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Sample records for aflatoxin biosynthesis genes

  1. Aflatoxin biosynthesis: current frontiers.

    PubMed

    Roze, Ludmila V; Hong, Sung-Yong; Linz, John E

    2013-01-01

    Aflatoxins are among the principal mycotoxins that contaminate economically important food and feed crops. Aflatoxin B1 is the most potent naturally occurring carcinogen known and is also an immunosuppressant. Occurrence of aflatoxins in crops has vast economic and human health impacts worldwide. Thus, the study of aflatoxin biosynthesis has become a focal point in attempts to reduce human exposure to aflatoxins. This review highlights recent advances in the field of aflatoxin biosynthesis and explores the functional connection between aflatoxin biosynthesis, endomembrane trafficking, and response to oxidative stress. Dissection of the regulatory mechanisms involves a complete comprehension of the aflatoxin biosynthetic process and the dynamic network of transcription factors that orchestrates coordinated expression of the target genes. Despite advancements in the field, development of a safe and effective multifaceted approach to solve the aflatoxin food contamination problem is still required.

  2. The potential effects of Zataria multiflora Boiss essential oil on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes of toxigenic Aspergillus parasiticus.

    PubMed

    Yahyaraeyat, R; Khosravi, A R; Shahbazzadeh, D; Khalaj, V

    2013-01-01

    This study aims at evaluating the effects of Zataria multiflora (Z. multiflora) essential oil (EO) on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes. Total RNAs of Aspergillus parasiticus (A.parasiticus) ATCC56775 grown in yeast extract sucrose (YES) broth medium treated with Z. multiflora EO were subjected to reverse transcription- polymerase chain reaction (RT-PCR). Specific primers of nor-1, ver-1, omt-A and aflR genes were used. In parallel mycelial dry weight of samples were measured and all the media were assayed by high-pressure liquid chromatography (HPLC) for aflatoxinB1 (AFB1), aflatoxinB2 (AFB2), aflatoxinG1 (AFG1), aflatoxinG2 (AFG2) and aflatoxin total (AFTotal) production. The results showed that mycelial dry weight and aflatoxin production reduce in the presence of Z. multiflora EO (100 ppm) on day 5 of growth. It was found that the expression of nor-1, ver-1, omt-A and aflR genes was correlated with the ability of fungus to produce aflatoxins on day 5 in YES medium. RT-PCR showed that in the presence of Z.multiflora EO (100 ppm) nor-1, ver-1 and omtA genes expression was reduced. It seems that toxin production inhibitory effects of Z. multiflora EO on day 5 may be at the transcription level and this herb may cause reduction in aflatoxin biosynthesis pathway genes activity.

  3. Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

  4. Inhibitory Effect of Cinnamaldehyde, Citral, and Eugenol on Aflatoxin Biosynthetic Gene Expression and Aflatoxin B1 Biosynthesis in Aspergillus flavus.

    PubMed

    Liang, Dandan; Xing, Fuguo; Selvaraj, Jonathan Nimal; Liu, Xiao; Wang, Limin; Hua, Huijuan; Zhou, Lu; Zhao, Yueju; Wang, Yan; Liu, Yang

    2015-12-01

    In order to reveal the inhibitory effects of cinnamaldehyde, citral, and eugenol on aflatoxin biosynthesis, the expression levels of 5 key aflatoxin biosynthetic genes were evaluated by real-time PCR. Aspergillus flavus growth and AFB1 production were completely inhibited by 0.80 mmol/L of cinnamaldehyde and 2.80 mmol/L of citral. However, at lower concentration, cinnamaldehyde (0.40 mmol/L), eugenol (0.80 mmol/L), and citral (0.56 mmol/L) significantly reduced AFB1 production with inhibition rate of 68.9%, 95.4%, and 41.8%, respectively, while no effect on fungal growth. Real-time PCR showed that the expressions of aflR, aflT, aflD, aflM, and aflP were down-regulated by cinnamaldehyde (0.40 mmol/L), eugenol (0.80 mmol/L), and citral (0.56 mmol/L). In the presence of cinnamaldehyde, AflM was highly down-regulated (average of 5963 folds), followed by aflP, aflR, aflD, and aflT with the average folds of 55, 18, 6.5, and 5.8, respectively. With 0.80 mmol/L of eugenol, aflP was highly down-regulated (average of 2061-folds), followed by aflM, aflR, aflD, and aflT with average of 138-, 15-, 5.2-, and 4.8-folds reduction, respectively. With 0.56 mmol/L of citral, aflT was completely inhibited, followed by aflM, aflP, aflR, and aflD with average of 257-, 29-, 3.5-, and 2.5-folds reduction, respectively. These results suggest that the reduction in AFB1 production by cinnamaldehyde, eugenol, and citral at low concentration may be due to the down-regulations of the transcription level of aflatoxin biosynthetic genes. Cinnamaldehyde and eugenol may be employed successfully as a good candidate in controlling of toxigenic fungi and subsequently contamination with aflatoxins in practice.

  5. Use of Selected Essential Oils to Control Aflatoxin Contaminated Stored Cashew and Detection of Aflatoxin Biosynthesis Gene

    PubMed Central

    Abd El-Aziz, Abeer R. M.; Mahmoud, Mohamed A.; Al-Othman, Monira R.; Al-Gahtani, Munirah F.

