration of light and phytohormones, including jasmonates (JAs). To address the molecular mechanisms of possible interactions between blue light and JA signaling in Arabidopsis thaliana, we used molecular and transgenic approaches to understand the regulatory relationships between FAR-RED INSENSITIVE 219 (FIN219)/JASMONATE RESISTANT1 (JAR1) and the blue-light photoreceptor cryptochrome1 (CRY1). FIN219 overexpression in the wild type resulted in a short-hypocotyl phenotype under blue light. However, FIN219 overexpression in cry1, cry2 and cry1cry2 double mutant backgrounds resulted in phenotypes similar to their respective mutant backgrounds, which suggests that FIN219 function may require blue light photoreceptors. Intriguingly, FIN219 overexpression in transgenic plants harboring ectopic expression of the C terminus of CRY1 (GUS-CCT1), which exhibits a hypersensitive short-hypocotyl phenotype in all light conditions including darkness, led to a rescued phenotype under all light conditions except red light. Further expression studies showed mutual suppression between FIN219 and CRY1 under blue light. Strikingly, FIN219 overexpression in GUS-CCT1 transgenic lines (FIN219-OE/GUS-CCT1) abolished GUS-CCT1 fusion protein under blue light, whereas GUS-CCT1 fusion protein was stable in the fin219-2 mutant background (fin219-2/GUS-CCT1). Moreover, FIN219 strongly interacted with COP1 under blue light, and methyl JA (MeJA) treatment enhanced the interaction between FIN219 and GUS-CCT1 under blue light. Furthermore, FIN219 level affected GUS-CCT1 seedling responses such as anthocyanin accumulation and bacterial resistance under various light conditions and MeJA treatment. Thus, FIN219/JAR1 and CRY1 antagonize each other to modulate photomorphogenic development of seedlings and stress responses in Arabidopsis. PMID:29561841

FIN219/JAR1 and cryptochrome1 antagonize each other to modulate photomorphogenesis under blue light in Arabidopsis.

PubMed

Chen, Huai-Ju; Fu, Tsu-Yu; Yang, Shao-Li; Hsieh, Hsu-Liang

2018-03-01

Plant development is affected by the integration of light and phytohormones, including jasmonates (JAs). To address the molecular mechanisms of possible interactions between blue light and JA signaling in Arabidopsis thaliana, we used molecular and transgenic approaches to understand the regulatory relationships between FAR-RED INSENSITIVE 219 (FIN219)/JASMONATE RESISTANT1 (JAR1) and the blue-light photoreceptor cryptochrome1 (CRY1). FIN219 overexpression in the wild type resulted in a short-hypocotyl phenotype under blue light. However, FIN219 overexpression in cry1, cry2 and cry1cry2 double mutant backgrounds resulted in phenotypes similar to their respective mutant backgrounds, which suggests that FIN219 function may require blue light photoreceptors. Intriguingly, FIN219 overexpression in transgenic plants harboring ectopic expression of the C terminus of CRY1 (GUS-CCT1), which exhibits a hypersensitive short-hypocotyl phenotype in all light conditions including darkness, led to a rescued phenotype under all light conditions except red light. Further expression studies showed mutual suppression between FIN219 and CRY1 under blue light. Strikingly, FIN219 overexpression in GUS-CCT1 transgenic lines (FIN219-OE/GUS-CCT1) abolished GUS-CCT1 fusion protein under blue light, whereas GUS-CCT1 fusion protein was stable in the fin219-2 mutant background (fin219-2/GUS-CCT1). Moreover, FIN219 strongly interacted with COP1 under blue light, and methyl JA (MeJA) treatment enhanced the interaction between FIN219 and GUS-CCT1 under blue light. Furthermore, FIN219 level affected GUS-CCT1 seedling responses such as anthocyanin accumulation and bacterial resistance under various light conditions and MeJA treatment. Thus, FIN219/JAR1 and CRY1 antagonize each other to modulate photomorphogenic development of seedlings and stress responses in Arabidopsis.

The impact of long-term water stress on tree architecture and production is related to changes in transitions between vegetative and reproductive growth in the 'Granny Smith' apple cultivar.

PubMed

Yang, Weiwei; Pallas, Benoît; Durand, Jean-Baptiste; Martinez, Sébastien; Han, Mingyu; Costes, Evelyne

2016-11-01

Water stress (WS) generates a number of physiological and morphological responses in plants that depend on the intensity and duration of stress as well as the plant species and development stage. In perennial plants, WS may affect plant development through cumulative effects that modify plant functions, architecture and production over time. Plant architecture depends on the fate of the terminal and axillary buds that can give rise, in the particular case of apple, to reproductive or vegetative growth units (GUs) of different lengths. In this study, the impact of long-term WS (7 years) on the fate of terminal and axillary buds was investigated in relation to flowering occurrence and production pattern (biennial vs regular) in the 'Granny Smith' cultivar. It was observed that WS decreased the total number of GUs per branch, regardless of their type. Conversely, WS did not modify the timing of the two successive developmental phases characterized by the production of long and medium GUs and an alternation of floral GUs over time, respectively. The analysis of GU successions over time using a variable-order Markov chain that included both the effects of the predecessor and water treatment revealed that WS reduced the transition towards long and medium GUs and increased the transition toward floral, short and dead GUs. WS also slightly increased the proportion of axillary floral GUs. The higher relative frequency of floral GUs compared with vegetative ones reduced the tendency to biennial bearing under WS. The accelerated ontogenetic trend observed under WS suggests lower vegetative growth that could, in turn, be beneficial to floral induction and fruit set. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Induction of hypoxic root metabolism results from physical limitations in O2 bio-availability in microgravity

NASA Astrophysics Data System (ADS)

Liao, J.; Monje, O.; Porterfield, D.

Numerous spaceflight experiments have noted changes in the roots that are consistent with hypoxia in the rootzone. These observations range from general ultrastructure analysis and biochemical measurements to direct measurements of stress specific enzymes. In experiments that have monitored alcohol dehydrogenase (ADH) the data shows this hypoxically responsive gene is induced and ADH activity is elevated in microgravity. These changes in ADH could be induced either by spaceflight hypoxia resulting from inhibition of gravity mediated O 2 transport, or by a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in two series of experiments. The objective of the first experiment was to determine if physical changes in gravity mediated O 2 transport can be directly measured, while the second series of experiments tested whether disruption of gravisensing can induce a non-specific ADH response. To directly measure O 2 bioavailability as a function of gravity we designed a sensor that mimics metabolic O 2 consumption from the rhizosphere. Because of these design criteria the sensor is sensitive to any changes in root O 2 bioavailability that may occur in microgravity. In a KC-135 experiment the sensor was implanted in a moist granular clay media and exposed to microgravity during parabolic flight. The resulting data indicated that root O 2 bioavailability decreased in phase with gravity. In experiments that tested for non-specific induction of ADH we compared the response of transgenic Arabidopsis plants (ADH promoted GUS marker gene) exposed to clinostat, control, and waterlogged conditions. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH enzyme activities

BnEPFL6, an EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) secreted peptide gene, is required for filament elongation in Brassica napus.

PubMed

Huang, Yi; Tao, Zhangsheng; Liu, Qiong; Wang, Xinfa; Yu, Jingyin; Liu, Guihua; Wang, Hanzhong

2014-07-01

Inflorescence architecture, pedicel length and stomata patterning in Arabidopsis thaliana are specified by inter-tissue communication mediated by ERECTA and its signaling ligands in the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family of secreted cysteine-rich peptides. Here, we identified and characterized BnEPFL6 from Brassica napus. Heterologous expression of this gene under the double enhanced CaMV promoter (D35S) in Arabidopsis resulted in shortened stamen filaments, filaments degradation, and reduced filament cell size that displayed down-regulated expression of AHK2, in which phenotypic variation of ahk2-1 mutant presented highly consistent with that of BnEPFL6 transgenic lines. Especially, the expression level of BnEPFL6 in the shortened filaments of four B. napus male sterile lines (98A, 86A, SA, and Z11A) was similar to that of BnEPFL6 in the transgenic Arabidopsis lines. The activity of pBnEPFL6.2::GUS was intensive in the filaments of transgenic lines. These observations reveal that BnEPFL6 plays an important role in filament elongation and may also affect organ morphology and floral organ specification via a BnEPFL6-mediated cascade.

Preservation and Faithful Expression of Transgene via Artificial Seeds in Alfalfa

PubMed Central

Liu, Wenting; Liang, Zongsuo; Wang, Xinhua; Sibbald, Susan; Hunter, David; Tian, Lining

2013-01-01

Proper preservation of transgenes and transgenic materials is important for wider use of transgenic technology in plants. Here, we report stable preservation and faithful expression of a transgene via artificial seed technology in alfalfa. DNA constructs containing the uid reporter gene coding for β-glucuronidase (GUS) driven by a 35S promoter or a tCUP promoter were introduced into alfalfa via Agrobacterium-mediated genetic transformation. Somatic embryos were subsequently induced from transgenic alfalfa plants via in vitro technology. These embryos were treated with abscisic acid to induce desiccation tolerance and were subjected to a water loss process. After the desiccation procedure, the water content in dried embryos, or called artificial seeds, was about 12–15% which was equivalent to that in true seeds. Upon water rehydration, the dried somatic embryos showed high degrees of viability and exhibited normal germination. Full plants were subsequently developed and recovered in a greenhouse. The progeny plants developed from artificial seeds showed GUS enzyme activity and the GUS expression level was comparable to that of plants developed from somatic embryos without the desiccation process. Polymerase chain reaction analysis indicated that the transgene was well retained in the plants and Southern blot analysis showed that the transgene was stably integrated in plant genome. The research showed that the transgene and the new trait can be well preserved in artificial seeds and the progeny developed. The research provides a new method for transgenic germplasm preservation in different plant species. PMID:23690914

Functional Characterization of Phalaenopsis aphrodite Flowering Genes PaFT1 and PaFD

PubMed Central

Jang, Seonghoe; Choi, Sang-Chul; Li, Hsing-Yi; An, Gynheung; Schmelzer, Elmon

2015-01-01

We show that the key flowering regulators encoded by Phalaenopsis aphrodite FLOWERING LOCUS T1 (PaFT1) and PaFD share high sequence homologies to these from long-day flowering Arabidopsis and short-day flowering rice. Interestingly, PaFT1 is specifically up-regulated during flowering inductive cooling treatment but is not subjected to control by photoperiod in P. aphrodite. Phloem or shoot apex-specific expression of PaFT1 restores the late flowering of Arabidopsis ft mutants. Moreover, PaFT1 can suppress the delayed flowering caused by SHORT VEGATATIVE PHASE (SVP) overexpression as well as an active FRIGIDA (FRI) allele, indicating the functional conservation of flowering regulatory circuit in different plant species. PaFT1 promoter:GUS in Arabidopsis showed similar staining pattern to that of Arabidopsis FT in the leaves and guard cells but different in the shoot apex. A genomic clone or heat shock-inducible expression of PaFT1 is sufficient to the partial complementation of the ft mutants. Remarkably, ectopic PaFT1 expression also triggers precocious heading in rice. To further demonstrate the functional conservation of the flowering regulators, we show that PaFD, a bZIP transcription factor involved in flowering promotion, interacts with PaFT1, and PaFD partially complemented Arabidopsis fd mutants. Transgenic rice expressing PaFD also flowered early with increased expression of rice homologues of APETALA1 (AP1). Consistently, PaFT1 knock-down Phalaenopsis plants generated by virus-induced gene silencing exhibit delayed spiking. These studies suggest functional conservation of FT and FD genes, which may have evolved and integrated into distinct regulatory circuits in monopodial orchids, Arabidopsis and rice that promote flowering under their own inductive conditions. PMID:26317412

The gene encoding the catalytically inactive beta-amylase BAM4 involved in starch breakdown in Arabidopsis leaves is expressed preferentially in vascular tissues in source and sink organs.

PubMed

Francisco, Perigio; Li, Jing; Smith, Steven M

2010-07-15

Genetic studies in Arabidopsis thaliana have shown that two members of the beta-amylase (BAM) family BAM3 and BAM4 are required for leaf starch breakdown at night. Both are plastid proteins and while BAM3 encodes an active BAM, BAM4 is not an active alpha-1,4-glucan hydrolase. To gain further insight into the possible function of BAM4 we constructed reporter genes using promoters for both BAM3 and BAM4 genes, driving beta-glucuronidase (GUS) and luciferase (LUC) expression in transgenic Arabidopsis plants. Both promoters directed expression in vascular tissue throughout the plant including cotyledons, leaves, petioles, stems, petals, siliques and roots. Tissue sections showed expression to be focused in phloem cells in stem and petiole. The BAM3 promoter was also expressed strongly throughout the photosynthetic tissues of leaves, sepals and siliques, whereas the BAM4 promoter was not. Conversely, the BAM4 promoter was active in root tip but the BAM3 promoter was not. To confirm these expression patterns and to compare with expression of other starch genes we carried-out RT-PCR analysis on RNA from vascular (replum) and non-vascular (valve) tissues of siliques. This confirmed that BAM4 expression together with RAM1 (BAM5) and GWD2 genes is stronger in the replum than the valve, whereas BAM3 is strong in both tissues. These results show that even though BAM3 and BAM4 genes apparently interact genetically in leaf starch metabolism, BAM4 is preferentially expressed in non-photosynthetic vascular tissue, so revealing a potentially greater level of complexity in the control of starch breakdown than had previously been recognised. Copyright (c) 2010 Elsevier GmbH. All rights reserved.

Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale

PubMed Central

Kui, Ling; Chen, Haitao; Zhang, Weixiong; He, Simei; Xiong, Zijun; Zhang, Yesheng; Yan, Liang; Zhong, Chaofang; He, Fengmei; Chen, Junwen; Zeng, Peng; Zhang, Guanghui; Yang, Shengchao; Dong, Yang; Wang, Wen; Cai, Jing

2017-01-01

Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D

Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale.

PubMed

Kui, Ling; Chen, Haitao; Zhang, Weixiong; He, Simei; Xiong, Zijun; Zhang, Yesheng; Yan, Liang; Zhong, Chaofang; He, Fengmei; Chen, Junwen; Zeng, Peng; Zhang, Guanghui; Yang, Shengchao; Dong, Yang; Wang, Wen; Cai, Jing

2016-01-01

Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale . In this study, based on the previously developed Agrobacterium -mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale . These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase ( GUS ) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium -mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale . By selecting five target genes ( C3H, C4H, 4CL, CCR, and IRX ) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous

Effects of plant downtime on the microbial community composition in the highly saline brine of a geothermal plant in the North German Basin.

PubMed

Westphal, Anke; Lerm, Stephanie; Miethling-Graff, Rona; Seibt, Andrea; Wolfgramm, Markus; Würdemann, Hilke

2016-04-01

The microbial biocenosis in highly saline fluids produced from the cold well of a deep geothermal heat store located in the North German Basin was characterized during regular plant operation and immediately after plant downtime phases. Genetic fingerprinting revealed the dominance of sulfate-reducing bacteria (SRB) and fermentative Halanaerobiaceae during regular plant operation, whereas after shutdown phases, sequences of sulfur-oxidizing bacteria (SOB) were also detected. The detection of SOB indicated oxygen ingress into the well during the downtime phase. High 16S ribosomal RNA (rRNA) and dsrA gene copy numbers at the beginning of the restart process showed an enrichment of bacteria, SRB, and SOB during stagnant conditions consistent with higher concentrations of dissolved organic carbon (DOC), sulfate, and hydrogen sulfide in the produced fluids. The interaction of SRB and SOB during plant downtimes might have enhanced the corrosion processes occurring in the well. It was shown that scale content of fluids was significantly increased after stagnant phases. Moreover, the sulfur isotopic signature of the mineral scales indicated microbial influence on scale formation.

Colonization by the arbuscular mycorrhizal fungus Glomus versiforme induces a defense response against the root-knot nematode Meloidogyne incognita in the grapevine (Vitis amurensis Rupr.), which includes transcriptional activation of the class III chitinase gene VCH3.

PubMed

Li, Hai-Yan; Yang, Guo-Dong; Shu, Huai-Rui; Yang, Yu-Tao; Ye, Bao-Xing; Nishida, Ikuo; Zheng, Cheng-Chao

2006-01-01

Inoculation of the grapevine (Vitis amurensis Rupr.) with the arbuscular mycorrhizal (AM) fungus Glomus versiforme significantly increased resistance against the root-knot nematode (RKN) Meloidogyne incognita. Studies using relative quantitative reverse transcription-PCR (RQRT-PCR) analysis of grapevine root inoculation with the AM fungus revealed an up-regulation of VCH3 transcripts. This increase was greater than that observed following infection with RKN. However, inoculation of the mycorrhizal grapevine roots with RKN was able to enhance VCH3 transcript expression further. Moreover, the increase in VCH3 transcripts appeared to result in a higher level of resistance against subsequent RKN infection. Constitutive expression of VCH3 cDNA in transgenic tobacco under the control of the cauliflower mosaic virus 35S promoter also conferred resistance against RKN, but had no significant effect on the growth of the AM fungus. We analyzed beta-glucuronidase (GUS) activity directed by a 1,216 bp VCH3 promoter in transgenic tobacco following inoculation with both the AM fungus and RKN. GUS activity was negligible in the root tissues before inoculation, and was more effectively induced after inoculation with the AM fungus than with RKN. Moreover, GUS staining in the mycorrhizal transgenic tobacco roots was enhanced by subsequent RKN infection, and was found ubiquitously throughout the whole root tissue. Together, these results suggest that AM fungus induced a defense response against RKN in the mycorrhizal grapevine roots, which appeared to involve transcriptional control of VCH3 expression throughout the whole root tissue.

AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xin, E-mail: fangfei6073@126.com; Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn; Zhai, Hong, E-mail: Zhai.h@neigaehrb.ac.cn

2012-06-15

Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, andmore » abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.« less

Isolation and characterization of the promoter sequence of a cassava gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in storage roots.

PubMed

de Souza, C R; Aragão, F J; Moreira, E C O; Costa, C N M; Nascimento, S B; Carvalho, L J

2009-03-24

Cassava is one of the most important tropical food crops for more than 600 million people worldwide. Transgenic technologies can be useful for increasing its nutritional value and its resistance to viral diseases and insect pests. However, tissue-specific promoters that guarantee correct expression of transgenes would be necessary. We used inverse polymerase chain reaction to isolate a promoter sequence of the Mec1 gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in cassava storage roots. In silico analysis revealed putative cis-acting regulatory elements within this promoter sequence, including root-specific elements that may be required for its expression in vascular tissues. Transient expression experiments showed that the Mec1 promoter is functional, since this sequence was able to drive GUS expression in bean embryonic axes. Results from our computational analysis can serve as a guide for functional experiments to identify regions with tissue-specific Mec1 promoter activity. The DNA sequence that we identified is a new promoter that could be a candidate for genetic engineering of cassava roots.

A structured interface to the object-oriented genomics unified schema for XML-formatted data.

PubMed

Clark, Terry; Jurek, Josef; Kettler, Gregory; Preuss, Daphe

2005-01-01

Data management systems are fast becoming required components in many biology laboratories as the role of computer-based information grows. Although the need for data management systems is on the rise, their inherent complexities can deter the full and routine use of their computational capabilities. The significant undertaking to implement a capable production system can be reduced in part by adapting an established data management system. In such a way, we are leveraging the Genomics Unified Schema (GUS) developed at the Computational Biology and Informatics Laboratory at the University of Pennsylvania as a foundation for managing and analysing DNA sequence data in centromere research projects around Arabidopsis thaliana and related species. Because GUS provides a core schema that includes support for genome sequences, mRNA and its expression, and annotated chromosomes, it is ideal for synthesising a variety of parameters to analyse these repetitive and highly dynamic portions of the genome. Despite this, production-strength data management frameworks are complex, requiring dedicated efforts to adapt and maintain. The work reported in this article addresses one component of such an effort, namely the pivotal task of marshalling data from various sources into GUS. In order to harness GUS for our project, and motivated by efficiency needs, we developed a structured framework for transferring data into GUS from outside sources. This technology is embodied in a GUS object-layer processor, XMLGUS. XMLGUS facilitates incorporating data into GUS by (i) formulating an XML interface that includes relational database key constraint definitions, (ii) regularising traversal through that XML, (iii) realising automatic processing of the XML with database key constraints and (iv) allowing for special processing of input data within the framework for automated processing. The application of XMLGUS to production pipeline processing for a sequencing project and inputting the

Plant genetic transformation efficiency of selected Malaysian rice based on selectable marker gene (hptII).

PubMed

Htwe, Nwe Nwe; Ling, Ho Chai; Zaman, Faridah Qamaruz; Maziah, Mahmood

2014-04-01

Rice is one of the most important cereal crops with great potential for biotechnology progress. In transformation method, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. In this study, the toxicity level of hygromycin was optimized for two selected mutant rice lines, MR219 line 4 and line 9. The mature embryos were isolated and cultured on an MS medium with different hygromycin concentrations (0, 20, 40, 60, 80 and 100 mg L(-1)). Evidently, above 60 mg L(-1) was effective for callus formation and observed completely dead. Further there were tested for specific concentration (0-60). Although, 21.28% calli survived on the medium containing 45 mg L(-1) hygromycin, it seemed suitable for the identification of putative transformants. These findings indicated that a system for rice transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants. Green shoots were regenerated from the explant under hygromycin stress. RT-PCR using hptII and gus sequence specific primer and Southern blot analysis were used to confirm the presence of the transgene and to determine the transformation efficiency for their stable integration in regenerated plants. This study demonstrated that the hygromycin resistance can be used as an effective marker for rice transformation.

Transformation of Morinda citrifolia via simple mature seed imbibition method.

PubMed

Lee, J J; Ahmad, S; Roslan, H A

2013-12-15

Morinda citrifolia, is a valuable medicinal plant with a wide range of therapeutic properties and extensive transformation study on this plant has yet been known. Present study was conducted to establish a simple and reliable transformation protocol for M. citrifolia utilising Agrobacterium tumefaciens via direct seed exposure. In this study, the seeds were processed by tips clipping and dried and subsequently incubated in inoculation medium. Four different parameters during the incubation such as incubation period, bacterial density, temperature and binary vectors harbouring beta-glucuronidase (GUS) gene (pBI121 and pGSA1131), were tested to examine its effect on transformation efficiency. The leaves from the treated and germinated seedlings were analysed via Polymerase Chain Reaction (PCR), histochemical assay of the GUS gene and reverse transcription-PCR (RT-PCR). Results of the study showed that Agrobacterium strain LBA4404 with optical density of 1.0 and 2 h incubation period were optimum for M. citrifolia transformation. It was found that various co-cultivation temperatures tested and type of vector used did not affect the transformation efficiency. The highest transformation efficiency for M. citrifolia direct seed transformation harbouring pBI121 and pGSA1131 was determined to be 96.8% with 2 h co-cultivation treatment and 80.4% when using bacterial density of 1.0, respectively. The transformation method can be applied for future characterization study of M. citrifolia.

Functional characterisation and cell specificity of BvSUT1, the transporter that loads sucrose into the phloem of sugar beet (Beta vulgaris L.) source leaves.

PubMed

Nieberl, P; Ehrl, C; Pommerrenig, B; Graus, D; Marten, I; Jung, B; Ludewig, F; Koch, W; Harms, K; Flügge, U-I; Neuhaus, H E; Hedrich, R; Sauer, N

2017-05-01

Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the β-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests.

PubMed

Saha, Prasenjit; Majumder, Pralay; Dutta, Indrajit; Ray, Tui; Roy, S C; Das, Sampa

2006-05-01

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be approximately 12.1%+/-0.351 (mean +/- SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T0 and T1 plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P < 0.01), 32% (P < 0.05) and 40.5, 29.5% (P < 0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T1 progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.

A gibberellin-stimulated transcript, OsGASR1, controls seedling growth and α-amylase expression in rice.

PubMed

Lee, Sang-Choon; Kim, Soo-Jin; Han, Soon-Ki; An, Gynheung; Kim, Seong-Ryong

2017-07-01

From a T-DNA-tagging population in rice, we identified OsGASR1 (LOC_Os03g55290), a member of the GAST (gibberellin (GA)-Stimulated Transcript) family that is induced by salt stress and ABA treatment. This gene was highly expressed in the regions of cell proliferation and panicle development, as revealed by a GUS assay of the mutant line. In the osgasr1 mutants, the second leaf blades were much longer than those of the segregating wild type due to an increase in cell length. In addition, five α-amylase genes were up-regulated in the mutants, implying that OsGASR1 is a negative regulator of those genes. These results suggest that OsGASR1 plays important roles in seedling growth and α-amylase gene expression. Copyright © 2017 Elsevier GmbH. All rights reserved.

Hypothalamic-Pituitary-Thyroid Axis Perturbations in Male Mice by CNS-Penetrating Thyromimetics.

PubMed

Ferrara, Skylar J; Bourdette, Dennis; Scanlan, Thomas S

2018-07-01

Thyromimetics represent a class of experimental drugs that can stimulate tissue-selective thyroid hormone action. As such, thyromimetics should have effects on the hypothalamic-pituitary-thyroid (HPT) axis, but details of this action and the subsequent effects on systemic thyroid hormone levels have not been reported to date. Here, we compare the HPT-axis effects of sobetirome, a well-studied thyromimetic, with Sob-AM2, a newly developed prodrug of sobetirome that targets sobetirome distribution to the central nervous system (CNS). Similar to endogenous thyroid hormone, administration of sobetirome and Sob-AM2 suppress HPT-axis gene transcript levels in a manner that correlates to their specific tissue distribution properties (periphery vs CNS, respectively). Dosing male C57BL/6 mice with sobetirome and Sob-AM2 at concentrations ≥10 μg/kg/d for 29 days induces a state similar to central hypothyroidism characterized by depleted circulating T4 and T3 and normal TSH levels. However, despite the systemic T4 and T3 depletion, the sobetirome- and Sob-AM2-treated mice do not show signs of hypothyroidism, which may result from the presence of the thyromimetic in the thyroid hormone-depleted background.

Labeled Azospirillum brasilense wild type and excretion-ammonium strains in association with barley roots.

PubMed

Santos, Adrian Richard Schenberger; Etto, Rafael Mazer; Furmam, Rafaela Wiegand; Freitas, Denis Leandro de; Santos, Karina Freire d'Eça Nogueira; Souza, Emanuel Maltempi de; Pedrosa, Fábio de Oliveira; Ayub, Ricardo Antônio; Steffens, Maria Berenice Reynaud; Galvão, Carolina Weigert

2017-09-01

Soil bacteria colonization in plants is a complex process, which involves interaction between many bacterial characters and plant responses. In this work, we labeled Azospirillum brasilense FP2 (wild type) and HM053 (excretion-ammonium) strains by insertion of the reporter gene gusA-kanamycin into the dinitrogenase reductase coding gene, nifH, and evaluated bacteria colonization in barley (Hordeum vulgare). In addition, we determined inoculation effect based on growth promotion parameters. We report an uncommon endophytic behavior of A. brasilense Sp7 derivative inside the root hair cells of barley and highlight the promising use of A. brasilense HM053 as plant growth-promoting bacterium. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis

USDA-ARS?s Scientific Manuscript database

Alfalfa (Medicago sativa L.) plants were transformed with two constructs: (1) a truncated phosphoenolpyruvate carboxylase promoter isolated from alfalfa nodules (PEPC-4) fused to GUS; and (2) PEPC-4 fused with sucrose synthase (SUS) isolated from alfalfa nodules. Histochemical staining for GUS in st...

Long-Term Administration of Conjugated Estrogen and Bazedoxifene Decreased Murine Fecal β-Glucuronidase Activity Without Impacting Overall Microbiome Community.

PubMed

Chen, Karen Lee Ann; Liu, Xiaoji; Zhao, Yiru Chen; Hieronymi, Kadriye; Rossi, Gianluigi; Auvil, Loretta Sue; Welge, Michael; Bushell, Colleen; Smith, Rebecca Lee; Carlson, Kathryn E; Kim, Sung Hoon; Katzenellenbogen, John A; Miller, Michael Joseph; Madak-Erdogan, Zeynep

2018-05-25

Conjugated estrogens (CE) and Bazedoxifene (BZA) combination is used to alleviate menopause-associated symptoms in women. CE+BZA undergo first-pass-metabolism in the liver and deconjugation by gut microbiome via β-glucuronidase (GUS) enzyme inside the distal gut. To date, the impact of long-term exposure to CE+BZA on the gut microbiome or GUS activity has not been examined. Our study using an ovariectomized mouse model showed that CE+BZA administration did not affect the overall cecal or fecal microbiome community except that it decreased the abundance of Akkermansia, which was identified as a fecal biomarker correlated with weight gain. The fecal GUS activity was reduced significantly and was positively correlated with the abundance of Lactobacillaceae in the fecal microbiome. We further confirmed in Escherichia coli K12 and Lactobacillus gasseri ADH that Tamoxifen-, 4-hydroxy-Tamoxifen- and Estradiol-Glucuronides competed for GUS activity. Our study for the first time demonstrated that long-term estrogen supplementation directly modulated gut microbial GUS activity. Our findings implicate that long-term estrogen supplementation impacts composition of gut microbiota and microbial activity, which affects estrogen metabolism in the gut. Thus, it is possible to manipulate such activity to improve the efficacy and safety of long-term administered estrogens for postmenopausal women or breast cancer patients.

The Arabidopsis vacuolar malate channel is a member of the ALMT family.

PubMed

Kovermann, Peter; Meyer, Stefan; Hörtensteiner, Stefan; Picco, Cristiana; Scholz-Starke, Joachim; Ravera, Silvia; Lee, Youngsook; Martinoia, Enrico

2007-12-01

In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.

Molecular genetic analysis of activation-tagged transcription factors thought to be involved in photomorphogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neff, Michael M.

This is a final report for Department of Energy Grant No. DE-FG02-08ER15927 entitled “Molecular Genetic Analysis of Activation-Tagged Transcription Factors Thought to be Involved in Photomorphogenesis”. Based on our preliminary photobiological and genetic analysis of the sob1-D mutant, we hypothesized that OBP3 is a transcription factor involved in both phytochrome and cryptochrome-mediated signal transduction. In addition, we hypothesized that OBP3 is involved in auxin signaling and root development. Based on our preliminary photobiological and genetic analysis of the sob2-D mutant, we also hypothesized that a related gene, LEP, is involved in hormone signaling and seedling development.

Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass

DOE PAGES

Liu, Wusheng; Mazarei, Mitra; Ye, Rongjian; ...

2018-04-24

Genetic engineering of switchgrass (Panicum virgatum L.) for reduced cell wall recalcitrance and improved biofuel production has been a long pursued goal. Up to now, constitutive promoters have been used to direct the expression of cell wall biosynthesis genes toward attaining that goal. While generally sufficient to gauge a transgene's effects in the heterologous host, constitutive overexpression often leads to undesirable plant phenotypic effects. Green tissue-specific promoters from switchgrass are potentially valuable to directly alter cell wall traits exclusively in harvestable aboveground biomass while not changing root phenotypes. We identified and functionally characterized three switchgrass green tissue-specific promoters and assessedmore » marker gene expression patterns and intensity in stably transformed rice (Oryza sativa L.), and then used them to direct the expression of the switchgrass MYB4 (PvMYB4) transcription factor gene in transgenic switchgrass to endow reduced recalcitrance in aboveground biomass. These promoters correspond to photosynthesis-related light-harvesting complex II chlorophyll-a/b binding gene (PvLhcb), phosphoenolpyruvate carboxylase (PvPEPC), and the photosystem II 10 kDa R subunit (PvPsbR). Real-time RT-PCR analysis detected their strong expression in the aboveground tissues including leaf blades, leaf sheaths, internodes, inflorescences, and nodes of switchgrass, which was tightly up-regulated by light. Stable transgenic rice expressing the GUS reporter under the control of each promoter (756-2005 bp in length) further confirmed their strong expression patterns in leaves and stems. With the exception of the serial promoter deletions of PvLhcb, all GUS marker patterns under the control of each 5'-end serial promoter deletion were not different from that conveyed by their respective promoters. All of the shortest promoter fragments (199-275 bp in length) conveyed strong green tissue-specific GUS expression in transgenic rice

Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Wusheng; Mazarei, Mitra; Ye, Rongjian

Genetic engineering of switchgrass (Panicum virgatum L.) for reduced cell wall recalcitrance and improved biofuel production has been a long pursued goal. Up to now, constitutive promoters have been used to direct the expression of cell wall biosynthesis genes toward attaining that goal. While generally sufficient to gauge a transgene's effects in the heterologous host, constitutive overexpression often leads to undesirable plant phenotypic effects. Green tissue-specific promoters from switchgrass are potentially valuable to directly alter cell wall traits exclusively in harvestable aboveground biomass while not changing root phenotypes. We identified and functionally characterized three switchgrass green tissue-specific promoters and assessedmore » marker gene expression patterns and intensity in stably transformed rice (Oryza sativa L.), and then used them to direct the expression of the switchgrass MYB4 (PvMYB4) transcription factor gene in transgenic switchgrass to endow reduced recalcitrance in aboveground biomass. These promoters correspond to photosynthesis-related light-harvesting complex II chlorophyll-a/b binding gene (PvLhcb), phosphoenolpyruvate carboxylase (PvPEPC), and the photosystem II 10 kDa R subunit (PvPsbR). Real-time RT-PCR analysis detected their strong expression in the aboveground tissues including leaf blades, leaf sheaths, internodes, inflorescences, and nodes of switchgrass, which was tightly up-regulated by light. Stable transgenic rice expressing the GUS reporter under the control of each promoter (756-2005 bp in length) further confirmed their strong expression patterns in leaves and stems. With the exception of the serial promoter deletions of PvLhcb, all GUS marker patterns under the control of each 5'-end serial promoter deletion were not different from that conveyed by their respective promoters. All of the shortest promoter fragments (199-275 bp in length) conveyed strong green tissue-specific GUS expression in transgenic rice

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.

Promoter isolation and characterization of GhAO-like1, a Gossypium hirsutum gene similar to multicopper oxidases that is highly expressed in reproductive organs.

PubMed

Lambret-Frotté, Julia; Artico, Sinara; Muniz Nardeli, Sarah; Fonseca, Fernando; Brilhante Oliveira-Neto, Osmundo; Grossi-de-Sá, Maria Fatima; Alves-Ferreira, Marcio

2016-01-01

Cotton is one of the most economically important cultivated crops. It is the major source of natural fiber for the textile industry and an important target for genetic modification for both biotic stress and herbicide tolerance. Therefore, the characterization of genes and regulatory regions that might be useful for genetic transformation is indispensable. The isolation and characterization of new regulatory regions is of great importance to drive transgene expression in genetically modified crops. One of the major drawbacks in cotton production is pest damage; therefore, the most promising, cost-effective, and sustainable method for pest control is the development of genetically resistant cotton lines. Considering this scenario, our group isolated and characterized the promoter region of a MCO (multicopper oxidase) from Gossypium hirsutum, named GhAO-like1 (ascorbate oxidase-like1). The quantitative expression, together with the in vivo characterization of the promoter region reveals that GhAO-like1 has a flower- and fruit-specific expression pattern. The GUS activity is mainly observed in stamens, as expected considering that the GhAO-like1 regulatory sequence is enriched in cis elements, which have been characterized as a target of reproductive tissue specific transcription factors. Both histological and quantitative analyses in Arabidopsis thaliana have confirmed flower (mainly in stamens) and fruit expression of GhAO-like1. In the present paper, we isolated and characterized both in silico and in vivo the promoter region of the GhAO-like1 gene. The regulatory region of GhAO-like1 might be useful to confer tissue-specific expression in genetically modified plants.