    2015-01-01

    Aspergillus spp. associated with cashew from the regions of Riyadh, Dammam, and Abha were isolated and three different culture media were used to qualitatively measure aflatoxin production by Aspergillus via UV light (365 nm), which was expressed as positive or negative. The obtained data showed that six isolates of A. flavus and four isolates of A. parasiticus were positive for aflatoxin production, while all isolates of A. niger were negative. Five commercially essential oils (thyme, garlic, cinnamon, mint, and rosemary) were tested to determine their influence on growth and aflatoxin production in A. flavus and A. parasiticus by performing high-performance liquid chromatography (HPLC). The results showed that the tested essential oils caused highly significant inhibition of fungal growth and aflatoxin production in A. flavus and A. parasiticus. The extent of the inhibition of fungal growth and aflatoxin production was dependent on the type and concentration of essential oils applied. The results indicate that cinnamon and thyme oils show strong antimicrobial potential. PCR was used with four sets of primer pairs for nor-1, omt-1, ver-1, and aflR genes, enclosed in the aflatoxin biosynthetic pathway. The interpretation of the results revealed that PCR is a rapid and sensitive method. PMID:25705718

  6. Use of selected essential oils to control aflatoxin contaminated stored cashew and detection of aflatoxin biosynthesis gene.

    PubMed

    Abd El-Aziz, Abeer R M; Mahmoud, Mohamed A; Al-Othman, Monira R; Al-Gahtani, Munirah F

    2015-01-01

    Aspergillus spp. associated with cashew from the regions of Riyadh, Dammam, and Abha were isolated and three different culture media were used to qualitatively measure aflatoxin production by Aspergillus via UV light (365 nm), which was expressed as positive or negative. The obtained data showed that six isolates of A. flavus and four isolates of A. parasiticus were positive for aflatoxin production, while all isolates of A. niger were negative. Five commercially essential oils (thyme, garlic, cinnamon, mint, and rosemary) were tested to determine their influence on growth and aflatoxin production in A. flavus and A. parasiticus by performing high-performance liquid chromatography (HPLC). The results showed that the tested essential oils caused highly significant inhibition of fungal growth and aflatoxin production in A. flavus and A. parasiticus. The extent of the inhibition of fungal growth and aflatoxin production was dependent on the type and concentration of essential oils applied. The results indicate that cinnamon and thyme oils show strong antimicrobial potential. PCR was used with four sets of primer pairs for nor-1, omt-1, ver-1, and aflR genes, enclosed in the aflatoxin biosynthetic pathway. The interpretation of the results revealed that PCR is a rapid and sensitive method.

  7. Effect of temperature and water activity on gene expression and aflatoxin biosynthesis in Aspergillus flavus on almond medium.

    PubMed

    Gallo, Antonia; Solfrizzo, Michele; Epifani, Filomena; Panzarini, Giuseppe; Perrone, Giancarlo

    2016-01-18

    Almonds are among the commodities at risk of aflatoxin contamination by Aspergillus flavus. Temperature and water activity are the two key determinants in pre and post-harvest environments influencing both the rate of fungal spoilage and aflatoxin production. Varying the combination of these parameters can completely inhibit or fully activate the biosynthesis of aflatoxin, so it is fundamental to know which combinations can control or be conducive to aflatoxin contamination. Little information is available about the influence of these parameters on aflatoxin production on almonds. The objective of this study was to determine the influence of different combinations of temperature (20 °C, 28 °C, and 37 °C) and water activity (0.90, 0.93, 0.96, 0.99 aw) on growth, aflatoxin B1 (AFB1) production and expression of the two regulatory genes, aflR and aflS, and two structural genes, aflD and aflO, of the aflatoxin biosynthetic cluster in A. flavus grown on an almond medium solidified with agar. Maximum accumulation of fungal biomass and AFB1 production was obtained at 28 °C and 0.96 aw; no fungal growth and AFB1 production were observed at 20 °C at the driest tested conditions (0.90 and 0.93 aw). At 20° and 37 °C AFB1 production was 70-90% lower or completely suppressed, depending on aw. Reverse transcriptase quantitative PCR showed that the two regulatory genes (aflR and aflS) were highly expressed at maximum (28 °C) and minimum (20 °C and 37 °C) AFB1 production. Conversely the two structural genes (aflD and aflO) were highly expressed only at maximum AFB1 production (28 °C and 0.96-0.99 aw). It seems that temperature acts as a key factor influencing aflatoxin production which is strictly correlated to the induction of expression of structural biosynthesis genes (aflD and aflO), but not to that of aflatoxin regulatory genes (aflR and aflS), whose functional products are most likely subordinated to other regulatory processes acting at post-translational level

  8. Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates.

    PubMed

    Chang, Perng-Kuang; Horn, Bruce W; Dorner, Joe W

    2005-11-01

    Aspergillus flavus populations are genetically diverse. Isolates that produce either, neither, or both aflatoxins and cyclopiazonic acid (CPA) are present in the field. We investigated defects in the aflatoxin gene cluster in 38 nonaflatoxigenic A. flavus isolates collected from southern United States. PCR assays using aflatoxin-gene-specific primers grouped these isolates into eight (A-H) deletion patterns. Patterns C, E, G, and H, which contain 40 kb deletions, were examined for their sequence breakpoints. Pattern C has one breakpoint in the cypA 3' untranslated region (UTR) and another in the verA coding region. Pattern E has a breakpoint in the amdA coding region and another in the ver1 5'UTR. Pattern G contains a deletion identical to the one found in pattern C and has another deletion that extends from the cypA coding region to one end of the chromosome as suggested by the presence of telomeric sequence repeats, CCCTAATGTTGA. Pattern H has a deletion of the entire aflatoxin gene cluster from the hexA coding region in the sugar utilization gene cluster to the telomeric region. Thus, deletions in the aflatoxin gene cluster among A. flavus isolates are not rare, and the patterns appear to be diverse. Genetic drift may be a driving force that is responsible for the loss of the entire aflatoxin gene cluster in nonaflatoxigenic A. flavus isolates when aflatoxins have lost their adaptive value in nature.

  9. Structural and functional analysis of the nor-1 gene involved in the biosynthesis of aflatoxins by Aspergillus parasiticus.