Transcriptional regulation of ABI3- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis.

PubMed

Nakashima, Kazuo; Fujita, Yasunari; Katsura, Koji; Maruyama, Kyonoshin; Narusaka, Yoshihiro; Seki, Motoaki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-01-01

ABA-responsive elements (ABREs) are cis-acting elements and basic leucine zipper (bZIP)-type ABRE-binding proteins (AREBs) are transcriptional activators that function in the expression of RD29B in vegetative tissue of Arabidopsis in response to abscisic acid (ABA) treatment. Dehydration-responsive elements (DREs) function as coupling elements of ABRE in the expression of RD29A in response to ABA. Expression analysis using abi3 and abi5 mutants showed that ABI3 and ABI5 play important roles in the expression of RD29B in seeds. Base-substitution analysis showed that two ABREs function strongly and one ABRE coupled with DRE functions weakly in the expression of RD29A in embryos. In a transient transactivation experiment, ABI3, ABI5 and AREB1 activated transcription of a GUS reporter gene driven by the RD29B promoter strongly but these proteins activated the transcription driven by the RD29A promoter weakly. In 35S::ABI3 Arabidopsis plants, the expression of RD29B was up-regulated strongly, but that of RD29A was up-regulated weakly. These results indicate that the expression of RD29B having ABREs in the promoter is up-regulated strongly by ABI3, whereas that of RD29A having one ABRE coupled with DREs in the promoter is up-regulated weakly by ABI3. We compared the expression of 7000 Arabidopsis genes in response to ABA treatment during germination and in the vegetative growth stage, and that in 35S::ABI3 plants using a full-length cDNA microarray. The expression of ABI3- and/or ABA-responsive genes and cis-elements in the promoters are discussed.

Growing Up Strong: A Mental Wellness and Life Skills Development Program. Spanish Bilingual Supplement for Preschoolers.

ERIC Educational Resources Information Center

Hammer, Judith McGowan; O'Bar, Angelina Merenda

Growing Up Strong (GUS), a mental health and substance abuse prevention program, was developed to help preschoolers through sixth graders gain improved mental and physical health and establish positive relationships with significant adults. The Spanish Bilingual Supplement to the preschool-level GUS program also promotes respect for Spanish…

Hexokinase 1 is required for glucose-induced repression of bZIP63, At5g22920, and BT2 in Arabidopsis

DOE PAGES

Kunz, Sabine; Gardestrom, Per; Pesquet, Edouard; ...

2015-07-14

Simple sugars, like glucose (Glc) and sucrose (Suc), act as signals to modulate the expression of hundreds of genes in plants. Frequently, however, it remains unclear whether this regulation is induced by the sugars themselves or by their derivatives generated in the course of carbohydrate (CH) metabolism. In the present study, we tested the relevance of different CH metabolism and allocation pathways affecting expression patterns of five selected sugar-responsive genes ( bZIP63, At5g22920, BT2, MGD2, and TPS9) in Arabidopsis thaliana. In general, the expression followed diurnal changes in the overall sugar availability. However, under steady growth conditions, this response wasmore » hardly impaired in the mutants for CH metabolizing/ transporting proteins ( adg1, sex1, sus1-4, sus5/6, and tpt2), including also hexokinase1 (HXK1) loss- and gain-of-function plants— gin2.1 and oe3.2, respectively. In addition, transgenic plants carrying pbZIP63::GUS showed no changes in reporter-gene-expression when grown on sugar under steady-state conditions. In contrast, short-term treatments of agar-grown seedlings with 1% Glc or Suc induced pbZIP63::GUS repression, which became even more apparent in seedlings grown in liquid media. Subsequent analyses of liquid-grown gin2.1 and oe3.2 seedlings revealed that Glc -dependent regulation of the five selected genes was not affected in gin2.1, whereas it was enhanced in oe3.2 plants for bZIP63, At5g22920, and BT. The sugar treatments had no effect on ATP/ADP ratio, suggesting that changes in gene expression were not linked to cellular energy status. Altogether, the data suggest that HXK1 does not act as Glc sensor controlling bZIP63, At5g22920, and BT2 expression, but it is nevertheless required for the production of a downstream metabolic signal regulating their expression« less

Hexokinase 1 is required for glucose-induced repression of bZIP63, At5g22920, and BT2 in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kunz, Sabine; Gardestrom, Per; Pesquet, Edouard

Simple sugars, like glucose (Glc) and sucrose (Suc), act as signals to modulate the expression of hundreds of genes in plants. Frequently, however, it remains unclear whether this regulation is induced by the sugars themselves or by their derivatives generated in the course of carbohydrate (CH) metabolism. In the present study, we tested the relevance of different CH metabolism and allocation pathways affecting expression patterns of five selected sugar-responsive genes ( bZIP63, At5g22920, BT2, MGD2, and TPS9) in Arabidopsis thaliana. In general, the expression followed diurnal changes in the overall sugar availability. However, under steady growth conditions, this response wasmore » hardly impaired in the mutants for CH metabolizing/ transporting proteins ( adg1, sex1, sus1-4, sus5/6, and tpt2), including also hexokinase1 (HXK1) loss- and gain-of-function plants— gin2.1 and oe3.2, respectively. In addition, transgenic plants carrying pbZIP63::GUS showed no changes in reporter-gene-expression when grown on sugar under steady-state conditions. In contrast, short-term treatments of agar-grown seedlings with 1% Glc or Suc induced pbZIP63::GUS repression, which became even more apparent in seedlings grown in liquid media. Subsequent analyses of liquid-grown gin2.1 and oe3.2 seedlings revealed that Glc -dependent regulation of the five selected genes was not affected in gin2.1, whereas it was enhanced in oe3.2 plants for bZIP63, At5g22920, and BT. The sugar treatments had no effect on ATP/ADP ratio, suggesting that changes in gene expression were not linked to cellular energy status. Altogether, the data suggest that HXK1 does not act as Glc sensor controlling bZIP63, At5g22920, and BT2 expression, but it is nevertheless required for the production of a downstream metabolic signal regulating their expression« less

An improved purification method for the lysosomal storage disease protein β-glucuronidase produced in CHO cells.

PubMed

Fratz-Berilla, Erica J; Ketcham, Stephanie A; Parhiz, Hamideh; Ashraf, Muhammad; Madhavarao, Chikkathur N

2017-12-01

Human β-glucuronidase (GUS; EC 3.2.1.31) is a lysosomal enzyme that catalyzes the hydrolysis of β-d-glucuronic acid residues from the non-reducing termini of glycosaminoglycans. Impairment in GUS function leads to the metabolic disorder mucopolysaccharidosis type VII, also known as Sly syndrome. We produced GUS from a CHO cell line grown in suspension in a 15 L perfused bioreactor and developed a three step purification procedure that yields ∼99% pure enzyme with a recovery of more than 40%. The method can be completed in two days and has the potential to be integrated into a continuous manufacturing scheme. Published by Elsevier Inc.

Growing Up Strong: A Mental Wellness and Life Skills Development Program. Spanish Bilingual Supplement for Kindergarten through Third Grade.

ERIC Educational Resources Information Center

Hammer, Judith McGowan; O'Bar, Angelina Merenda

The Growing Up Strong (GUS) program was developed for preschool through sixth grade students to promote mental and physical health and prevent substance abuse. The Spanish Bilingual Supplement to the kindergarten through third grade GUS materials also promotes respect for Spanish language and cultures. This document includes an English-language…

Sunflower (Helianthus annuus L.).

PubMed

Radonic, Laura M; Lewi, Dalia M; López, Nilda E; Hopp, H Esteban; Escandón, Alejandro S; Bilbao, Marisa López

2015-01-01

Sunflower (Helianthus annuus L.) is still considered as a recalcitrant species to in vitro culture and transformation in spite of the publication of different protocols. Here we describe a routine transformation system of this crop which requires mature HA89 genotype seeds and Agrobacterium tumefaciens EHA105 strain for gene delivery, being both easily available. Selection of transformed shoots depends on root development in kanamycin-selective media, instead of shoot color, avoiding selection of escapes. The establishment of this protocol proved successful for the incorporation of both reporter and agronomic important genes and also for the evaluation of the specific expression patterns of different promoters in transgenic sunflower plants. Stable expression of the incorporated transgenes was confirmed by RT-PCR and GUS reporter gene visualization. Stable inheritance of transgenes was successfully followed until T2 generation in several independent lines.

Succession of Sulfur-Oxidizing Bacteria in the Microbial Community on Corroding Concrete in Sewer Systems† ▿

PubMed Central

Okabe, Satoshi; Odagiri, Mitsunori; Ito, Tsukasa; Satoh, Hisashi

2007-01-01

Microbially induced concrete corrosion (MICC) in sewer systems has been a serious problem for a long time. A better understanding of the succession of microbial community members responsible for the production of sulfuric acid is essential for the efficient control of MICC. In this study, the succession of sulfur-oxidizing bacteria (SOB) in the bacterial community on corroding concrete in a sewer system in situ was investigated over 1 year by culture-independent 16S rRNA gene-based molecular techniques. Results revealed that at least six phylotypes of SOB species were involved in the MICC process, and the predominant SOB species shifted in the following order: Thiothrix sp., Thiobacillus plumbophilus, Thiomonas intermedia, Halothiobacillus neapolitanus, Acidiphilium acidophilum, and Acidithiobacillus thiooxidans. A. thiooxidans, a hyperacidophilic SOB, was the most dominant (accounting for 70% of EUB338-mixed probe-hybridized cells) in the heavily corroded concrete after 1 year. This succession of SOB species could be dependent on the pH of the concrete surface as well as on trophic properties (e.g., autotrophic or mixotrophic) and on the ability of the SOB to utilize different sulfur compounds (e.g., H2S, S0, and S2O32−). In addition, diverse heterotrophic bacterial species (e.g., halo-tolerant, neutrophilic, and acidophilic bacteria) were associated with these SOB. The microbial succession of these microorganisms was involved in the colonization of the concrete and the production of sulfuric acid. Furthermore, the vertical distribution of microbial community members revealed that A. thiooxidans was the most dominant throughout the heavily corroded concrete (gypsum) layer and that A. thiooxidans was most abundant at the highest surface (1.5-mm) layer and decreased logarithmically with depth because of oxygen and H2S transport limitations. This suggested that the production of sulfuric acid by A. thiooxidans occurred mainly on the concrete surface and the

Finger millet [Eleusine coracana (L.) Gaertn].

PubMed

Ceasar, Stanislaus Antony; Ignacimuthu, Savarimuthu

2015-01-01

Millets are the primary food source for millions of people in tropical regions of the world supplying mineral nutrition and protein. In this chapter, we describe an optimized protocol for the Agrobacterium-mediated transformation of finger millet variety GPU 45. Agrobacterium strain LBA4404 harboring plasmid pCAMBIA1301 which contains hygromycin phosphotransferase (hph) as selectable marker gene and β-glucuronidase (GUS) as reporter gene has been used. This protocol utilizes the shoot apex explants for the somatic embryogenesis and regeneration of finger millet after the transformation by Agrobacterium. Desiccation of explants during cocultivation helps for the better recovery of transgenic plants. This protocol is very useful for the efficient production of transgenic plants in finger millet through Agrobacterium-mediated transformation.

Continuous fluorometric method for measuring β-glucuronidase activity: comparative analysis of three fluorogenic substrates.

PubMed

Briciu-Burghina, Ciprian; Heery, Brendan; Regan, Fiona

2015-09-07

E. coli β-glucuronidase (GUS) activity assays are routinely used in fields such as plant molecular biology, applied microbiology and healthcare. Methods based on the optical detection of GUS using synthetic fluorogenic substrates are widely employed since they don't require expensive instrumentation and are easy to perform. In this study three fluorogenic substrates and their respective fluorophores were studied for the purpose of developing a continuous fluorometric method for GUS. The fluorescence intensity of 6-chloro-4-methyl-umbelliferone (6-CMU) at pH 6.8 was found to be 9.5 times higher than that of 4-methyl umbelliferone (4-MU) and 3.2 times higher than the fluorescence of 7-hydroxycoumarin-3-carboxylic acid (3-CU). Michaelis-Menten kinetic parameters of GUS catalysed hydrolysis of 6-chloro-4-methyl-umbelliferyl-β-D-glucuronide (6-CMUG) were determined experimentally (Km = 0.11 mM, Kcat = 74 s(-1), Kcat/Km = 6.93 × 10(5) s(-1) M(-1)) and compared with the ones found for 4-methyl-umbelliferyl-β-D-glucuronide (4-MUG) (Km = 0.07 mM, Kcat = 92 s(-1), Kcat/Km = 1.29 × 10(6) s(-1) M(-1)) and 3-carboxy-umbelliferyl-β-D-glucuronide (3-CUG) (Km = 0.48 mM, Kcat = 35 s(-1), Kcat/Km = 7.40 × 10(4) s(-1) M(-1)). Finally a continuous fluorometric method based on 6-CMUG as a fluorogenic substrate has been developed for measuring GUS activity. When compared with the highly used discontinuous method based on 4-MUG as a substrate it was found that the new method is more sensitive and reproducible (%RSD = 4.88). Furthermore, the developed method is less laborious, faster and more economical and should provide an improved alternative for GUS assays and kinetic studies.

The Vaccinium corymbosum FLOWERING LOCUS T-like gene (VcFT): a flowering activator reverses photoperiodic and chilling requirements in blueberry.

PubMed

Song, Guo-qing; Walworth, Aaron; Zhao, Dongyan; Jiang, Ning; Hancock, James F

2013-11-01

The blueberry FLOWERING LOCUS T ( FT )-like gene ( VcFT ) cloned from the cDNA of a tetraploid, northern highbush blueberry ( Vaccinium corymbosum L.) is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering. Blueberry is a woody perennial bush with a longer juvenile period than annual crops, requiring vernalization to flower normally. Few studies have been reported on the molecular mechanism of flowering in blueberry or other woody plants. Because FLOWERING LOCUS T (FT) from Arabidopsis thaliana plays a multifaceted role in generating mobile molecular signals to regulate plant flowering time, isolation and functional analysis of the blueberry (Vaccinium corymbosum L.) FT-like gene (VcFT) will facilitate the elucidation of molecular mechanisms of flowering in woody plants. Based on EST sequences, a 525-bpVcFT was identified and cloned from the cDNA of a tetraploid, northern highbush blueberry cultivar, Bluecrop. Ectopic expression of 35S:VcFT in tobacco induced flowering an average of 28 days earlier than wild-type plants. Expression of the 35S:VcFT in the blueberry cultivar Aurora resulted in an extremely early flowering phenotype, which flowered not only during in vitro culture, a growth stage when nontransgenic shoots had not yet flowered, but also in 6-10-week old, soil-grown transgenic plants, in contrast to the fact that at least 1 year and 800 chilling hours are required for the appearance of the first flower of both nontransgenic 'Aurora' and transgenic controls with the gusA. These results demonstrate that the VcFT is a functional floral activator and overexpression of the VcFT is able to reverse the photoperiodic and chilling requirements and drive early and continuous flowering.

Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues.

PubMed

Viana, Antonio A B; Fragoso, Rodrigo R; Guimarães, Luciane M; Pontes, Naiara; Oliveira-Neto, Osmundo B; Artico, Sinara; Nardeli, Sarah M; Alves-Ferreira, Marcio; Batista, João A N; Silva, Maria C M; Grossi-de-Sa, Maria F

2011-11-24

Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.

Enhanced transgene expression in rice following selection controlled by weak promoters.

PubMed

Zhou, Jie; Yang, Yong; Wang, Xuming; Yu, Feibo; Yu, Chulang; Chen, Juan; Cheng, Ye; Yan, Chenqi; Chen, Jianping

2013-03-27

Techniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. The conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. Although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent. In this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tCUP1, a fragment derived from the tobacco cryptic promoter (tCUP), was tested for its activity in rice by fusion to both a β-glucuronidase (GUS) reporter and a hygromycin phosphotransferase (HPT) selectable marker. The tCUP1 promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the GUS reporter, but was much weaker than the CaMV 35S promoter in driving a selectable marker for growth of resistant calli. However, in the resistant calli and regenerated transgenic plants selected by the use of tCUP1, the constitutive expression of green fluorescent protein (GFP) was dramatically increased as a result of the additive effect of multiple T-DNA insertions. The correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase (Nos). The use of weak promoter derived selectable markers leads to a high T-DNA copy number and then greatly increases the expression of the foreign gene. The method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice.

Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris.

PubMed

Cha, Thye San; Yee, Willy; Aziz, Ahmad

2012-04-01

The successful establishment of an Agrobacterium-mediated transformation method and optimisation of six critical parameters known to influence the efficacy of Agrobacterium T-DNA transfer in the unicellular microalga Chlorella vulgaris (UMT-M1) are reported. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1304 containing the gfp:gusA fusion reporter and a hygromycin phosphotransferase (hpt) selectable marker driven by the CaMV35S promoter were used for transformation. Transformation frequency was assessed by monitoring transient β-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24°C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 μM acetosyringone, Agrobacterium density of 1.0 units (OD(600)) and 2 days of pre-culture were optimum variables which produced the highest number of GUS-positive cells (8.8-20.1%) when each of these parameters was optimised individually. Transformation conducted with the combination of all optimal parameters above produced 25.0% of GUS-positive cells, which was almost a threefold increase from 8.9% obtained from un-optimised parameters. Evidence of transformation was further confirmed in 30% of 30 randomly-selected hygromycin B (20 mg L(-1)) resistant colonies by polymerase chain reaction (PCR) using gfp:gusA and hpt-specific primers. The developed transformation method is expected to facilitate the genetic improvement of this commercially-important microalga.

The boron transporter BnaC4.BOR1;1c is critical for inflorescence development and fertility under boron limitation in Brassica napus.

PubMed

Zhang, Quan; Chen, Haifei; He, Mingliang; Zhao, Zhuqing; Cai, Hongmei; Ding, Guangda; Shi, Lei; Xu, Fangsen

2017-09-01

Boron (B) is an essential micronutrient for plants, but the molecular mechanisms underlying the uptake and distribution of B in allotetraploid rapeseed (Brassica napus) are unclear. Here, we identified a B transporter of rapeseed, BnaC4.BOR1;1c, which is expressed in shoot nodes and involved in distributing B to the reproductive organs. Transgenic Arabidopsis plants containing a BnaC4.BOR1;1c promoter-driven GUS reporter gene showed strong GUS activity in roots, nodal regions of the shoots and immature floral buds. Overexpressing BnaC4.BOR1;1c in Arabidopsis wild type or in bor1-1 mutants promoted wild-type growth and rescued the bor1-1 mutant phenotype. Conversely, knockdown of BnaC4.BOR1;1c in a B-efficient rapeseed line reduced B accumulation in flower organs, eventually resulting in severe sterility and seed yield loss. BnaC4.BOR1;1c RNAi plants exhibited large amounts of disintegrated stigma papilla cells with thickened cell walls accompanied by abnormal proliferation of lignification under low-B conditions, indicating that the sterility may be a result of altered cell wall properties in flower organs. Taken together, our results demonstrate that BnaC4.BOR1;1c is a AtBOR1-homologous B transporter gene expressing in both roots and shoot nodes that is essential for the developing inflorescence tissues, which highlights its diverse functions in allotetraploid rapeseed compared with diploid model plant Arabidopsis. © 2017 John Wiley & Sons Ltd.

In vitro and in vivo inoculation of four endophytic bacteria on Lycopersicon esculentum.

PubMed

Botta, Anna Lucia; Santacecilia, Alessandra; Ercole, Claudia; Cacchio, Paola; Del Gallo, Maddalena

2013-09-25

Four bacteria selected on the basis of their capability of fixing atmospheric nitrogen, stimulating plant-growth, and protecting the host plant from pathogens - Azospirillum brasilense, Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, Burkholderia ambifaria - were inoculated on tomato seeds either singularly, in couple and in a four bacteria mixer. Aim of this research was to evaluate: (1) effect of single and mixed cultures on the inoculated plant - plant growth, dry weight, root length and surface, number of leaves, among others; (2) colonization and interactions of the bacteria inside the host plant; (3) localization inside the host of single bacterial strains marked with the gusA reporter gene. The results obtained indicate that all selected microbial strains have colonized Lycopersicon esculentum but in a different way, depending on the single species. A. brasilense, G. diazotrophicus inoculated in vitro singularly and together were the best plant colonizers. In vivo essays, instead, B. ambifaria and the four-bacteria mixer gave the best results. It was possible to localize both A. brasilense and H. seropedicae inside the plant by the gusA reporter gene. The bacterial strains occur along the root axis from the apical zone until to the basal stem, on the shoot from the base up to the leaves. The four bacteria actively colonize tomato seeds and establish an endophytic community inside the plant. This review gives new information about colonization processes, in particular how bacteria interact with plants and whether they are likely to establish themselves in the plant environment after field application as biofertilizers or biocontrol agents. Copyright © 2013 Elsevier B.V. All rights reserved.

Rha1, a new mutant of Arabidopsis disturbed in root slanting, gravitropism and auxin physiology.

PubMed

Fortunati, Alessio; Piconese, Silvia; Tassone, Paola; Ferrari, Simone; Migliaccio, Fernando

2008-11-01

A new Arabidopsis mutant is characterized (rha1) that shows, in the roots, reduced right-handed slanting, reduced gravitropism and resistance to 2,4-D, TIBA, NPA and ethylene. It also shows reduced length in the shoot and root, reduced number of lateral roots and shorter siliques. The gene was cloned through TAIL-PCR and resulted in a HSF. Because none of the known gravitropic and auxinic mutants result from damage in a HSF, rha1 seems to belong to a new class of this group of mutants. Quantitative PCR analysis showed that the expression of the gene is increased by heat and cold shock, and by presence of 2,4-D in the media. Study of the expression through the GUS reporter gene revealed increased expression in clinostated and gravistimulated plants, but only in adult tissues, and not in the apical meristems of shoots and roots.

Rha1, a new mutant of Arabidopsis disturbed in root slanting, gravitropism and auxin physiology

PubMed Central

Fortunati, Alessio; Piconese, Silvia; Tassone, Paola; Ferrari, Simone

2008-01-01

A new Arabidopsis mutant is characterized (rha1) that shows, in the roots, reduced right-handed slanting, reduced gravitropism and resistance to 2,4-D, TIBA, NPA and ethylene. It also shows reduced length in the shoot and root, reduced number of lateral roots and shorter siliques. The gene was cloned through TAIL-PCR and resulted in a HSF. Because none of the known gravitropic and auxinic mutants result from damage in a HSF, rha1 seems to belong to a new class of this group of mutants. Quantitative PCR analysis showed that the expression of the gene is increased by heat and cold shock, and by presence of 2,4-D in the media. Study of the expression through the GUS reporter gene revealed increased expression in clinostated and gravistimulated plants, but only in adult tissues, and not in the apical meristems of shoots and roots. PMID:19704429

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.

Isolation and functional characterization of Lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites

PubMed Central

2010-01-01

Background Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061. Results A 908 bp region upstream to the initiation codon of the Lycopene β-cyclase gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the ShCYC-B gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of GUS reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to Agrobacterium. Agrobacterium-mediated transient and stable expression systems were used to study the GUS expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the ShCYC-B promoter strength, while deletion of -908 to -437 bp 5' to ATG led to

FOREVER YOUNG FLOWER Negatively Regulates Ethylene Response DNA-Binding Factors by Activating an Ethylene-Responsive Factor to Control Arabidopsis Floral Organ Senescence and Abscission1

PubMed Central

Li, Pei-Fang; Lee, Yung-I; Yang, Chang-Hsien

2015-01-01

In this study of Arabidopsis (Arabidopsis thaliana), we investigated the relationship between FOREVER YOUNG FLOWER (FYF) and Ethylene Response DNA-binding Factors (EDFs) and functionally analyzed a key FYF target, an Ethylene-Responsive Factor (ERF), that controls flower senescence/abscission. Ectopic expression of EDF1/2/3/4 caused promotion of flower senescence/abscission and the activation of the senescence-associated genes. The presence of a repressor domain in EDFs and the enhancement of the promotion of senescence/abscission in EDF1/2/3/4+SRDX (converting EDFs to strong repressors by fusion with the ERF-associated amphiphilic repression motif repression domain SRDX) transgenic plants suggested that EDFs act as repressors. The significant reduction of β-glucuronidase (GUS) expression by 35S:FYF in EDF1/2/3/4:GUS plants indicates that EDF1/2/3/4 functions downstream of FYF in regulating flower senescence/abscission. In this study, we also characterized an ERF gene, FOREVER YOUNG FLOWER UP-REGULATING FACTOR1 (FUF1), which is up-regulated by FYF during flower development. Ectopic expression of FUF1 caused similar delayed flower senescence/abscission as seen in 35S:FYF plants. This phenotype was correlated with deficient abscission zone formation, ethylene insensitivity, and down-regulation of EDF1/2/3/4 and abscission-associated genes in 35S:FUF1 flowers. In contrast, significant promotion of flower senescence/abscission and up-regulation of EDF1/2/3/4 were observed in 35S:FUF1+SRDX transgenic dominant-negative plants, in which FUF1 is converted to a potent repressor by fusion to an SRDX-suppressing motif. Thus, FUF1 acts as an activator in suppressing EDF1/2/3/4 function and senescence/abscission of the flowers. Our results reveal that FYF regulates flower senescence/abscission by negatively regulating EDF1/2/3/4, which is the downstream gene in the ethylene response, by activating FUF1 in Arabidopsis. PMID:26063506

FOREVER YOUNG FLOWER Negatively Regulates Ethylene Response DNA-Binding Factors by Activating an Ethylene-Responsive Factor to Control Arabidopsis Floral Organ Senescence and Abscission.

PubMed

Chen, Wei-Han; Li, Pei-Fang; Chen, Ming-Kun; Lee, Yung-I; Yang, Chang-Hsien

2015-08-01

In this study of Arabidopsis (Arabidopsis thaliana), we investigated the relationship between FOREVER YOUNG FLOWER (FYF) and Ethylene Response DNA-binding Factors (EDFs) and functionally analyzed a key FYF target, an Ethylene-Responsive Factor (ERF), that controls flower senescence/abscission. Ectopic expression of EDF1/2/3/4 caused promotion of flower senescence/abscission and the activation of the senescence-associated genes. The presence of a repressor domain in EDFs and the enhancement of the promotion of senescence/abscission in EDF1/2/3/4+SRDX (converting EDFs to strong repressors by fusion with the ERF-associated amphiphilic repression motif repression domain SRDX) transgenic plants suggested that EDFs act as repressors. The significant reduction of β-glucuronidase (GUS) expression by 35S:FYF in EDF1/2/3/4:GUS plants indicates that EDF1/2/3/4 functions downstream of FYF in regulating flower senescence/abscission. In this study, we also characterized an ERF gene, FOREVER YOUNG FLOWER UP-REGULATING FACTOR1 (FUF1), which is up-regulated by FYF during flower development. Ectopic expression of FUF1 caused similar delayed flower senescence/abscission as seen in 35S:FYF plants. This phenotype was correlated with deficient abscission zone formation, ethylene insensitivity, and down-regulation of EDF1/2/3/4 and abscission-associated genes in 35S:FUF1 flowers. In contrast, significant promotion of flower senescence/abscission and up-regulation of EDF1/2/3/4 were observed in 35S:FUF1+SRDX transgenic dominant-negative plants, in which FUF1 is converted to a potent repressor by fusion to an SRDX-suppressing motif. Thus, FUF1 acts as an activator in suppressing EDF1/2/3/4 function and senescence/abscission of the flowers. Our results reveal that FYF regulates flower senescence/abscission by negatively regulating EDF1/2/3/4, which is the downstream gene in the ethylene response, by activating FUF1 in Arabidopsis. © 2015 American Society of Plant Biologists. All Rights

A Non-specific Setaria italica Lipid Transfer Protein Gene Plays a Critical Role under Abiotic Stress.

PubMed

Pan, Yanlin; Li, Jianrui; Jiao, Licong; Li, Cong; Zhu, Dengyun; Yu, Jingjuan

2016-01-01

Lipid transfer proteins (LTPs) are a class of cysteine-rich soluble proteins having small molecular weights. LTPs participate in flower and seed development, cuticular wax deposition, also play important roles in pathogen and abiotic stress responses. A non-specific LTP gene ( SiLTP ) was isolated from a foxtail millet ( Setaria italica ) suppression subtractive hybridization library enriched for differentially expressed genes after abiotic stress treatments. A semi-quantitative reverse transcriptase PCR analysis showed that SiLTP was expressed in all foxtail millet tissues. Additionally, the SiLTP promoter drove GUS expression in root tips, stems, leaves, flowers, and siliques of transgenic Arabidopsis . Quantitative real-time PCR indicated that the SiLTP expression was induced by NaCl, polyethylene glycol, and abscisic acid (ABA). SiLTP was localized in the cytoplasm of tobacco leaf epidermal cells and maize protoplasts. The ectopic expression of SiLTP in tobacco resulted in higher levels of salt and drought tolerance than in the wild type (WT). To further assess the function of SiLTP, SiLTP overexpression (OE) and RNA interference (RNAi)-based transgenic foxtail millet were obtained. SiLTP -OE lines performed better under salt and drought stresses compared with WT plants. In contrast, the RNAi lines were much more sensitive to salt and drought compared than WT. Electrophoretic mobility shift assays and yeast one-hybrids indicated that the transcription factor ABA-responsive DRE-binding protein (SiARDP) could bind to the dehydration-responsive element of SiLTP promoter in vitro and in vivo , respectively. Moreover, the SiLTP expression levels were higher in SiARDP -OE plants compared than the WT. These results confirmed that SiLTP plays important roles in improving salt and drought stress tolerance of foxtail millet, and may partly be upregulated by SiARDP. SiLTP may provide an effective genetic resource for molecular breeding in crops to enhance salt and drought

The Post-transcriptional Regulator rsmA/csrA Activates T3SS by Stabilizing the 5′ UTR of hrpG, the Master Regulator of hrp/hrc Genes, in Xanthomonas

PubMed Central

Andrade, Maxuel O.; Farah, Chuck S.; Wang, Nian

2014-01-01

The RsmA/CsrA family of the post-transcriptional regulators of bacteria is involved in the regulation of many cellular processes, including pathogenesis. In this study, we demonstrated that rsmA not only is required for the full virulence of the phytopathogenic bacterium Xanthomonas citri subsp. citri (XCC) but also contributes to triggering the hypersensitive response (HR) in non-host plants. Deletion of rsmA resulted in significantly reduced virulence in the host plant sweet orange and a delayed and weakened HR in the non-host plant Nicotiana benthamiana. Microarray, quantitative reverse-transcription PCR, western-blotting, and GUS assays indicated that RsmA regulates the expression of the type 3 secretion system (T3SS) at both transcriptional and post-transcriptional levels. The regulation of T3SS by RsmA is a universal phenomenon in T3SS-containing bacteria, but the specific mechanism seems to depend on the interaction between a particular bacterium and its hosts. For Xanthomonads, the mechanism by which RsmA activates T3SS remains unknown. Here, we show that RsmA activates the expression of T3SS-encoding hrp/hrc genes by directly binding to the 5′ untranslated region (UTR) of hrpG, the master regulator of the hrp/hrc genes in XCC. RsmA stabilizes hrpG mRNA, leading to increased accumulation of HrpG proteins and subsequently, the activation of hrp/hrc genes. The activation of the hrp/hrc genes by RsmA via HrpG was further supported by the observation that ectopic overexpression of hrpG in an rsmA mutant restored its ability to cause disease in host plants and trigger HR in non-host plants. RsmA also stabilizes the transcripts of another T3SS-associated hrpD operon by directly binding to the 5′ UTR region. Taken together, these data revealed that RsmA primarily activates T3SS by acting as a positive regulator of hrpG and that this regulation is critical to the pathogenicity of XCC. PMID:24586158

Monolignol radical-radical coupling networks in western red cedar and Arabidopsis and their evolutionary implications

NASA Technical Reports Server (NTRS)

Kim, Myoung K.; Jeon, Jae-Heung; Davin, Laurence B.; Lewis, Norman G.

2002-01-01

The discovery of a nine-member multigene dirigent family involved in control of monolignol radical-radical coupling in the ancient gymnosperm, western red cedar, suggested that a complex multidimensional network had evolved to regulate such processes in vascular plants. Accordingly, in this study, the corresponding promoter regions for each dirigent multigene member were obtained by genome-walking, with Arabidopsis being subsequently transformed to express each promoter fused to the beta-glucuronidase (GUS) reporter gene. It was found that each component gene of the proposed network is apparently differentially expressed in individual tissues, organs and cells at all stages of plant growth and development. The data so obtained thus further support the hypothesis that a sophisticated monolignol radical-radical coupling network exists in plants which has been highly conserved throughout vascular plant evolution.

Salt tolerance and activity of antioxidative enzymes of transgenic finger millet overexpressing a vacuolar H(+)-pyrophosphatase gene (SbVPPase) from Sorghum bicolor.

PubMed

Anjaneyulu, Ediga; Reddy, Palle Surender; Sunita, Merla Srilakshmi; Kishor, Polavarapu B Kavi; Meriga, Balaji

2014-06-15

A vacuolar proton pyrophosphatase cDNA clone was isolated from Sorghum bicolor (SbVPPase) using end-to-end gene-specific primer amplification. It showed 80-90% homology at the nucleotide and 85-95% homology at the amino acid level with other VPPases. The gene was introduced into expression vector pCAMBIA1301 under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter and transformed into Agrobacterium tumifaciens strain LBA4404 to infect embryogenic calli of finger millet (Eleusine coracana). Successful transfer of SbVPPase was confirmed by a GUS histochemical assay and PCR analysis. Both, controls and transgenic plants were subjected to 100 and 200mM NaCl and certain biochemical and physiological parameters were studied. Relative water content (RWC), plant height, leaf expansion, finger length and width and grain weight were severely reduced (50-70%), and the flowering period was delayed by 20% in control plants compared to transgenic plants under salinity stress. With increasing salt stress, the proline and chlorophyll contents as well as the enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) increased by 25-100% in transgenics, while malondialdehyde (MDA) showed a 2-4-fold decrease. The increased activities of antioxidant enzymes and the reduction in the MDA content suggest efficient scavenging of reactive oxygen species (ROS) in transgenics and, as a consequence, probably alleviation of salt stress. Also, the leaf tissues of the transgenics accumulated 1.5-2.5-fold higher Na(+) and 0.4-0.8-fold higher K(+) levels. Together, these results clearly demonstrate that overexpression of SbVPPase in transgenic finger millet enhances the plant's performance under salt stress. Copyright © 2014 Elsevier GmbH. All rights reserved.