    PubMed Central

    Trail, F; Chang, P K; Cary, J; Linz, J E

    1994-01-01

    The nor-1 gene was cloned previously by complementation of a mutation (nor-1) in Aspergillus parasiticus SU-1 which blocked aflatoxin B1 biosynthesis, resulting in the accumulation of norsolorinic acid (NA). In this study, the nucleotide sequences of the cDNA and genomic DNA clones encompassing the coding region of the nor-1 gene were determined. The transcription initiation and polyadenylation sites of nor-1 were located by primer extension and RNase protection analyses and by comparison of the nucleotide sequences of the nor-1 genomic and cDNA clones. A plasmid, pNA51-82, was created for one-step disruption of the nor-1 gene by inserting a functional copy of the nitrate reductase (niaD) gene from A. parasiticus into the coding region of the nor-1 gene. Transformation of A. parasiticus NR-3 (niaD Afl+) with pNA51-82 resulted in niaD+ transformants that accumulated NA and produced reduced levels of aflatoxin as determined by thin-layer chromatography and enzyme-linked immunosorbent assay analyses of extracts from mycelia and the growth medium. Southern analysis of genomic DNA isolated from the NA-accumulating transformants indicated that the wild-type nor-1 gene in the chromosome had been replaced by the nonfunctional allele carried on pNA51-82. This recombinational inactivation event provides direct evidence that the nor-1 gene is functionally involved in aflatoxin biosynthesis. Comparison of the predicted nor-1 amino acid sequence with sequences in the GenBank and EMBL databases suggested that the protein is a member of the family of short-chain alcohol dehydrogenases, consistent with its proposed function as a keto reductase. Images PMID:7993094

  10. HypC, the anthrone oxidase involved in aflatoxin biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Based on gene disruption and enzyme activity, hypC, an open reading frame in the pksA (aflC)/nor-1 (aflD) intergenic region in the aflatoxin biosynthesis cluster, encodes a 17 kDa oxidase that catalyzes the conversion of norsolorinic acid anthrone to norsolorinic acid....

  11. How Peroxisomes Affect Aflatoxin Biosynthesis in Aspergillus Flavus

    PubMed Central

    Reverberi, Massimo; Punelli, Marta; Smith, Carrie A.; Zjalic, Slaven; Scarpari, Marzia; Scala, Valeria; Cardinali, Giorgia; Aspite, Nicaela; Pinzari, Flavia; Payne, Gary A.; Fabbri, Anna A.; Fanelli, Corrado

    2012-01-01

    In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids β-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal β-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal β-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids β-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived), an hyper-oxidant cell environment and of hormone-like signals (oxylipins) enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis. PMID:23094106

  12. Autoxidated linolenic acid inhibits aflatoxin biosynthesis in Aspergillus flavus via oxylipin species.

    PubMed

    Yan, Shijuan; Liang, Yating; Zhang, Jindan; Chen, Zhuang; Liu, Chun-Ming

    2015-08-01

    Aflatoxins produced by Aspergillus species are among the most toxic and carcinogenic compounds in nature. Although it has been known for a long time that seeds with high oil content are more susceptible to aflatoxin contamination, the role of fatty acids in aflatoxin biosynthesis remains controversial. Here we demonstrate in A. flavus that both the saturated stearic acid (C18:0) and the polyunsaturated linolenic acid (C18:3) promoted aflatoxin production, while C18:3, but not C18:0, inhibited aflatoxin biosynthesis after exposure to air for several hours. Further experiments showed that autoxidated C18:3 promoted mycelial growth, sporulation, and kojic acid production, but inhibited the expression of genes in the AF biosynthetic gene cluster. Mass spectrometry analyses of autoxidated C18:3 fractions that were able to inhibit aflatoxin biosynthesis led to the identification of multiple oxylipin species. These results may help to clarify the role of fatty acids in aflatoxin biosynthesis, and may explain why controversial results have been obtained for fatty acids in the past.

  13. Degeneration of aflatoxin gene cluster in Aspergillus flavus from Africa and North America

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus is the primary causal agent of food and feed contamination with the toxic fungal metabolites aflatoxins. Aflatoxin-producing potential of A. flavus is known to vary among isolates. The genes involved in aflatoxin biosynthesis are clustered together and the order of genes within th...

  14. Aflatoxin biosynthesis is a novel source of reactive oxygen species—a potential redox signal to initiate resistance to oxidative stress?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aflatoxin biosynthesis in the filamentous fungus Aspergillus parasiticus involves a minimum of 21 enzymes, encoded by genes located in a 70 kb gene cluster. For aflatoxin biosynthesis to be completed, the required enzymes must be transported to specialized early and late endosomes called aflatoxisom...

  15. Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus

    PubMed Central

    Yang, Kunlong; Qin, Qiuping; Liu, Yinghang; Zhang, Limei; Liang, Linlin; Lan, Huahui; Chen, Chihao; You, Yunchao; Zhang, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus. PMID:28066725

  16. The Aspergillus flavus Histone Acetyltransferase AflGcnE Regulates Morphogenesis, Aflatoxin Biosynthesis, and Pathogenicity

    PubMed Central

    Lan, Huahui; Sun, Ruilin; Fan, Kun; Yang, Kunlong; Zhang, Feng; Nie, Xin Y.; Wang, Xiunai; Zhuang, Zhenhong; Wang, Shihua

    2016-01-01

    Histone acetyltransferases (HATs) help regulate fungal development and the production of secondary metabolites. In this study, we determined that the HAT AflGcnE influenced morphogenesis and aflatoxin biosynthesis in Aspergillus flavus. We observed that AflGcnE localized to the nucleus and cytoplasm during the conidial production and germination stages, while it was located mainly in the nucleus during the hyphal development stage. Deletion of AflgcnE inhibited the growth of A. flavus and decreased the hydrophobicity of the cell surface. The ΔAflgcnE mutant exhibited a lack of asexual sporulation and was unable to generate sclerotia. Additionally, AflgcnE was required to maintain cell wall integrity and genotoxic stress responses. Importantly, the ΔAflgcnE mutant did not produce aflatoxins, which was consistent with a significant down-regulation of aflatoxin gene expression levels. Furthermore, our data revealed that AflgcnE is a pathogenicity factor required for colonizing maize seeds. In summary, we revealed that A. flavus AflGcnE is crucial for morphological development, aflatoxin biosynthesis, stress responses, and pathogenicity. Our findings help clarify the functional divergence of GcnE orthologs, and may provide a possible target for controlling A. flavus infections of agriculturally important crops. PMID:27625637