Characterization of HbWRKY1, a WRKY transcription factor from Hevea brasiliensis that negatively regulates HbSRPP.

PubMed

Wang, Ying; Guo, Dong; Li, Hui-Liang; Peng, Shi-Qing

2013-10-01

Small rubber particle protein (SRPP) is a major component of Hevea brasiliensis (H. brasiliensis) latex, which is involved in natural rubber (NR) biosynthesis. However, little information is available on the regulation of SRPP gene (HbSRPP) expression. To study the transcriptional regulation of HbSRPP, the yeast one-hybrid experiment was performed to screen the latex cDNA library using the HbSRPP promoter as bait. One cDNA that encodes the WRKY transcription factor, designated as HbWRKY1, was isolated from H. brasiliensis. HbWRKY1 contains a 1437 bp open reading frame that encodes 478 amino acids. The deduced HbWRKY1 protein was predicted to possess two conserved WRKY domains and a C2H2 zinc-finger motif. HbWRKY1 was expressed at different levels, with the highest transcription in the flower, followed by the bark, latex, and leaf. Furthermore, the co-expression of pHbSRP::GUS with CaMV35S::HbWRKY1 significantly decreased the GUS activity in transgenic tobacco, indicating that HbWRKY1 significantly suppressed the HbSRPP promoter. These results suggested that HbWRKY1 maybe a negative transcription regulator of HbSRPP involved in NR biosynthesis in H. brasiliensis. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Constitutive expression of a salinity-induced wheat WRKY transcription factor enhances salinity and ionic stress tolerance in transgenic Arabidopsis thaliana.

PubMed

Qin, Yuxiang; Tian, Yanchen; Han, Lu; Yang, Xinchao

2013-10-25

The isolation and characterization of TaWRKY79, a wheat class II WRKY transcription factor, is described. Its 1297 bp coding region includes a 987 bp long open reading frame. TaWRKY79 was induced by stressing seedlings with either NaCl or abscisic acid (ABA). When a fusion between an 843 bp segment upstream of the TaWRKY79 coding sequence and GUS was introduced into Arabidopsis thaliana, GUS staining indicated that this upstream segment captured the sequence(s) required to respond to ABA or NaCl treatment. When TaWRKY79 was constitutively expressed as a transgene in A. thaliana, the transgenic plants showed an improved capacity to extend their primary root in the presence of either 100 mM NaCl, 10 mM LiCl or 2 μM ABA. The inference was that TaWRKY79 enhanced the level of tolerance to both salinity and ionic stress, while reducing the level of sensitivity to ABA. The ABA-related genes ABA1, ABA2 ABI1 and ABI5 were all up-regulated in the TaWRKY79 transgenic plants, suggesting that the transcription factor operates in an ABA-dependent pathway. Copyright © 2013. Published by Elsevier Inc.

Unique β-Glucuronidase Locus in Gut Microbiomes of Crohn's Disease Patients and Unaffected First-Degree Relatives.

PubMed

Gloux, Karine; Anba-Mondoloni, Jamila

2016-01-01

Crohn's disease, an incurable chronic inflammatory bowel disease, has been attributed to both genetic predisposition and environmental factors. A dysbiosis of the gut microbiota, observed in numerous patients but also in at least one hundred unaffected first-degree relatives, was proposed to have a causal role. Gut microbiota β-D-glucuronidases (EC 3.2.1.33) hydrolyse β-D-glucuronate from glucuronidated compounds. They include a GUS group, that is homologous to the Escherichia coli GusA, and a BG group, that is homologous to metagenomically identified H11G11 BG and has unidentified natural substrates. H11G11 BG is part of the functional core of the human gut microbiota whereas GusA, known to regenerate various toxic products, is variably found in human subjects. We investigated potential risk markers for Crohn's disease using DNA-sequence-based exploration of the β-D-glucuronidase loci (GUS or Firmicute H11G11-BG and the respective co-encoded glucuronide transporters). Crohn's disease-related microbiomes revealed a higher frequency of a C7D2 glucuronide transporter (12/13) compared to unrelated healthy subjects (8/32). This transporter was in synteny with the potential harmful GUS β-D-glucuronidase as only observed in a Eubacterium eligens plasmid. A conserved NH2-terminal sequence in the transporter (FGDFGND motif) was found in 83% of the disease-related subjects and only in 12% of controls. We propose a microbiota-pathology hypothesis in which the presence of this unique β-glucuronidase locus may contribute to an increase risk for Crohn's disease.

Old drug new use--amoxapine and its metabolites as potent bacterial β-glucuronidase inhibitors for alleviating cancer drug toxicity.

PubMed

Kong, Ren; Liu, Timothy; Zhu, Xiaoping; Ahmad, Syed; Williams, Alfred L; Phan, Alexandria T; Zhao, Hong; Scott, John E; Yeh, Li-An; Wong, Stephen T C

2014-07-01

Irinotecan (CPT-11) induced diarrhea occurs frequently in patients with cancer and limits its usage. Bacteria β-glucuronidase (GUS) enzymes in intestines convert the nontoxic metabolite of CPT-11, SN-38G, to toxic SN-38, and finally lead to damage of intestinal epithelial cells and diarrhea. We previously reported amoxapine as a potent GUS inhibitor in vitro. To further understand the molecular mechanism of amoxapine and its potential for treatment of CPT-11-induced diarrhea, we studied the binding modes of amoxapine and its metabolites by docking and molecular dynamics simulation, and tested the in vivo efficacy on mice in combination with CPT-11. The binding of amoxapine, its metabolites, 7-hydroxyamoxapine and 8-hydroxyamoxapine, and a control drug loxapine with GUS was explored by computational protocols. The in vitro potencies of metabolites were measured by Escherichia coli GUS enzyme and cell-based assay. Low-dosage daily oral administration was designed to use along with CPT-11 to treat tumor-bearing mice. Computational modeling results indicated that amoxapine and its metabolites bound in the active site of GUS and satisfied critical pharmacophore features: aromatic features near bacterial loop residue F365' and hydrogen bond toward E413. Amoxapine and its metabolites were demonstrated as potent in vitro. Administration of low dosages of amoxapine with CPT-11 in mice achieved significant suppression of diarrhea and reduced tumor growth. Amoxapine has great clinical potential to be rapidly translated to human subjects for irinotecan-induced diarrhea. ©2014 American Association for Cancer Research.

Old drug new use - Amoxapine and its metabolites as potent bacterial β-glucuronidase inhibitors for alleviating cancer drug toxicity

PubMed Central

Kong, Ren; Liu, Timothy; Zhu, Xiaoping; Ahmad, Syed; Williams, Alfred L.; Phan, Alexandria T; Zhao, Hong; Scott, John E.; Yeh, Li-An; Wong, Stephen TC

2014-01-01

Purpose Irinotecan (CPT-11) induced diarrhea occurs frequently in cancer patients and limits its usage. Bacteria β-glucuronidase (GUS) enzymes in intestines convert the non-toxic metabolite of CPT-11, SN-38G, to toxic SN-38, and finally lead to damage of intestinal epithelial cells and diarrhea. We previously reported amoxapine as potent GUS inhibitor in vitro. To further understand the molecular mechanism of amoxapine and its potential for treatment of CPT-11 induced diarrhea, we studied the binding modes of amoxapine and its metabolites by docking and molecular dynamics simulation, and tested the in vivo efficacy on mice in combination with CPT-11. Experimental Design The binding of amoxapine, its metabolites, 7-hydroxyamoxapine and 8-hydroxyamoxapine, and a control drug loxapine with GUS was explored by computational protocols. The in vitro potencies of metabolites were measured by E. Coli GUS enzyme and cell-based assay. Low dosage daily oral administration was designed to use along with CPT-11 to treat tumor-bearing mice. Results Computational modeling results indicated that amoxapine and its metabolites bound in the active site of GUS and satisfied critical pharmacophore features: aromatic features near bacterial loop residue F365’ and hydrogen bond toward E413. Amoxapine and its metabolites were demonstrated as potent in vitro. Administration of low dosages of amoxapine with CPT-11 in mice achieved significant suppression of diarrhea and reduced tumor growth. Conclusions Amoxapine has great clinical potential to be rapidly translated to human subjects for irinotecan induced diarrhea. PMID:24780296

Female Reproductive Tissues Are the Primary Target of Agrobacterium-Mediated Transformation by the Arabidopsis Floral-Dip Method1

PubMed Central

Desfeux, Christine; Clough, Steven J.; Bent, Andrew F.

2000-01-01

The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding β-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved. PMID:10889238

Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method.

PubMed

Desfeux, C; Clough, S J; Bent, A F

2000-07-01

The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding beta-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.

The C2 protein of tomato leaf curl Taiwan virus is a pathogenicity determinant that interferes with expression of host genes encoding chromomethylases.

PubMed

Tu, Yu-Ching; Tsai, Wen-Shi; Wei, Jyuan-Yu; Chang, Kai-Ya; Tien, Chang-Ching; Hsiao, Hui-Yu; Fu, Shih-Feng

2017-12-01

Tomato (Solanum lycopersicum) is one of the most important crops worldwide and is severely affected by geminiviruses. Tomato leaf curl Taiwan virus (ToLCTWV), belonging to the geminiviruses, was isolated in Taiwan and causes tremendous crop loss. The geminivirus-encoded C2 proteins are crucial for a successful interaction between the virus and host plants. However, the exact functions of the viral C2 protein of ToLCTWV have not been investigated. We analyzed the molecular function(s) of the C2 protein by transient or stable expression in tomato cv. Micro-Tom and Nicotiana benthamiana. Severe stunting of tomato and N. benthamiana plants infected with ToLCTWV was observed. Expression of ToLCTWV C2-green fluorescent protein (GFP) fusion protein was predominately located in the nucleus and contributed to activation of a coat protein promoter. Notably, the C2-GFP fluorescence was distributed in nuclear aggregates. Tomato and N. benthamiana plants inoculated with potato virus X (PVX)-C2 displayed chlorotic lesions and stunted growth. PVX-C2 elicited hypersensitive responses accompanied by production of reactive oxygen species in N. benthamiana plants, which suggests that the viral C2 was a potential recognition target to induce host-defense responses. In tomato and N. benthamiana, ToLCTWV C2 was found to interfere with expression of genes encoding chromomethylases. N. benthamiana plants with suppressed NbCMT3-2 expression were more susceptible to ToLCTWV infection. Transgenic N. benthamiana plants expressing the C2 protein showed decreased expression of the NbCMT3-2 gene and pNbCMT3-2::GUS (β-glucuronidase) promoter activity. C2 protein is an important pathogenicity determinant of ToLCTWV and interferes with host components involved in DNA methylation. © 2017 Scandinavian Plant Physiology Society.

Orchids (Cymbidium spp., Oncidium, and Phalaenopsis).

PubMed

Chan, Ming-Tsair; Chan, Yuan-Li; Sanjaya

2006-01-01

Recent advances in genetic engineering have made the transformation and regeneration of plants into a powerful tool for orchid improvement. This chapter presents a simple and reproducible Agrobacterium tumefaciens-mediated transformation protocol and molecular screening technique of transgenics for two orchid species, Oncidium and Phalaenopsis. The target tissues for gene transfer were protocorm-like bodies (PLBs) derived from protocorms, into which constructed foreign genes were successfully introduced. To establish stable transformants, two stages of selection were applied on the PLBs co-cultivated with A. tumefaciens. About 10% transformation efficiency was achieved in Oncidium orchid, as 108 antibiotic resistant independent PLBs were proliferated from 1000 infected PLBs. In Phalaenopsis orchid about 11 to 12% of transformation efficiency was achieved by using the present protocol. Different molecular methods and GUS-staining used to screen putative transgenic plants to confirm the integration of foreign DNA into the orchid genome were also described in detail. The methods described would also be useful for transformation of desired genes into other orchid species.

Rice GH3 gene family: regulators of growth and development.

PubMed

Fu, Jing; Yu, Huihui; Li, Xianghua; Xiao, Jinghua; Wang, Shiping

2011-04-01

Auxin is an indispensable hormone throughout the lifetime of nearly all plant species. Several aspects of plant growth and development are rigidly governed by auxin, from micro to macro hierarchies; auxin also has a close relationship with plant-pathogen interactions. Undoubtedly, precise auxin levels are vitally important to plants, which have many effective mechanisms to maintain auxin homeostasis. One mechanism is conjugating amino acid to excessive indole-3-acetic acid (IAA; main form of auxin) through some GH3 family proteins to inactivate it. Our previous study demonstrated that GH3-2 mediated broad-spectrum resistance in rice (Oryza sativa L.) by suppressing pathogen-induced IAA accumulation and downregulating auxin signaling. Here, we further investigated the expression pattern of GH3-2 and other GH3 family paralogues in the life cycle of rice and presented the possible function of GH3-2 on rice root development by histochemical analysis of GH3-2 promoter:GUS reporter transgenic plants.

RabGAP22 Is Required for Defense to the Vascular Pathogen Verticillium longisporum and Contributes to Stomata Immunity

PubMed Central

Roos, Jonas; Bejai, Sarosh; Oide, Shinichi; Dixelius, Christina

2014-01-01

Verticillium longisporum is a soil-borne pathogen with a preference for plants within the family Brassicaceae. Following invasion of the roots, the fungus proliferates in the plant vascular system leading to stunted plant growth, chlorosis and premature senescence. RabGTPases have been demonstrated to play a crucial role in regulating multiple responses in plants. Here, we report on the identification and characterization of the Rab GTPase-activating protein RabGAP22 gene from Arabidopsis, as an activator of multiple components in the immune responses to V. longisporum. RabGAP22Pro:GUS transgenic lines showed GUS expression predominantly in root meristems, vascular tissues and stomata, whereas the RabGAP22 protein localized in the nucleus. Reduced RabGAP22 transcript levels in mutants of the brassinolide (BL) signaling gene BRI1-ASSOCIATED RECEPTOR KINASE 1, together with a reduction of fungal proliferation following BL pretreatment, suggested RabGAP22 to be involved in BL-mediated responses. Pull-down assays revealed SERINE:GLYOXYLATE AMINOTRANSFERASE (AGT1) as an interacting partner during V. longisporum infection and bimolecular fluorescence complementation (BiFC) showed the RabGAP22-AGT1 protein complex to be localized in the peroxisomes. Further, fungal-induced RabGAP22 expression was found to be associated with elevated endogenous levels of the plant hormones jasmonic acid (JA) and abscisic acid (ABA). An inadequate ABA response in rabgap22-1 mutants, coupled with a stomata-localized expression of RabGAP22 and impairment of guard cell closure in response to V. longisporum and Pseudomonas syringae, suggest that RabGAP22 has multiple roles in innate immunity. PMID:24505423

Localization in roots and flowers of pea chloroplastic thioredoxin f and thioredoxin m proteins reveals new roles in nonphotosynthetic organs.

PubMed

de Dios Barajas-López, Juan; Serrato, Antonio Jesús; Olmedilla, Adela; Chueca, Ana; Sahrawy, Mariam

2007-11-01

Plant thioredoxins (TRXs) are involved in redox regulation of a wide variety processes and usually exhibit organ specificity. We report strong evidence that chloroplastic TRXs are localized in heterotrophic tissues and suggest some ways in which they might participate in several metabolic and developmental processes. The promoter regions of the chloroplastic f and m1 TRX genes were isolated from a pea (Pisum sativum) plant genomic bank. Histochemical staining for beta-glucuronidase (GUS) in transgenic homozygous Arabidopsis (Arabidopsis thaliana) plants showed preferential expression of the 444-bp PsTRXf1 promoter in early seedlings, stems, leaves, and roots, as well as in flowers, stigma, pollen grains, and filaments. GUS activity under the control of the 1,874-bp PsTRXm1 promoter was restricted to the leaves, roots, seeds, and flowers. To gain insight into the translational regulation of these genes, a series of deletions of 5' elements in both TRX promoters were analyzed. The results revealed that a 126-bp construct of the PsTRXf2 promoter was unable to reproduce the expression pattern observed with the full promoter. The differences in expression and tissue specificity between PsTRXm1 and the deleted promoters PsTRXm2 and PsTRXm3 suggest the existence of upstream positive or negative regulatory regions that affect tissue specificity, sucrose metabolism, and light regulation. PsTRXm1 expression is finely regulated by light and possibly by other metabolic factors. In situ hybridization experiments confirmed new localizations of these chloroplastic TRX transcripts in vascular tissues and flowers, and therefore suggest possible new functions in heterotrophic tissues related to cell division, germination, and plant reproduction.

A novel plant ferredoxin-like protein and the regulator Hor are quorum-sensing targets in the plant pathogen Erwinia carotovora.

PubMed

Sjöblom, Solveig; Harjunpää, Heidi; Brader, Günter; Palva, E Tapio

2008-07-01

Quorum sensing (QS), a population-density-sensing mechanism, controls the production of the main virulence determinants, the plant cell-wall-degrading enzymes (PCWDEs) of the soft-rot phytopathogen Erwinia carotovora subsp. carotovora. In this study, we used random transposon mutagenesis with a gusA reporter construct to identify two new QS-controlled genes encoding the regulator Hor and a plant ferredoxin-like protein, FerE. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and mediated by the global repressor RsmA. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production. Our results showed that FerE contributes to oxidative stress tolerance and in planta fitness of the bacteria and suggest that QS could be central to control of oxidative stress tolerance. The presence of the FerE protein appears to be rather unique in heterotrophic bacteria and suggests an acquisition of the corresponding gene from plant host by horizontal gene transfer.

Unique β-Glucuronidase Locus in Gut Microbiomes of Crohn’s Disease Patients and Unaffected First-Degree Relatives

PubMed Central

Gloux, Karine; Anba-Mondoloni, Jamila

2016-01-01

Crohn’s disease, an incurable chronic inflammatory bowel disease, has been attributed to both genetic predisposition and environmental factors. A dysbiosis of the gut microbiota, observed in numerous patients but also in at least one hundred unaffected first-degree relatives, was proposed to have a causal role. Gut microbiota β-D-glucuronidases (EC 3.2.1.33) hydrolyse β-D-glucuronate from glucuronidated compounds. They include a GUS group, that is homologous to the Escherichia coli GusA, and a BG group, that is homologous to metagenomically identified H11G11 BG and has unidentified natural substrates. H11G11 BG is part of the functional core of the human gut microbiota whereas GusA, known to regenerate various toxic products, is variably found in human subjects. We investigated potential risk markers for Crohn’s disease using DNA-sequence-based exploration of the β-D-glucuronidase loci (GUS or Firmicute H11G11-BG and the respective co-encoded glucuronide transporters). Crohn’s disease-related microbiomes revealed a higher frequency of a C7D2 glucuronide transporter (12/13) compared to unrelated healthy subjects (8/32). This transporter was in synteny with the potential harmful GUS β-D-glucuronidase as only observed in a Eubacterium eligens plasmid. A conserved NH2-terminal sequence in the transporter (FGDFGND motif) was found in 83% of the disease-related subjects and only in 12% of controls. We propose a microbiota-pathology hypothesis in which the presence of this unique β-glucuronidase locus may contribute to an increase risk for Crohn’s disease. PMID:26824357

Beta-glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells.

PubMed

Izagirre, U; Angulo, E; Wade, S C; ap Gwynn, I; Marigómez, I

2009-02-01

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase (beta-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that beta-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas beta-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, beta-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that beta-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed.

Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver

PubMed Central

Schreiber, Renate; Taschler, Ulrike; Wolinski, Heimo; Seper, Andrea; Tamegger, Stefanie N.; Graf, Maria; Kohlwein, Sepp D.; Haemmerle, Guenter; Zimmermann, Robert; Zechner, Rudolf; Lass, Achim

2009-01-01

Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with β-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl β-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism. PMID:19723663

ApiEST-DB: analyzing clustered EST data of the apicomplexan parasites.

PubMed

Li, Li; Crabtree, Jonathan; Fischer, Steve; Pinney, Deborah; Stoeckert, Christian J; Sibley, L David; Roos, David S

2004-01-01

ApiEST-DB (http://www.cbil.upenn.edu/paradbs-servlet/) provides integrated access to publicly available EST data from protozoan parasites in the phylum Apicomplexa. The database currently incorporates a total of nearly 100,000 ESTs from several parasite species of clinical and/or veterinary interest, including Eimeria tenella, Neospora caninum, Plasmodium falciparum, Sarcocystis neurona and Toxoplasma gondii. To facilitate analysis of these data, EST sequences were clustered and assembled to form consensus sequences for each organism, and these assemblies were then subjected to automated annotation via similarity searches against protein and domain databases. The underlying relational database infrastructure, Genomics Unified Schema (GUS), enables complex biologically based queries, facilitating validation of gene models, identification of alternative splicing, detection of single nucleotide polymorphisms, identification of stage-specific genes and recognition of phylogenetically conserved and phylogenetically restricted sequences.

Cucumber (Cucumis sativus L.) Nitric Oxide Synthase Associated Gene1 (CsNOA1) Plays a Role in Chilling Stress

PubMed Central

Liu, Xingwang; Liu, Bin; Xue, Shudan; Cai, Yanlinq; Qi, Wenzhu; Jian, Chen; Xu, Shuo; Wang, Ting; Ren, Huazhong

2016-01-01

Nitric oxide (NO) is a gaseous signaling molecule in plants, transducing information as a result of exposure to low temperatures. However, the underlying molecular mechanism linking NO with chilling stress is not well understood. Here, we functionally characterized the cucumber (Cucumis sativus L.) nitric oxide synthase-associated gene, NITRIC OXIDE ASSOCIATED 1 (CsNOA1). Expression analysis of CsNOA1, using quantitative real-time PCR, in situ hybridization, and a promoter::β-glucuronidase (GUS) reporter assay, revealed that it is expressed mainly in the root and shoot apical meristem (SAM), and that expression is up-regulated by low temperatures. A CsNOA1-GFP fusion protein was found to be localized in the mitochondria, and ectopic expression of CsNOA1 in the A. thaliana noa1 mutant partially rescued the normal phenotype. When overexpressing CsNOA1 in the Atnoa1 mutant under normal condition, no obvious phenotypic differences was observed between its wild type and transgenic plants. However, the leaves from mutant plant grown under chilling conditions showed hydrophanous spots and wilting. Physiology tolerance markers, chlorophyll fluorescence parameter (Fv/Fm), and electrolyte leakage, were observed to dramatically change, compared mutant to overexpressing lines. Transgenic cucumber plants revealed that the gene is required by seedlings to tolerate chilling stress: constitutive over-expression of CsNOA1 led to a greater accumulation of soluble sugars, starch, and an up-regulation of Cold-regulatory C-repeat binding factor3 (CBF3) expression as well as a lower chilling damage index (CI). Conversely, suppression of CsNOA1 expression resulted in the opposite phenotype and a reduced NO content compared to wild type plants. Those results suggest that CsNOA1 regulates cucumber seedlings chilling tolerance. Additionally, under normal condition, we took several classic inhibitors to perform, and detect endogenous NO levels in wild type cucumber seedling. The results

Genes and Gene Therapy

MedlinePlus

... a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

Gene network biological validity based on gene-gene interaction relevance.

PubMed

Gómez-Vela, Francisco; Díaz-Díaz, Norberto

2014-01-01

In recent years, gene networks have become one of the most useful tools for modeling biological processes. Many inference gene network algorithms have been developed as techniques for extracting knowledge from gene expression data. Ensuring the reliability of the inferred gene relationships is a crucial task in any study in order to prove that the algorithms used are precise. Usually, this validation process can be carried out using prior biological knowledge. The metabolic pathways stored in KEGG are one of the most widely used knowledgeable sources for analyzing relationships between genes. This paper introduces a new methodology, GeneNetVal, to assess the biological validity of gene networks based on the relevance of the gene-gene interactions stored in KEGG metabolic pathways. Hence, a complete KEGG pathway conversion into a gene association network and a new matching distance based on gene-gene interaction relevance are proposed. The performance of GeneNetVal was established with three different experiments. Firstly, our proposal is tested in a comparative ROC analysis. Secondly, a randomness study is presented to show the behavior of GeneNetVal when the noise is increased in the input network. Finally, the ability of GeneNetVal to detect biological functionality of the network is shown.

A stress-associated protein, AtSAP13, from Arabidopsis thaliana provides tolerance to multiple abiotic stresses.

PubMed

Dixit, Anirudha; Tomar, Parul; Vaine, Evan; Abdullah, Hesham; Hazen, Samuel; Dhankher, Om Parkash

2018-05-01

Members of Stress-Associated Protein (SAP) family in plants have been shown to impart tolerance to multiple abiotic stresses, however, their mode of action in providing tolerance to multiple abiotic stresses is largely unknown. There are 14 SAP genes in Arabidopsis thaliana containing A20, AN1, and Cys2-His2 zinc finger domains. AtSAP13, a member of the SAP family, carries two AN1 zinc finger domains and an additional Cys2-His2 domain. AtSAP13 transcripts showed upregulation in response to Cd, ABA, and salt stresses. AtSAP13 overexpression lines showed strong tolerance to toxic metals (AsIII, Cd, and Zn), drought, and salt stress. Further, transgenic lines accumulated significantly higher amounts of Zn, but less As and Cd accumulation in shoots and roots. AtSAP13 promoter-GUS fusion studies showed GUS expression predominantly in the vascular tissue, hydathodes, and the apical meristem and region of root maturation and elongation as well as the root hairs. At the subcellular level, the AtSAP13-eGFP fusion protein was found to localize in both nucleus and cytoplasm. Through yeast one-hybrid assay, we identified several AP2/EREBP family transcription factors that interacted with the AtSAP13 promoter. AtSAP13 and its homologues will be highly useful for developing climate resilient crops. © 2017 John Wiley & Sons Ltd.

Jasmonate and Phytochrome A Signaling in Arabidopsis Wound and Shade Responses Are Integrated through JAZ1 Stability[C][W

PubMed Central

Robson, Frances; Okamoto, Haruko; Patrick, Elaine; Harris, Sue-Ré; Wasternack, Claus; Brearley, Charles; Turner, John G.

2010-01-01

Jasmonate (JA) activates plant defense, promotes pollen maturation, and suppresses plant growth. An emerging theme in JA biology is its involvement in light responses; here, we examine the interdependence of the JA- and light-signaling pathways in Arabidopsis thaliana. We demonstrate that mutants deficient in JA biosynthesis and signaling are deficient in a subset of high irradiance responses in far-red (FR) light. These mutants display exaggerated shade responses to low, but not high, R/FR ratio light, suggesting a role for JA in phytochrome A (phyA) signaling. Additionally, we demonstrate that the FR light–induced expression of transcription factor genes is dependent on CORONATINE INSENSITIVE1 (COI1), a central component of JA signaling, and is suppressed by JA. phyA mutants had reduced JA-regulated growth inhibition and VSP expression and increased content of cis-(+)-12-oxophytodienoic acid, an intermediate in JA biosynthesis. Significantly, COI1-mediated degradation of JASMONATE ZIM DOMAIN1-β-glucuronidase (JAZ1-GUS) in response to mechanical wounding and JA treatment required phyA, and ectopic expression of JAZ1-GUS resulted in exaggerated shade responses. Together, these results indicate that JA and phyA signaling are integrated through degradation of the JAZ1 protein, and both are required for plant responses to light and stress. PMID:20435902

Abscisic Acid Regulates Auxin Homeostasis in Rice Root Tips to Promote Root Hair Elongation

PubMed Central

Wang, Tao; Li, Chengxiang; Wu, Zhihua; Jia, Yancui; Wang, Hong; Sun, Shiyong; Mao, Chuanzao; Wang, Xuelu

2017-01-01

Abscisic acid (ABA) plays an essential role in root hair elongation in plants, but the regulatory mechanism remains to be elucidated. In this study, we found that exogenous ABA can promote rice root hair elongation. Transgenic rice overexpressing SAPK10 (Stress/ABA-activated protein kinase 10) had longer root hairs; rice plants overexpressing OsABIL2 (OsABI-Like 2) had attenuated ABA signaling and shorter root hairs, suggesting that the effect of ABA on root hair elongation depends on the conserved PYR/PP2C/SnRK2 ABA signaling module. Treatment of the DR5-GUS and OsPIN-GUS lines with ABA and an auxin efflux inhibitor showed that ABA-induced root hair elongation depends on polar auxin transport. To examine the transcriptional response to ABA, we divided rice root tips into three regions: short root hair, long root hair and root tip zones; and conducted RNA-seq analysis with or without ABA treatment. Examination of genes involved in auxin transport, biosynthesis and metabolism indicated that ABA promotes auxin biosynthesis and polar auxin transport in the root tip, which may lead to auxin accumulation in the long root hair zone. Our findings shed light on how ABA regulates root hair elongation through crosstalk with auxin biosynthesis and transport to orchestrate plant development. PMID:28702040

Overexpression of PhEXPA1 increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida.

PubMed

Zenoni, Sara; Fasoli, Marianna; Tornielli, Giovanni Battista; Dal Santo, Silvia; Sanson, Andrea; de Groot, Peter; Sordo, Sara; Citterio, Sandra; Monti, Francesca; Pezzotti, Mario

2011-08-01

• Expansins are cell wall proteins required for cell enlargement and cell wall loosening during many developmental processes. The involvement of the Petunia hybrida expansin A1 (PhEXPA1) gene in cell expansion, the control of organ size and cell wall polysaccharide composition was investigated by overexpressing PhEXPA1 in petunia plants. • PhEXPA1 promoter activity was evaluated using a promoter-GUS assay and the protein's subcellular localization was established by expressing a PhEXPA1-GFP fusion protein. PhEXPA1 was overexpressed in transgenic plants using the cauliflower mosaic virus (CaMV) 35S promoter. Fourier transform infrared (FTIR) and chemical analysis were used for the quantitative analysis of cell wall polymers. • The GUS and GFP assays demonstrated that PhEXPA1 is present in the cell walls of expanding tissues. The constitutive overexpression of PhEXPA1 significantly affected expansin activity and organ size, leading to changes in the architecture of petunia plants by initiating premature axillary meristem outgrowth. Moreover, a significant change in cell wall polymer composition in the petal limbs of transgenic plants was observed. • These results support a role for expansins in the determination of organ shape, in lateral branching, and in the variation of cell wall polymer composition, probably reflecting a complex role in cell wall metabolism. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

Cold Shock Domain Protein 3 Regulates Freezing Tolerance in Arabidopsis thaliana*

PubMed Central

Kim, Myung-Hee; Sasaki, Kentaro; Imai, Ryozo

2009-01-01

In response to cold, Escherichia coli produces cold shock proteins (CSPs) that have essential roles in cold adaptation as RNA chaperones. Here, we demonstrate that Arabidopsis cold shock domain protein 3 (AtCSP3), which shares a cold shock domain with bacterial CSPs, is involved in the acquisition of freezing tolerance in plants. AtCSP3 complemented a cold-sensitive phenotype of the E. coli CSP quadruple mutant and displayed nucleic acid duplex melting activity, suggesting that AtCSP3 also functions as an RNA chaperone. Promoter-GUS transgenic plants revealed tissue-specific expression of AtCSP3 in shoot and root apical regions. When exposed to low temperature, GUS activity was extensively induced in a broader region of the roots. In transgenic plants expressing an AtCSP3-GFP fusion, GFP signals were detected in both the nucleus and cytoplasm. An AtCSP3 knock-out mutant (atcsp3-2) was sensitive to freezing compared with wild-type plants under non-acclimated and cold-acclimated conditions, whereas expression of C-repeat-binding factors and their downstream genes during cold acclimation was not altered in the atcsp3-2 mutant. Overexpression of AtCSP3 in transgenic plants conferred enhanced freezing tolerance over wild-type plants. Together, the data demonstrated an essential role of RNA chaperones for cold adaptation in higher plants. PMID:19556243

An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars.

PubMed

Shri, Manju; Rai, Arti; Verma, Pankaj Kumar; Misra, Prashant; Dubey, Sonali; Kumar, Smita; Verma, Sikha; Gautam, Neelam; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar; Chakrabarty, Debasis

2013-04-01

Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of L-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD600 nm = 0.5-0.8) promoted the highest frequency of transformation (83.04 %) in medium containing L-cysteine (400 mg l(-1)). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of L-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.

Evolvement of transgenic male-sterility and fertility-restoration system in rice for production of hybrid varieties.

PubMed

Rao, Gundra Sivakrishna; Deveshwar, Priyanka; Sharma, Malini; Kapoor, Sanjay; Rao, Khareedu Venkateswara

2018-01-01

We have developed a unique male-sterility and fertility-restoration system in rice by combining Brassica napus cysteine-protease gene (BnCysP1) with anther-specific P12 promoter of rice for facilitating production of hybrid varieties. In diverse crop plants, male-sterility has been exploited as a useful approach for production of hybrid varieties to harness the benefits of hybrid vigour. The promoter region of Os12bglu38 gene of rice has been isolated from the developing panicles and was designated as P12. The promoter was fused with gusA reporter gene and was expressed in Arabidopsis and rice systems. Transgenic plants exhibited GUS activity in tapetal cells and pollen of the developing anthers indicating anther/pollen-specific expression of the promoter. For engineering nuclear male sterility, the coding region of Brassica napus cysteine protease1 (BnCysP1) was isolated from developing seeds and fused to P12 promoter. Transgenic rice plants obtained with P12-BnCysP1 failed to produce functional pollen grains. The F 1 seeds obtained from BnCysP1 male-sterile plants and untransformed controls showed 1:1 (tolerant:sensitive) ratio when germinated on the MS medium supplemented with phosphinothricin (5 mg/l), confirming that the male sterility has been successfully engineered in rice. For male fertility restoration, transgenic rice plants carrying BnCysP1Si silencing system were developed. The pollination of BnCysP1 male-sterile (female-fertile) plants with BnCysP1Si pollen resulted in normal grain filling. The F 1 seeds of BnCysP1 × BnCysP1Si when germinated on the MS basal medium containing PPT (5 mg/l) and hygromycin (70 mg/l) exhibited 1:1 (tolerant:sensitive) ratio and the tolerant plants invariably showed normal grain filling. The overall results clearly suggest that the customized male-sterility & fertility-restoration system can be exploited for quality hybrid seed production in various crops.

Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues

PubMed Central

2011-01-01

Background Cotton (Gossypium spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization. Results Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2. Conclusions uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues. PMID:22115195

Capture of a recombinant protein from unclarified canola extract using streamline expanded bed anion exchange.