  17. Effects of nitrogen metabolism on growth and aflatoxin biosynthesis in Aspergillus flavus.

    PubMed

    Wang, Bin; Han, Xiaoyun; Bai, Youhuang; Lin, Zhenguo; Qiu, Mengguang; Nie, Xinyi; Wang, Sen; Zhang, Feng; Zhuang, Zhenhong; Yuan, Jun; Wang, Shihua

    2017-02-15

    Aflatoxins (AFs), produced mainly by Aspergillus flavus and Aspergillus parasiticus, are strongly toxic and carcinogenic. Here, we showed that glutamine is the optimal nitrogen source for AF-production in A. flavus grown in Czapek Dox medium. Additionally, 4mM glutamine was the threshold for high production of aflatoxin B1. However, no significant impact of glutamine synthetase inhibitor was detected for on AF biosynthesis. In contrast, rapamycin could significantly suppress the glutamine inducing effect on AFs production, simultaneously inhibiting the fungal growth and conidiation. To identify the genes and regulatory networks involved in AFs biosynthesis, especially concerning the nitrogen source metabolism pathway and the target of rapamycin (TOR) signaling pathway, we obtained transcriptomes for A. flavus under treatment of three nitrogen sources by RNA-sequencing. We identified 1429 differentially expressed genes. Through GO and KEGG pathway analyses, the relationship between nitrogen metabolism and AFs biosynthesis was revealed, and the effects of TOR inhibitor were confirmed. Additionally, the quantitative real-time PCR results verified the credibility and reliability of the RNA-seq data, and were consistent with the other experimental results. Our research laid the foundation for a primary study on the involvement of the nitrogen regulatory network and TOR signaling pathway in AF biosynthesis.

  18. Association with AflR in Endosomes Reveals New Functions for AflJ in Aflatoxin Biosynthesis

    PubMed Central

    Ehrlich, Kenneth C.; Mack, Brian M.; Wei, Qijian; Li, Ping; Roze, Ludmila V.; Dazzo, Frank; Cary, Jeffrey W.; Bhatnagar, Deepak; Linz, John E.

    2012-01-01

    Aflatoxins are the most potent naturally occurring carcinogens of fungal origin. Biosynthesis of aflatoxin involves the coordinated expression of more than 25 genes. The function of one gene in the aflatoxin gene cluster, aflJ, is not entirely understood but, because previous studies demonstrated a physical interaction between the Zn2Cys6 transcription factor AflR and AflJ, AflJ was proposed to act as a transcriptional co-activator. Image analysis revealed that, in the absence of aflJ in A. parasiticus, endosomes cluster within cells and near septa. AflJ fused to yellow fluorescent protein complemented the mutation in A. parasiticus ΔaflJ and localized mainly in endosomes. We found that AflJ co-localizes with AflR both in endosomes and in nuclei. Chromatin immunoprecipitation did not detect AflJ binding at known AflR DNA recognition sites suggesting that AflJ either does not bind to these sites or binds to them transiently. Based on these data, we hypothesize that AflJ assists in AflR transport to or from the nucleus, thus controlling the availability of AflR for transcriptional activation of aflatoxin biosynthesis cluster genes. AflJ may also assist in directing endosomes to the cytoplasmic membrane for aflatoxin export. PMID:23342682

  19. The DmtA methyltransferase contributes to Aspergillus flavus conidiation, sclerotial production, aflatoxin biosynthesis and virulence

    PubMed Central

    Yang, Kunlong; Liang, Linlin; Ran, Fanlei; Liu, Yinghang; Li, Zhenguo; Lan, Huahui; Gao, Peili; Zhuang, Zhenhong; Zhang, Feng; Nie, Xinyi; Kalayu Yirga, Shimuye; Wang, Shihua

    2016-01-01

    DNA methylation is essential for epigenetic regulation of gene transcription and development in many animals, plants and fungi. We investigated whether DNA methylation plays a role in the development and secondary metabolism of Aspergillus flavus, identified the DmtA methyltransferase from A. flavus, and produced a dmtA knock-out mutant by replacing the dmtA coding sequence with the pyrG selectable marker. The A. flavus dmtA null mutant lines produced white fluffy mycelium in liquid medium, and displayed a slightly flavescent conidial pigmentation compared with the normal yellow of the wild-type strain when grown on agar. The ΔdmtA lines exhibited decreased conidiation and aflatoxin (AF) biosynthesis, compared with the wild-type line, suggesting that the DmtA knock-out affected the transcriptional level of genes in the AF cluster. In particular, sclerotia development and host colonization were altered in the dmtA null mutants. Green fluorescent protein tagging at the C-terminus of DmtA showed that DmtA localized to the nucleus and cytoplasm. DNA methylation content measurements in the dmtA mutants revealed no widespread DNA methylation in the mutants or wild-type lines. Thus, our findings suggest that DmtA, apart from being a C-5 cytosine methyltransferase in A. flavus, contributes to asexual development, aflatoxin biosynthesis, sclerotial production and virulence. PMID:26979781

  20. Suppression of Aflatoxin Biosynthesis in Aspergillus flavus by 2-Phenylethanol Is Associated with Stimulated Growth and Decreased Degradation of Branched-Chain Amino Acids.