PubMed

Bai, Yun; Glatz, Charles E

2003-03-30

The feasibility of applying expanded bed adsorption technology to recombinant protein recovery from extracts of transgenic canola (rapeseed) was assessed. The extraction step results in a suspension of high solids content that is difficult to clarify. The coarse portion of the solids can be removed easily, and our aim was to operate the expanded bed in the presence of the recalcitrant particulates. Recombinant beta-glucuronidase (rGUS) produced in transgenic canola seed was the model system. Diethylaminoethyl (DEAE) and Streamline DEAE resin exhibited similar binding and elution properties for both rGUS and native canola proteins. More than 95% of native canola proteins did not bind to DEAE resins at pH 7.5, whereas the bound proteins were fractionated by two-step salt elution into two groups with the first peak, containing 70% of total bound proteins, at 20 mS/cm, followed by elution of rGUS at 50 mS/cm. The adsorption isotherm was only slightly influenced by the presence of up to 14 mg solids/mL extract; C(m) and K(d) changed by -1% and +39%, respectively. Bed expansion was semiquantitatively predictable from physical properties of the fluid together with Stokes's law and the Richardson-Zaki correlation for both clarified and partially clarified extracts. The presence of 1.4% solids did not change rGUS breakthrough behavior of the expanded bed; however, a small difference between expanded bed and packed bed was observed early in the sample loading stage, during which bed expansion adjusts. Canola solids moved through the column in approximately plug flow with no detriment to bed stability. Seventy-two percent recovery of 34-fold purified rGUS was obtained after initial loading of 1.4% (w/w) solids extract to 25% breakthrough. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 855-864, 2003.

Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Yuan; Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3; Wu, Keqiang

2010-05-28

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants.more » We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.« less

Two closely related members of Arabidopsis 13-lipoxygenases (13-LOXs), LOX3 and LOX4, reveal distinct functions in response to plant-parasitic nematode infection.

PubMed

Ozalvo, Rachel; Cabrera, Javier; Escobar, Carolina; Christensen, Shawn A; Borrego, Eli J; Kolomiets, Michael V; Castresana, Carmen; Iberkleid, Ionit; Brown Horowitz, Sigal

2014-05-01

The responses of two closely related members of Arabidopsis 13-lipoxygenases (13-LOXs), LOX3 and LOX4, to infection by the sedentary nematodes root-knot nematode (Meloidogyne javanica) and cyst nematode (Heterodera schachtii) were analysed in transgenic Arabidopsis seedlings. The tissue localization of LOX3 and LOX4 gene expression using β-glucuronidase (GUS) reporter gene constructs showed local induction of LOX3 expression when second-stage juveniles reached the vascular bundle and during the early stages of plant-nematode interaction through gall and syncytia formation. Thin sections of nematode-infested knots indicated LOX3 expression in mature giant cells, and high expression in neighbouring cells and those surrounding the female body. LOX4 promoter was also activated by nematode infection, although the GUS signal weakened as infection and disease progressed. Homozygous insertion mutants lacking LOX3 were less susceptible than wild-type plants to root-knot nematode infection, as reflected by a decrease in female counts. Conversely, deficiency in LOX4 function led to a marked increase in females and egg mass number and in the female to male ratio of M. javanica and H. schachtii, respectively. The susceptibility of lox4 mutants was accompanied by increased expression of allene oxide synthase, allene oxide cyclase and ethylene-responsive transcription factor 4, and the accumulation of jasmonic acid, measured in the roots of lox4 mutants. This response was not found in lox3 mutants. Taken together, our results reveal that LOX4 and LOX3 interfere differentially with distinct metabolic and signalling pathways, and that LOX4 plays a major role in controlling plant defence against nematode infection. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Isolation and characterization of the Jatropha curcas APETALA1 (JcAP1) promoter conferring preferential expression in inflorescence buds.

PubMed

Tao, Yan-Bin; He, Liang-Liang; Niu, Longjian; Xu, Zeng-Fu

2016-08-01

The 1.5 kb JcAP1 promoter from the biofuel plant Jatropha curcas is predominantly active in the inflorescence buds of transgenic plants, in which the -1313/-1057 region is essential for maintaining the activity. Arabidopsis thaliana APETALA1 (AP1) is a MADS-domain transcription factor gene that functions primarily in flower development. We isolated a homolog of AP1 from Jatropha curcas (designated JcAP1), which was shown to exhibit flower-specific expression in Jatropha. JcAP1 is first expressed in inflorescence buds and continues to be primarily expressed in the sepals. We isolated a 1.5 kb JcAP1 promoter and evaluated its activity in transgenic Arabidopsis and Jatropha using the β-glucuronidase (GUS) reporter gene. In transgenic Arabidopsis and Jatropha, the inflorescence buds exhibited notable GUS activity, whereas the sepals did not. Against expectations, the JcAP1 promoter was active in the anthers of Arabidopsis and Jatropha and was highly expressed in Jatropha seeds. An analysis of promoter deletions in transgenic Arabidopsis revealed that deletion of the -1313/-1057 region resulted in loss of JcAP1 promoter activity in the inflorescence buds and increased activity in the anthers. These results suggested that some regulatory sequences in the -1313/-1057 region are essential for maintaining promoter activity in inflorescence buds and can partly suppress activity in the anthers. Based on these findings, we hypothesized that other elements located upstream of the 1.5 kb JcAP1 promoter may be required for flower-specific activation. The JcAP1 promoter characterized in this study can be used to drive transgene expression in both the inflorescence buds and seeds of Jatropha.

Effects of the inoculation of Burkholderia vietnamensis and related endophytic diazotrophic bacteria on grain yield of rice.

PubMed

Govindarajan, Munusamy; Balandreau, Jacques; Kwon, Soon-Wo; Weon, Hang-Yeon; Lakshminarasimhan, Cunthipuram

2008-01-01

During a survey of endophytic diazotrophic bacteria associated with different rice varieties in Tamilnadu, some "endophytes" were obtained. Thirteen bacterial isolates from surface-sterilized roots and shoots were obtained in pure culture, which produced indole acetic acid (IAA) and reduced acetylene to ethylene. Polymerase chain reaction (PCR) amplification confirmed the presence of nif-H gene in all the isolates. Morphological, biochemical, and molecular characteristics indicated that all of them belonged to the genus Burkholderia One of them, MGK3, was consistently more active in reducing acetylene, and 16S rDNA sequences of isolate MGK3 confirmed its identification as Burkholderia vietnamiensis. Colonization of rice root was confirmed by strain MGK3 marked with gusA gene. The inoculated roots showed a blue color, which was most intense at the points of lateral root emergence and at the root tip. Transverse sections of roots, 15 days after inoculation, revealed beta-glucuronidase (GUS) activity within many of the cortical intercellular spaces next to the stele and within the aerenchyma. Nitrogen fixation was quantified by using (15)N isotope dilution method with two different cultivars grown in pot and field experiments. Higher nitrogen fixation was observed in variety Ponni than in ADT-43, where nearly 42% (field) and 40% (pot) of the nitrogen was derived from the atmosphere (% Ndfa). Isolate MGK3 was used to inoculate rice seedlings in a comparison with four other diazotrophs, viz., Gluconacetobacter diazotrophicus LMG7603, Herbaspirillum seropedicae LMG6513, Azospirillum lipoferum 4B LMG4348, and B. vietnamiensis LMG10929. They were used to conduct two pot and four field inoculation experiments. MGK3 alone, and combined with other diazotrophs, performed best under both pot and field conditions: combined inoculation produced yield increases between 9.5 and 23.6%, while MGK3 alone increased yield by 5.6 to 12.16% over the uninoculated control treatment.

A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis

PubMed Central

Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Chen, Chao; Qin, Zhiwei; Yang, Kejun; Shen, Yang; Meiping, Zhang; Mingyang, Cong; Zhu, Yanming

2015-01-01

It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis. PMID:26717241

The Promoter of AtUSP Is Co-regulated by Phytohormones and Abiotic Stresses in Arabidopsis thaliana.

PubMed

Bhuria, Monika; Goel, Parul; Kumar, Sanjay; Singh, Anil K

2016-01-01

Universal stress proteins (USPs) are known to be expressed in response to various abiotic stresses in a wide variety of organisms, such as bacteria, archaebacteria, protists, algae, fungi, plants, and animals. However, in plants, biological function of most of the USPs still remains obscure. In the present study, Arabidopsis USP gene ( AtUSP ) showed induction in response to abscisic acid (ABA) and various abiotic stresses viz . heat, dehydration, salt, osmotic, and cold stresses. Additionally, in silico analysis of AtUSP promoter identified several cis -elements responsive to phytohormones and abiotic stresses such as ABRE, ERE, DRE, and HSE, etc. To functionally validate the AtUSP promoter, the 1115 bp region of promoter was characterized under phytohormone and abiotic stress treatments. Deletion analysis of promoter was carried out by cloning the full length promoter (D0) and its three 5' deletion derivatives, D1 (964 bp), D2 (660 bp), and D3 (503 bp) upstream of the β-glucuronidase (GUS) reporter gene, which were then stably transformed in Arabidopsis plants. The AtUSP promoter (D0) showed minimal activity under non-stress conditions which was enhanced in response to phytohormone treatments (ABA and ACC) and abiotic stresses such as dehydration, heat, cold, salt, and osmotic stresses. The seedlings harboring D1 and D2 deletion fragments showed constitutive GUS expression even under control condition with increased activity almost under all the treatments. However, D3 seedlings exhibited complete loss of activity under control condition with induction under ACC treatment, dehydration, heat, oxidative, salt, and osmotic stresses. Thus, present study clearly showed that AtUSP promoter is highly inducible by phytohormones and multiple abiotic stresses and it can be exploited as stress inducible promoter to generate multi-stress tolerant crops with minimal effects on their other important traits.

Roles of Soybean Plasma Membrane Intrinsic Protein GmPIP2;9 in Drought Tolerance and Seed Development.

PubMed

Lu, Linghong; Dong, Changhe; Liu, Ruifang; Zhou, Bin; Wang, Chuang; Shou, Huixia

2018-01-01

Aquaporins play an essential role in water uptake and transport in vascular plants. The soybean genome contains a total of 22 plasma membrane intrinsic protein (PIP) genes. To identify candidate PIPs important for soybean yield and stress tolerance, we studied the transcript levels of all 22 soybean PIPs. We found that a GmPIP2 subfamily member, GmPIP2;9, was predominately expressed in roots and developing seeds. Here, we show that GmPIP2;9 localized to the plasma membrane and had high water channel activity when expressed in Xenopus oocytes. Using transgenic soybean plants expressing a native GmPIP2;9 promoter driving a GUS-reporter gene, it was found high GUS expression in the roots, in particular, in the endoderm, pericycle, and vascular tissues of the roots of transgenic plants. In addition, GmPIP2;9 was also highly expressed in developing pods. GmPIP2;9 expression significantly increased in short term of polyethylene glycol (PEG)-mediated drought stress treatment. GmPIP2;9 overexpression increased tolerance to drought stress in both solution cultures and soil plots. Drought stress in combination with GmPIP2;9 overexpression increased net CO 2 assimilation of photosynthesis, stomata conductance, and transpiration rate, suggesting that GmPIP2;9- overexpressing transgenic plants were less stressed than wild-type (WT) plants. Furthermore, field experiments showed that GmPIP2;9 -overexpressing plants had significantly more pod numbers and larger seed sizes than WT plants. In summary, the study demonstrated that GmPIP2;9 has water transport activity. Its relative high expression levels in roots and developing pods are in agreement with the phenotypes of GmPIP2;9 -overexpressing plants in drought stress tolerance and seed development.

Roles of Soybean Plasma Membrane Intrinsic Protein GmPIP2;9 in Drought Tolerance and Seed Development

PubMed Central

Lu, Linghong; Dong, Changhe; Liu, Ruifang; Zhou, Bin; Wang, Chuang; Shou, Huixia

2018-01-01

Aquaporins play an essential role in water uptake and transport in vascular plants. The soybean genome contains a total of 22 plasma membrane intrinsic protein (PIP) genes. To identify candidate PIPs important for soybean yield and stress tolerance, we studied the transcript levels of all 22 soybean PIPs. We found that a GmPIP2 subfamily member, GmPIP2;9, was predominately expressed in roots and developing seeds. Here, we show that GmPIP2;9 localized to the plasma membrane and had high water channel activity when expressed in Xenopus oocytes. Using transgenic soybean plants expressing a native GmPIP2;9 promoter driving a GUS-reporter gene, it was found high GUS expression in the roots, in particular, in the endoderm, pericycle, and vascular tissues of the roots of transgenic plants. In addition, GmPIP2;9 was also highly expressed in developing pods. GmPIP2;9 expression significantly increased in short term of polyethylene glycol (PEG)-mediated drought stress treatment. GmPIP2;9 overexpression increased tolerance to drought stress in both solution cultures and soil plots. Drought stress in combination with GmPIP2;9 overexpression increased net CO2 assimilation of photosynthesis, stomata conductance, and transpiration rate, suggesting that GmPIP2;9-overexpressing transgenic plants were less stressed than wild-type (WT) plants. Furthermore, field experiments showed that GmPIP2;9-overexpressing plants had significantly more pod numbers and larger seed sizes than WT plants. In summary, the study demonstrated that GmPIP2;9 has water transport activity. Its relative high expression levels in roots and developing pods are in agreement with the phenotypes of GmPIP2;9-overexpressing plants in drought stress tolerance and seed development. PMID:29755491

Transformation of pecan and regeneration of transgenic plants.

PubMed

McGranahan, G H; Leslie, C A; Dandekar, A M; Uratsu, S L; Yates, I E

1993-09-01

A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of 'Elliott', 'Wichita', and 'Schley' were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line 'Elliott'6, 3 from 'Schley'5/3, and 3 from 'Wichita'9. Transgenic embryos of 'Wichita'9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.

Identification and expression pattern analysis of BoMYB51 involved in indolic glucosinolate biosynthesis from broccoli (Brassica oleracea var. italica).

PubMed

Yu, Qingyue; Hao, Guodong; Zhou, Jianxin; Wang, Jingying; Evivie, Ejiroghene Ruona; Li, Jing

2018-06-22

Glucosinolates are a class of amino acid-derived specialized metabolites characteristic of the Brassicales order. Trp derived indolic glucosinolates are essential for the effective plant defense responses to a wide range of pathogens and herbivores. In Arabidopsis, MYB51 is the key transcription factor positively regulates indolic glucosinolate production by activating certain biosynthetic genes. In this study, we report the isolation and identification of a MYB51 from broccoli designated as BoMYB51. Overexpression of BoMYB51 in Arabidopsis increased indolic glucosinolate production by upregulating biosynthetic genes and resulted in enhanced flagellin22 (Flg22) induced callose deposition. The spatial expression pattern and responsive expression of BoMYB51 to several hormones and stress treatments were investigated by expressing the β-glucuronidase (GUS) reporter gene driven by BoMYB51 promotor in Arabidopsis and quantitative real-time PCR analysis in broccoli. Our study provides information on molecular characteristics of BoMYB51 and possible physiological process BoMYB51 may involve. Copyright © 2018 Elsevier Inc. All rights reserved.

Avirulence Genes in Cereal Powdery Mildews: The Gene-for-Gene Hypothesis 2.0.

PubMed

Bourras, Salim; McNally, Kaitlin E; Müller, Marion C; Wicker, Thomas; Keller, Beat

2016-01-01

The gene-for-gene hypothesis states that for each gene controlling resistance in the host, there is a corresponding, specific gene controlling avirulence in the pathogen. Allelic series of the cereal mildew resistance genes Pm3 and Mla provide an excellent system for genetic and molecular analysis of resistance specificity. Despite this opportunity for molecular research, avirulence genes in mildews remain underexplored. Earlier work in barley powdery mildew (B.g. hordei) has shown that the reaction to some Mla resistance alleles is controlled by multiple genes. Similarly, several genes are involved in the specific interaction of wheat mildew (B.g. tritici) with the Pm3 allelic series. We found that two mildew genes control avirulence on Pm3f: one gene is involved in recognition by the resistance protein as demonstrated by functional studies in wheat and the heterologous host Nicotiana benthamiana. A second gene is a suppressor, and resistance is only observed in mildew genotypes combining the inactive suppressor and the recognized Avr. We propose that such suppressor/avirulence gene combinations provide the basis of specificity in mildews. Depending on the particular gene combinations in a mildew race, different genes will be genetically identified as the "avirulence" gene. Additionally, the observation of two LINE retrotransposon-encoded avirulence genes in B.g. hordei further suggests that the control of avirulence in mildew is more complex than a canonical gene-for-gene interaction. To fully understand the mildew-cereal interactions, more knowledge on avirulence determinants is needed and we propose ways how this can be achieved based on recent advances in the field.

Avirulence Genes in Cereal Powdery Mildews: The Gene-for-Gene Hypothesis 2.0

PubMed Central

Bourras, Salim; McNally, Kaitlin E.; Müller, Marion C.; Wicker, Thomas; Keller, Beat

2016-01-01

The gene-for-gene hypothesis states that for each gene controlling resistance in the host, there is a corresponding, specific gene controlling avirulence in the pathogen. Allelic series of the cereal mildew resistance genes Pm3 and Mla provide an excellent system for genetic and molecular analysis of resistance specificity. Despite this opportunity for molecular research, avirulence genes in mildews remain underexplored. Earlier work in barley powdery mildew (B.g. hordei) has shown that the reaction to some Mla resistance alleles is controlled by multiple genes. Similarly, several genes are involved in the specific interaction of wheat mildew (B.g. tritici) with the Pm3 allelic series. We found that two mildew genes control avirulence on Pm3f: one gene is involved in recognition by the resistance protein as demonstrated by functional studies in wheat and the heterologous host Nicotiana benthamiana. A second gene is a suppressor, and resistance is only observed in mildew genotypes combining the inactive suppressor and the recognized Avr. We propose that such suppressor/avirulence gene combinations provide the basis of specificity in mildews. Depending on the particular gene combinations in a mildew race, different genes will be genetically identified as the “avirulence” gene. Additionally, the observation of two LINE retrotransposon-encoded avirulence genes in B.g. hordei further suggests that the control of avirulence in mildew is more complex than a canonical gene-for-gene interaction. To fully understand the mildew–cereal interactions, more knowledge on avirulence determinants is needed and we propose ways how this can be achieved based on recent advances in the field. PMID:26973683

Mutation in xyloglucan 6-xylosytransferase results in abnormal root hair development in Oryza sativa

PubMed Central

Wang, Chuang; Li, Shuai; Ng, Sophia; Zhang, Baocai; Zhou, Yihua; Whelan, James; Wu, Ping; Shou, Huixia

2014-01-01

Root hairs are important for nutrient uptake, anchorage, and plant–microbe interactions. From a population of rice (Oryza sativa) mutagenized by ethyl methanesulfonate (EMS), a short root hair2 (srh2) mutant was identified. In hydroponic culture, srh2 seedlings were significantly reduced in root hair length. Bubble-like extrusions and irregular epidermal cells were observed at the tips of srh2 root hairs when grown under acidic conditions, suggesting the possible reduction of the tensile strength of the cell wall in this mutant. Map-based cloning identified a mutation in the gene encoding xyloglucan (XyG) 6-xylosyltransferase (OsXXT1). OsXXT1 displays more than 70% amino acid sequence identity with the previously characterized Arabidopsis thaliana XYG XYLOSYL TRANSFERASE 1 (AtXXT1) and XYG XYLOSYL TRANSFERASE 2 (AtXXT2), which catalyse the transfer of xylose onto β-1,4-glucan chains. Furthermore, expression of the full-length coding sequence of OsXXT1 could complement the root hair defect, and slow growth and XyG synthesis in the Arabidopsis xxt1 xxt2 double mutant. Transgenic plants expressing the β-glucuronidase (GUS) reporter under the control of the OsXXT1 promoter displayed GUS expression in multiple tissues, most prominently in root epidermal cells. These results demonstrate the importance of OsXXT1 in maintaining cell wall structure and tensile strength in rice, a typical grass species that contains relatively low XyG content in cell walls. PMID:24834920

Identification and functional characterization of the soybean GmaPPO12 promoter conferring Phytophthora sojae induced expression.

PubMed

Chai, Chunyue; Lin, Yanling; Shen, Danyu; Wu, Yuren; Li, Hongjuan; Dou, Daolong

2013-01-01

Identification of pathogen-inducible promoters largely lags behind cloning of the genes for disease resistance. Here, we cloned the soybean GmaPPO12 gene and found that it was rapidly and strongly induced by Phytophthorasojae infection. Computational analysis revealed that its promoter contained many known cis-elements, including several defense related transcriptional factor-binding boxes. We showed that the promoter could mediate induction of GUS expression upon infection in both transient expression assays in Nicotianabenthamiana and stable transgenic soybean hairy roots. Importantly, we demonstrated that pathogen-induced expression of the GmaPPO12 promoter was higher than that of the soybean GmaPR1a promoter. A progressive 5' and 3' deletion analysis revealed two fragments that were essential for promoter activity. Thus, the cloned promoter could be used in transgenic plants to enhance resistance to phytophthora pathogens, and the identified fragment could serve as a candidate to produce synthetic pathogen-induced promoters.

Converting cancer genes into killer genes.

PubMed Central

Da Costa, L T; Jen, J; He, T C; Chan, T A; Kinzler, K W; Vogelstein, B

1996-01-01

Over the past decade, it has become clear that tumorigenesis is driven by alterations in genes that control cell growth or cell death. Theoretically, the proteins encoded by these genes provide excellent targets for new therapeutic agents. Here, we describe a gene therapy approach to specifically kill tumor cells expressing such oncoproteins. In outline, the target oncoprotein binds to exogenously introduced gene products, resulting in transcriptional activation of a toxic gene. As an example, we show that this approach can be used to specifically kill cells overexpressing a mutant p53 gene in cell culture. The strategy may be generally applicable to neoplastic diseases in which the underlying patterns of genetic alterations or abnormal gene expression are known. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8633039

GoGene: gene annotation in the fast lane.

PubMed

Plake, Conrad; Royer, Loic; Winnenburg, Rainer; Hakenberg, Jörg; Schroeder, Michael

2009-07-01

High-throughput screens such as microarrays and RNAi screens produce huge amounts of data. They typically result in hundreds of genes, which are often further explored and clustered via enriched GeneOntology terms. The strength of such analyses is that they build on high-quality manual annotations provided with the GeneOntology. However, the weakness is that annotations are restricted to process, function and location and that they do not cover all known genes in model organisms. GoGene addresses this weakness by complementing high-quality manual annotation with high-throughput text mining extracting co-occurrences of genes and ontology terms from literature. GoGene contains over 4,000,000 associations between genes and gene-related terms for 10 model organisms extracted from more than 18,000,000 PubMed entries. It does not cover only process, function and location of genes, but also biomedical categories such as diseases, compounds, techniques and mutations. By bringing it all together, GoGene provides the most recent and most complete facts about genes and can rank them according to novelty and importance. GoGene accepts keywords, gene lists, gene sequences and protein sequences as input and supports search for genes in PubMed, EntrezGene and via BLAST. Since all associations of genes to terms are supported by evidence in the literature, the results are transparent and can be verified by the user. GoGene is available at http://gopubmed.org/gogene.

The Relationship Between Endogenous β-Glucuronidase Activity and Biologically Active Flavones-Aglycone Contents in Hairy Roots of Baikal Skullcap.

PubMed

Dikaya, Varvara S; Solovyeva, Aleksandra I; Sidorov, Roman A; Solovyev, Pavel A; Stepanova, Anna Yu

2018-02-01

Here, we examine the relationship between contents of principal flavones in hairy roots of Scutellaria baicalensis with the activity of the β-glucuronidase (sGUS) enzyme during a culturing cycle. Using RP-HPLC, we show that the highest contents of aglycones, baicalin and wogonin is observed at the growth days 8, 14, and 71 and reach 45, 41, and 62% (based on the total weight of hairy roots of the Baikal skullcap), correspondingly. Their accumulation is accompanied by increase of the sGUS activity, which we determined fluorometrically. Moreover, the enzyme activity is characterized by significant and reasonable correlation only with the wogonin contents. Our results confirm a significant role of sGUS at the final steps of the metabolism in root-specific flavones of Baikal skullcap and suggest how one can optimize the conditions of culturing the hairy roots for biotechnological production of individual flavonoids. For example, at the culturing day 71 wogonin constituted over 80% of all flavones extracted from cells. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

Down-weighting overlapping genes improves gene set analysis

PubMed Central

2012-01-01

Background The identification of gene sets that are significantly impacted in a given condition based on microarray data is a crucial step in current life science research. Most gene set analysis methods treat genes equally, regardless how specific they are to a given gene set. Results In this work we propose a new gene set analysis method that computes a gene set score as the mean of absolute values of weighted moderated gene t-scores. The gene weights are designed to emphasize the genes appearing in few gene sets, versus genes that appear in many gene sets. We demonstrate the usefulness of the method when analyzing gene sets that correspond to the KEGG pathways, and hence we called our method Pathway Analysis with Down-weighting of Overlapping Genes (PADOG). Unlike most gene set analysis methods which are validated through the analysis of 2-3 data sets followed by a human interpretation of the results, the validation employed here uses 24 different data sets and a completely objective assessment scheme that makes minimal assumptions and eliminates the need for possibly biased human assessments of the analysis results. Conclusions PADOG significantly improves gene set ranking and boosts sensitivity of analysis using information already available in the gene expression profiles and the collection of gene sets to be analyzed. The advantages of PADOG over other existing approaches are shown to be stable to changes in the database of gene sets to be analyzed. PADOG was implemented as an R package available at: http://bioinformaticsprb.med.wayne.edu/PADOG/or http://www.bioconductor.org. PMID:22713124

Gene-gene interactions and gene polymorphisms of VEGFA and EG-VEGF gene systems in recurrent pregnancy loss.

PubMed

Su, Mei-Tsz; Lin, Sheng-Hsiang; Chen, Yi-Chi; Kuo, Pao-Lin

2014-06-01

Both vascular endothelial growth factor A (VEGFA) and endocrine gland-derived vascular endothelial growth factor (EG-VEGF) systems play major roles in angiogenesis. A body of evidence suggests VEGFs regulate critical processes during pregnancy and have been associated with recurrent pregnancy loss (RPL). However, little information is available regarding the interaction of these two major major angiogenesis-related systems in early human pregnancy. This study was conducted to investigate the association of gene polymorphisms and gene-gene interaction among genes in VEGFA and EG-VEGF systems and idiopathic RPL. A total of 98 women with history of idiopathic RPL and 142 controls were included, and 5 functional SNPs selected from VEGFA, KDR, EG-VEGF (PROK1), PROKR1 and PROKR2 were genotyped. We used multifactor dimensionality reduction (MDR) analysis to choose a best model and evaluate gene-gene interactions. Ingenuity pathways analysis (IPA) was introduced to explore possible complex interactions. Two receptor gene polymorphisms [KDR (Q472H) and PROKR2 (V331M)] were significantly associated with idiopathic RPL (P<0.01). The MDR test revealed that the KDR (Q472H) polymorphism was the best loci to be associated with RPL (P=0.02). IPA revealed EG-VEGF and VEGFA systems shared several canonical signaling pathways that may contribute to gene-gene interactions, including the Akt, IL-8, EGFR, MAPK, SRC, VHL, HIF-1A and STAT3 signaling pathways. Two receptor gene polymorphisms [KDR (Q472H) and PROKR2 (V331M)] were significantly associated with idiopathic RPL. EG-VEGF and VEGFA systems shared several canonical signaling pathways that may contribute to gene-gene interactions, including the Akt, IL-8, EGFR, MAPK, SRC, VHL, HIF-1A and STAT3.

Arabidopsis thaliana gonidialess A/Zuotin related factors (GlsA/ZRF) are essential for maintenance of meristem integrity.

PubMed

Guzmán-López, José Alfredo; Abraham-Juárez, María Jazmín; Lozano-Sotomayor, Paulina; de Folter, Stefan; Simpson, June

2016-05-01

Observation of a differential expression pattern, including strong expression in meristematic tissue of an Agave tequilana GlsA/ZRF ortholog suggested an important role for this gene during bulbil formation and developmental changes in this species. In order to better understand this role, the two GlsA/ZFR orthologs present in the genome of Arabidopsis thaliana were functionally characterized by analyzing expression patterns, double mutant phenotypes, promoter-GUS fusions and expression of hormone related or meristem marker genes. Patterns of expression for A. thaliana show that GlsA/ZFR genes are strongly expressed in SAMs and RAMs in mature plants and developing embryos and double mutants showed multiple changes in morphology related to both SAM and RAM tissues. Typical double mutants showed stunted growth of aerial and root tissue, formation of multiple ectopic meristems and effects on cotyledons, leaves and flowers. The KNOX genes STM and BP were overexpressed in double mutants whereas CLV3, WUSCHEL and AS1 were repressed and lack of AtGlsA expression was also associated with changes in localization of auxin and cytokinin. These results suggest that GlsA/ZFR is an essential component of the machinery that maintains the integrity of SAM and RAM tissue and underline the potential to identify new genes or gene functions based on observations in non-model plants.

FunGene: the functional gene pipeline and repository.

PubMed

Fish, Jordan A; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C Titus; Tiedje, James M; Cole, James R

2013-01-01

Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.

Mining disease genes using integrated protein-protein interaction and gene-gene co-regulation information.

PubMed

Li, Jin; Wang, Limei; Guo, Maozu; Zhang, Ruijie; Dai, Qiguo; Liu, Xiaoyan; Wang, Chunyu; Teng, Zhixia; Xuan, Ping; Zhang, Mingming

2015-01-01

In humans, despite the rapid increase in disease-associated gene discovery, a large proportion of disease-associated genes are still unknown. Many network-based approaches have been used to prioritize disease genes. Many networks, such as the protein-protein interaction (PPI), KEGG, and gene co-expression networks, have been used. Expression quantitative trait loci (eQTLs) have been successfully applied for the determination of genes associated with several diseases. In this study, we constructed an eQTL-based gene-gene co-regulation network (GGCRN) and used it to mine for disease genes. We adopted the random walk with restart (RWR) algorithm to mine for genes associated with Alzheimer disease. Compared to the Human Protein Reference Database (HPRD) PPI network alone, the integrated HPRD PPI and GGCRN networks provided faster convergence and revealed new disease-related genes. Therefore, using the RWR algorithm for integrated PPI and GGCRN is an effective method for disease-associated gene mining.

Gene-gene, gene-environment, gene-nutrient interactions and single nucleotide polymorphisms of inflammatory cytokines.

PubMed

Nadeem, Amina; Mumtaz, Sadaf; Naveed, Abdul Khaliq; Aslam, Muhammad; Siddiqui, Arif; Lodhi, Ghulam Mustafa; Ahmad, Tausif

2015-05-15

Inflammation plays a significant role in the etiology of type 2 diabetes mellitus (T2DM). The rise in the pro-inflammatory cytokines is the essential step in glucotoxicity and lipotoxicity induced mitochondrial injury, oxidative stress and beta cell apoptosis in T2DM. Among the recognized markers are interleukin (IL)-6, IL-1, IL-10, IL-18, tissue necrosis factor-alpha (TNF-α), C-reactive protein, resistin, adiponectin, tissue plasminogen activator, fibrinogen and heptoglobins. Diabetes mellitus has firm genetic and very strong environmental influence; exhibiting a polygenic mode of inheritance. Many single nucleotide polymorphisms (SNPs) in various genes including those of pro and anti-inflammatory cytokines have been reported as a risk for T2DM. Not all the SNPs have been confirmed by unifying results in different studies and wide variations have been reported in various ethnic groups. The inter-ethnic variations can be explained by the fact that gene expression may be regulated by gene-gene, gene-environment and gene-nutrient interactions. This review highlights the impact of these interactions on determining the role of single nucleotide polymorphism of IL-6, TNF-α, resistin and adiponectin in pathogenesis of T2DM.

Modeling month-season of birth as a risk factor in mouse models of chronic disease: from multiple sclerosis to autoimmune encephalomyelitis.

PubMed

Reynolds, Jacob D; Case, Laure K; Krementsov, Dimitry N; Raza, Abbas; Bartiss, Rose; Teuscher, Cory

2017-06-01

Month-season of birth (M-SOB) is a risk factor in multiple chronic diseases, including multiple sclerosis (MS), where the lowest and greatest risk of developing MS coincide with the lowest and highest birth rates, respectively. To determine whether M-SOB effects in such chronic diseases as MS can be experimentally modeled, we examined the effect of M-SOB on susceptibility of C57BL/6J mice to experimental autoimmune encephalomyelitis (EAE). As in MS, mice that were born during the M-SOB with the lowest birth rate were less susceptible to EAE than mice born during the M-SOB with the highest birth rate. We also show that the M-SOB effect on EAE susceptibility is associated with differential production of multiple cytokines/chemokines by neuroantigen-specific T cells that are known to play a role in EAE pathogenesis. Taken together, these results support the existence of an M-SOB effect that may reflect seasonally dependent developmental differences in adaptive immune responses to self-antigens independent of external stimuli, including exposure to sunlight and vitamin D. Moreover, our documentation of an M-SOB effect on EAE susceptibility in mice allows for modeling and detailed analysis of mechanisms that underlie the M-SOB effect in not only MS but in numerous other diseases in which M-SOB impacts susceptibility.-Reynolds, J. D., Case, L. K., Krementsov, D. N., Raza, A., Bartiss, R., Teuscher, C. Modeling month-season of birth as a risk factor in mouse models of chronic disease: from multiple sclerosis to autoimmune encephalomyelitis. © FASEB.

Neighboring Genes Show Correlated Evolution in Gene Expression.

PubMed

Ghanbarian, Avazeh T; Hurst, Laurence D

2015-07-01

When considering the evolution of a gene's expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Neighboring Genes Show Correlated Evolution in Gene Expression

PubMed Central

Ghanbarian, Avazeh T.; Hurst, Laurence D.

2015-01-01

When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

Gene doping: gene delivery for olympic victory

PubMed Central

Gould, David

2013-01-01

With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place. PMID:23082866

GeneSigDB—a curated database of gene expression signatures

PubMed Central

Culhane, Aedín C.; Schwarzl, Thomas; Sultana, Razvan; Picard, Kermshlise C.; Picard, Shaita C.; Lu, Tim H.; Franklin, Katherine R.; French, Simon J.; Papenhausen, Gerald; Correll, Mick; Quackenbush, John

2010-01-01

The primary objective of most gene expression studies is the identification of one or more gene signatures; lists of genes whose transcriptional levels are uniquely associated with a specific biological phenotype. Whilst thousands of experimentally derived gene signatures are published, their potential value to the community is limited by their computational inaccessibility. Gene signatures are embedded in published article figures, tables or in supplementary materials, and are frequently presented using non-standard gene or probeset nomenclature. We present GeneSigDB (http://compbio.dfci.harvard.edu/genesigdb) a manually curated database of gene expression signatures. GeneSigDB release 1.0 focuses on cancer and stem cells gene signatures and was constructed from more than 850 publications from which we manually transcribed 575 gene signatures. Most gene signatures (n = 560) were successfully mapped to the genome to extract standardized lists of EnsEMBL gene identifiers. GeneSigDB provides the original gene signature, the standardized gene list and a fully traceable gene mapping history for each gene from the original transcribed data table through to the standardized list of genes. The GeneSigDB web portal is easy to search, allows users to compare their own gene list to those in the database, and download gene signatures in most common gene identifier formats. PMID:19934259