    PubMed

    Chang, Perng-Kuang; Hua, Sui Sheng T; Sarreal, Siov Bouy L; Li, Robert W

    2015-09-24

    The saprophytic soil fungus Aspergillus flavus infects crops and produces aflatoxin. Pichia anomala, which is a biocontrol yeast and produces the major volatile 2-phenylethanol (2-PE), is able to reduce growth of A. flavus and aflatoxin production when applied onto pistachio trees. High levels of 2-PE are lethal to A. flavus and other fungi. However, at low levels, the underlying mechanism of 2-PE to inhibit aflatoxin production remains unclear. In this study, we characterized the temporal transcriptome response of A. flavus to 2-PE at a subinhibitory level (1 μL/mL) using RNA-Seq technology and bioinformatics tools. The treatment during the entire 72 h experimental period resulted in 131 of the total A. flavus 13,485 genes to be significantly impacted, of which 82 genes exhibited decreased expression. They included those encoding conidiation proteins and involved in cyclopiazonic acid biosynthesis. All genes in the aflatoxin gene cluster were also significantly decreased during the first 48 h treatment. Gene Ontology (GO) analyses showed that biological processes with GO terms related to catabolism of propionate and branched-chain amino acids (valine, leucine and isoleucine) were significantly enriched in the down-regulated gene group, while those associated with ribosome biogenesis, translation, and biosynthesis of α-amino acids OPEN ACCESS Toxins 2015, 7 3888 were over-represented among the up-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways negatively impacted among the down-regulated genes parallel to those active at 30 °C, a condition conducive to aflatoxin biosynthesis. In contrast, metabolic pathways positively related to the up-regulated gene group resembled those at 37 °C, which favors rapid fungal growth and is inhibitory to aflatoxin biosynthesis. The results showed that 2-PE at a low level stimulated active growth of A. flavus but concomitantly rendered decreased activities in

  1. Aflatoxin-free transgenic maize using host-induced gene silencing

    PubMed Central

    Thakare, Dhiraj; Zhang, Jianwei; Wing, Rod A.; Cotty, Peter J.; Schmidt, Monica A.

    2017-01-01

    Aflatoxins, toxic secondary metabolites produced by some Aspergillus species, are a universal agricultural economic problem and a critical health issue. Despite decades of control efforts, aflatoxin contamination is responsible for a global loss of millions of tons of crops each year. We show that host-induced gene silencing is an effective method for eliminating this toxin in transgenic maize. We transformed maize plants with a kernel-specific RNA interference (RNAi) gene cassette targeting the aflC gene, which encodes an enzyme in the Aspergillus aflatoxin biosynthetic pathway. After pathogen infection, aflatoxin could not be detected in kernels from these RNAi transgenic maize plants, while toxin loads reached thousands of parts per billion in nontransgenic control kernels. A comparison of transcripts in developing aflatoxin-free transgenic kernels with those from nontransgenic kernels showed no significant differences between these two groups. These results demonstrate that small interfering RNA molecules can be used to silence aflatoxin biosynthesis in maize, providing an attractive and precise engineering strategy that could also be extended to other crops to improve food security. PMID:28345051

  2. Malonate as a precursor in the biosynthesis of aflatoxins.

    PubMed

    Gupta, S R; Prasanna, H R; Viswanathan, L; Venkitasurbramanian, T A

    1975-06-01

    Incorporation of [I-14C]acetate and [2-14C]malonate into aflatoxins by resting mycelia of Aspergillus parasiticus resuspended in different buffers was studied. A decrease in pH from 5-8 to 2-8, as well as addition of EDTA, markedly stimulated the incorporation of malonate but the effect on acetate incorporation was less pronounced. Mycelia took up comparatively more acetate than malonate, but more malonate (4-3%) entering mycelia was incorporated into aflatoxins than was acetate (1-6%). Furthermore, the addition of unlabelled acetate reduced the incorporation of label from [I-14C]acetate by 75% but from [2-14C]malonate by only 25%. These results suggest that malonate is an intermediate in aflatoxin synthesis and that is can be incorporated without prior conversion to acetate.

  3. Two distinct O-methyltransferases in aflatoxin biosynthesis.

    PubMed Central

    Yabe, K; Ando, Y; Hashimoto, J; Hamasaki, T

    1989-01-01

    The substances belonging to the sterigmatocystin group bear a close structural relationship to aflatoxins. When demethylsterigmatocystin (DMST) was fed to Aspergillus parasiticus NIAH-26, which endogenously produces neither aflatoxins nor precursors in YES medium, aflatoxins B1 and G1 were produced. When dihydrodemethylsterigmatocystin (DHDMST) was fed to this mutant, aflatoxins B2 and G2 were produced. Results of the cell-free experiment with S-adenosyl-[methyl-3H]methionine showed that first the C-6-OH groups of DMST and DHDMST are methylated to produce sterigmatocystin and dihydrosterigmatocystin (O-methyltransferase I) and then the C-7-OH groups are methylated to produce O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin (DHOMST) (O-methyltransferase II). However, no methyltransferase activity was observed when either OMST, DHOMST, 5,6-dimethoxysterigmatocystin, 5-methoxysterigmatocystin, or sterigmatin was incubated with the cell extract. Treatment of the cell extract with N-ethylmaleimide inhibited O-methyltransferase I activity but not that of O-methyltransferase II. Furthermore, these O-methyltransferases were different in their protein molecules and were involved in both the reactions from DMST to OMST and DHDMST to DHOMST. The reactions described in this paper were not observed when the same mold had been cultured in YEP medium. Images PMID:2802602

  4. Comparison of expression of secondary metabolite biosynthesis cluster genes in Aspergillus flavus, A. parasiticus, and A. oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    More than 55 secondary metabolite biosynthesis gene clusters are predicted to be present in the Aspergillus flavus genome. In spite of this the biosynthesis of only a few metabolites, such as the aflatoxin, cyclopiazonic acid and aflatrem, has been correlated with a particular gene cluster. Using RN...

  5. ord1, an oxidoreductase gene responsible for conversion of O-methylsterigmatocystin to aflatoxin in Aspergillus flavus.

    PubMed Central

    Prieto, R; Woloshuk, C P

    1997-01-01

    Among the enzymatic steps in the aflatoxin biosynthetic pathway, the conversion of O-methylsterigmatocystin to aflatoxin has been proposed to be catalyzed by an oxidoreductase. Transformants of Aspergillus flavus 649WAF2 containing a 3.3-kb genomic DNA fragment and the aflatoxin biosynthesis regulatory gene aflR converted exogenously supplied O-methylsterigmatocystin to aflatoxin B1. A gene, ord1, corresponding to a transcript of about 2 kb was identified within the 3.3-kb DNA fragment. The promoter region presented a putative AFLR binding site and a TATA sequence. The nucleotide sequence of the gene revealed an open reading frame encoding a protein of 528 amino acids with a deduced molecular mass of 60.2 kDa. The gene contained six introns and seven exons. Heterologous expression of the ord1 open reading frame under the transcriptional control of the Saccharomyces cerevisiae galactose-inducible gal1 promoter results in the ability to convert O-methylsterigmatocystin to aflatoxin B1. The data indicate that ord1 is sufficient to accomplish the last step of the aflatoxin biosynthetic pathway. A search of various databases for similarity indicated that ord1 encodes a cytochrome P-450-type monooxygenase, and the gene has been assigned to a new P-450 gene family named CYP64. PMID:9143099

  6. Beyond aflatoxin: four distinct expression patterns and functional roles associated with Aspergillus flavus secondary metabolism gene clusters

    PubMed Central

    Georgianna, D. Ryan; Fedorova, Natalie D.; Burroughs, James L.; Dolezal, Andrea L.; Bok, J.; Horowitz-Brown, S.; Woloshuk, Charles P.; Yu, Jiujiang; Keller, Nancy P.; Payne, Gary A.

    2014-01-01

    SUMMARY Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis predicts that these species have the capacity to synthesize many more compounds. It has been possible to infer the presence of 55 gene clusters associated with secondary metabolism in A. flavus, however, only three metabolic pathways - aflatoxin, cyclopiazonic acid (CPA), and aflatrem - have been assigned to these clusters. To gain insight into the regulation of, and infer ecological significance for the 55 secondary metabolite gene clusters predicted in A. flavus, we examined their expression over 28 diverse conditions. Variables included culture media and temperature, fungal development, colonization of developing maize seeds, and misexpression of laeA, a global regulator of secondary metabolism. Hierarchical clustering analysis of expression profiles allowed us to categorize the gene clusters into four distinct clades. Gene clusters for the production of aflatoxins, CPA, and seven other unknown compound(s) were identified as belonging to one clade. To further explore the relationships found by gene expression analysis, aflatoxin and CPA production were quantified under five different cell culture environments known to be conducive or non-conducive for aflatoxin biosynthesis and during colonization of developing maize seeds. Results from these studies showed that secondary metabolism gene clusters have distinctive gene expression profiles. Aflatoxin and CPA were found to have unique regulation but are similar enough that they would be expected to co-occur in substrates colonized with A. flavus. PMID:20447271

  7. Aflatoxin

    MedlinePlus

    ... found in the following foods: Peanuts and peanut butter Tree nuts such as pecans Corn Wheat Oil ... foods that may contain aflatoxin. Peanuts and peanut butter are some of the most rigorously tested products ...

  8. Global Phosphoproteomic Analysis Reveals the Involvement of Phosphorylation in Aflatoxins Biosynthesis in the Pathogenic Fungus Aspergillus flavus

    PubMed Central

    Ren, Silin; Yang, Mingkun; Li, Yu; Zhang, Feng; Chen, Zhuo; Zhang, Jia; Yang, Guang; Yue, Yuewei; Li, Siting; Ge, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A. flavus. A total of 598 high-confidence phosphorylation sites were identified in 283 phosphoproteins. The identified phosphoproteins were involved in various biological processes, including signal transduction and aflatoxins biosynthesis. Five identified phosphoproteins associated with MAPK signal transduction and aflatoxins biosynthesis were validated by immunoblotting using phospho-specific antibodies. Further functional studies revealed that phosphorylation of the MAP kinase kinase kinase Ste11 affected aflatoxins biosynthesis in A. flavus. Our data represent the results of the first global survey of protein phosphorylation in A. flavus and reveal previously unappreciated roles for phosphorylation in the regulation of aflatoxins production. The generated dataset can serve as an important resource for the functional analysis of protein phosphorylation in A. flavus and facilitate the elucidation of phosphorylated signaling networks in this pathogen. PMID:27667718

  9. Targeting mycotoxin biosynthesis pathway genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chemical detoxification and physical destruction of aflatoxins in foods and feed commodities is mostly unattainable in a way that preserves the edibility of the food. Therefore, preventing mycotoxins in general and aflatoxins in particular from entering the food chain is a better approach. This requ...

  10. Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis

    SciTech Connect

    Skory, C.D.; Horng, J.S.; Pestka, J.J.; Linz, J.E. )

    1990-11-01

    The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per {mu}g of DNA was developed for A. parasiticus by using homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert ({sup 14}C)OMP to ({sup 14}C)UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert ({sup 14}C)OMP to ({sup 14}C)UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.

  11. Sulforaphane, a cancer chemopreventive agent, induces pathways associated with membrane biosynthesis in response to tissue damage by aflatoxin B{sub 1}

    SciTech Connect

    Techapiesancharoenkij, Nirachara; Fiala, Jeannette L.A.; Navasumrit, Panida; Croy, Robert G.; Wogan, Gerald N.; Groopman, John D.; Ruchirawat, Mathuros; Essigmann, John M.

    2015-01-01

    Aflatoxin B{sub 1} (AFB{sub 1}) is one of the major risk factors for liver cancer globally. A recent study showed that sulforaphane (SF), a potent inducer of phase II enzymes that occurs naturally in widely consumed vegetables, effectively induces hepatic glutathione S-transferases (GSTs) and reduces levels of hepatic AFB{sub 1}-DNA adducts in AFB{sub 1}-exposed Sprague Dawley rats. The present study characterized the effects of SF pre-treatment on global gene expression in the livers of similarly treated male rats. Combined treatment with AFB{sub 1} and SF caused reprogramming of a network of genes involved in signal transduction and transcription. Changes in gene regulation were observable 4 h after AFB{sub 1} administration in SF-pretreated animals and may reflect regeneration of cells in the wake of AFB{sub 1}-induced hepatotoxicity. At 24 h after AFB{sub 1} administration, significant induction of genes that play roles in cellular lipid metabolism and acetyl-CoA biosynthesis was detected in SF-pretreated AFB{sub 1}-dosed rats. Induction of this group of genes may indicate a metabolic shift toward glycolysis and fatty acid synthesis to generate and maintain pools of intermediate molecules required for tissue repair, cell growth and compensatory hepatic cell proliferation. Collectively, gene expression data from this study provide insights into molecular mechanisms underlying the protective effects of SF against AFB{sub 1} hepatotoxicity and hepatocarcinogenicity, in addition to the chemopreventive activity of this compound as a GST inducer. - Highlights: • This study revealed sulforaphane (SF)-deregulated gene sets in aflatoxin B{sub 1} (AFB{sub 1})-treated rat livers. • SF redirects biochemical networks toward lipid biosynthesis in AFB{sub 1}-dosed rats. • SF enhanced gene sets that would be expected to favor cell repair and regeneration.

  12. Understanding Nonaflatoxigenicity of Aspergillus sojae: A Windfall of Aflatoxin Biosynthesis Research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus section Flavi includes aflatoxin-producing and nonproducing fungi. A. sojae is unable to produce aflatoxins and is generally recognized as safe for food fermentation. However, because of its taxonomical relatedness to aflatoxin-producing A. parasiticus and A. flavus, it is necessary to...

  13. Genome-Wide Transcriptome Analysis of Cotton (Gossypium hirsutum L.) Identifies Candidate Gene Signatures in Response to Aflatoxin Producing Fungus Aspergillus flavus.

    PubMed

    Bedre, Renesh; Rajasekaran, Kanniah; Mangu, Venkata Ramanarao; Sanchez Timm, Luis Eduardo; Bhatnagar, Deepak; Baisakh, Niranjan

    2015-01-01

    Aflatoxins are toxic and potent carcinogenic metabolites produced from the fungi Aspergillus flavus and A. parasiticus. Aflatoxins can contaminate cottonseed under conducive preharvest and postharvest conditions. United States federal regulations restrict the use of aflatoxin contaminated cottonseed at >20 ppb for animal feed. Several strategies have been proposed for controlling aflatoxin contamination, and much success has been achieved by the application of an atoxigenic strain of A. flavus in cotton, peanut and maize fields. Development of cultivars resistant to aflatoxin through overexpression of resistance associated genes and/or knocking down aflatoxin biosynthesis of A. flavus will be an effective strategy for controlling aflatoxin contamination in cotton. In this study, genome-wide transcriptome profiling was performed to identify differentially expressed genes in response to infection with both toxigenic and atoxigenic strains of A. flavus on cotton (Gossypium hirsutum L.) pericarp and seed. The genes involved in antifungal response, oxidative burst, transcription factors, defense signaling pathways and stress response were highly differentially expressed in pericarp and seed tissues in response to A. flavus infection. The cell-wall modifying genes and genes involved in the production of antimicrobial substances were more active in pericarp as compared to seed. The genes involved in auxin and cytokinin signaling were also induced. Most of the genes involved in defense response in cotton were highly induced in pericarp than in seed. The global gene expression analysis in response to fungal invasion in cotton will serve as a source for identifying biomarkers for breeding, potential candidate genes for transgenic manipulation, and will help in understanding complex plant-fungal interaction for future downstream research.

  14. Production of M-/GM-group aflatoxins catalyzed by the OrdA enzyme in aflatoxin biosynthesis.

    PubMed

    Yabe, Kimiko; Chihaya, Naomi; Hatabayashi, Hidemi; Kito, Masako; Hoshino, Sachiko; Zeng, Hongmei; Cai, Jingjing; Nakajima, Hiromitsu

    2012-09-01

    Aspergillus parasiticus produces the minor aflatoxins M(1) (AFM(1)), M(2) (AFM(2)), GM(1) (AFGM(1)), and GM(2) (AFGM(2)), as well as the major aflatoxins B(1) (AFB(1)), B(2) (AFB(2)), G(1) (AFG(1)), and G(2) (AFG(2)). Feeding of A. parasiticus with aspertoxin (12c-hydroxyOMST) caused AFM(1) and AFGM(1), and cell-free experiments using the microsomal fraction of A. parasiticus and aspertoxin caused production of AFM(1), indicating that aspertoxin is a precursor of AFM(1) and AFGM(1). Feeding of the same fungus with O-methylsterigmatocystin (OMST) caused AFM(1) and AFGM(1) together with AFB(1) and AFG(1); feeding with dihydroOMST (DHOMST) caused AFM(2) and AFGM(2) together with AFB(2) and AFG(2). Incubation of either the microsomal fraction or OrdA enzyme-expressing yeast with OMST caused production of aspertoxin together with AFM(1) and AFB(1). These results demonstrated that the OrdA enzyme catalyzes both 12c-hydroxylation reaction from OMST to aspertoxin and the successive reaction from aspertoxin to AFM(1). In contrast, feeding of the fungus with AFB(1) did not produce any AFM(1), demonstrating that M-/GM-aflatoxins are not produced from B-/G-aflatoxins. Furthermore, AFM(1) together with AFB(1) and AFG(1) was also produced from 11-hydroxyOMST (HOMST) in feeding experiment of A. parasiticus, whereas no aflatoxins were produced when used the ordA deletion mutant. These results demonstrated that OrdA enzyme can also catalyze 12c-hydroxylation of HOMST to produce 11-hydroxyaspertoxin, which serves as a precursor for the production of AFM(1) and AFGM(1). The same pathway may work for the production of AFM(2) and AFGM(2) from DHOMST and dihydroHOMST through the formation of dihydroaspertoxin and dihydro-11-hydroxyaspertoxin, respectively.

  15. Dillapiol and Apiol as specific inhibitors of the biosynthesis of aflatoxin G1 in Aspergillus parasiticus.

    PubMed

    Razzaghi-Abyaneh, Mehdi; Yoshinari, Tomoya; Shams-Ghahfarokhi, Masoomeh; Rezaee, Mohammad-Bagher; Nagasawa, Hiromichi; Sakuda, Shohei

    2007-09-01

    Dillapiol was isolated from the essential oil of dill as a specific inhibitor of aflatoxin G1 production. It inhibited aflatoxin G1 production by Aspergillus parasiticus with an IC50 value of 0.15 microM without inhibiting aflatoxin B1 production or fungal growth. Apiol and myristicin, congeners of dillapiol, showed similar activity with IC50 values of 0.24 and 3.5 microM, respectively.

  16. PCR detection of aflatoxin producing fungi and its limitations.

    PubMed

    Levin, Robert E

    2012-05-01

    Unlike bacterial toxins that are primarily peptides and are therefore encoded by a single gene, fungal toxins such as the aflatoxins are multi-ring structures and therefore require a sequence of structural genes for their biological synthesis. There is therefore no specific PCR for any one of the four biologically produced aflatoxins. Unfortunately, the structural genes presently in use for PCR detection of aflatoxin producing fungi are also involved in the synthesis of other fungal toxins such as sterigmatocystin by Aspergillus versicolor and Aspergillus nidulans and therefore lack absolute specificity for aflatoxin producing fungi (Table 1). In addition, the genomic presence of several structural genes involved in aflatoxin biosynthesis does not guarantee the production of aflatoxin by all isolates of Aspergillus flavus and Aspergillus parasiticus. The most widely used DNA target regions for discriminating Aspergillus species are those of the rDNA complex, mainly the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) and the variable regions in the 5'-end of the 28S rRNA gene. Since these sequence regions are unrelated to the structural genes involved in aflatoxin biosynthesis there successful amplification can be used for species identification but do not confirm aflatoxin production. This review therefore presents the various approaches and limitations in the use of the PCR in attempting to detect aflatoxin producing fungi.

  17. The potential role of oxidative stress in Aspergillus flavus survivability and aflatoxin biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aflatoxin contamination of food and feed occurs due to growth of Aspergillus flavus. This poses a serious health risk because of aflatoxin’s toxic and carcinogenic properties which negatively impact human and livestock health. Colonization and subsequent aflatoxin production by A. flavus is typicall...

  18. Regulation of the aflatoxin-like toxin dothistromin by AflJ

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biosynthesis by Aspergillus parasiticus of aflatoxin, one of the most potent known naturally occurring carcinogens, requires the activity of two regulatory proteins, AflR and AflJ, which are encoded by divergently transcribed genes within the aflatoxin gene cluster. Although the Zn2Cys6 transcriptio...

  19. Characterization of Actinobacillus pleuropneumoniae riboflavin biosynthesis genes.

    PubMed Central

    Fuller, T E; Mulks, M H

    1995-01-01

    In this paper, we report the identification, cloning, and complete nucleotide sequence of four genes from Actinobacillus pleuropneumoniae that are involved in riboflavin biosynthesis. The cloned genes can specify production of large amounts of riboflavin in Escherichia coli, can complement several defined genetic mutations in riboflavin biosynthesis in E. coli, and are homologous to riboflavin biosynthetic genes from E. coli, Haemophilus influenzae, and Bacillus subtilis. The genes have been designated A. pleuropneumoniae ribGBAH because of their similarity in both sequence and arrangement to the B. subtilis ribGBAH operon. PMID:8522537

  20. Cyclopiazonic acid biosynthesis gene cluster gene cpaM is required for speradine A biosynthesis.

    PubMed

    Tokuoka, Masafumi; Kikuchi, Tomoki; Shinohara, Yasutomo; Koyama, Akifumi; Iio, Shin-Ichiro; Kubota, Takaaki; Kobayashi, Jun'ichi; Koyama, Yasuji; Totsuka, Akira; Shindo, Hitoshi; Sato, Kazuo

    2015-01-01

    Speradine A is a derivative of cyclopiazonic acid (CPA) found in culture of an Aspergillus tamarii isolate. Heterologous expression of a predicted methyltransferase gene, cpaM, in the cpa biosynthesis gene cluster of A. tamarii resulted in the speradine A production in a 2-oxoCPA producing A. oryzae strain, indicating cpaM is involved in the speradine A biosynthesis.

  1. Cinnamaldehyde inhibits fungal growth and aflatoxin B1 biosynthesis by modulating the oxidative stress response of Aspergillus flavus.

    PubMed

    Sun, Qi; Shang, Bo; Wang, Ling; Lu, Zhisong; Liu, Yang

    2016-02-01

    Cinnamaldehyde (CIN) is a promising natural preservative and generally recognized as safe for commodities as well as consumers. In this work, the antifungal effects of CIN on Aspergillus flavus were evaluated both in solid and in liquid culture conditions. Our results indicated that CIN effectively inhibited radial growth, spore production, mycelium formation, and aflatoxin B1 biosynthesis by A. flavus in a dose-dependent manner. At the concentration of 104 mg L(-1), CIN exposure was able to completely inhibit fungal growth as well as aflatoxin B1 production. Furthermore, the inhibitory activities of CIN were closely connected with the treatment period and the tested fungal species. Compared with the control strains, CIN dose dependently changed the morphology and ultrastructure of mycelium in different degree. Especially, the reduction of hydrogen peroxide was considered to follow the destruction of mitochondrial. Meanwhile, CIN significantly cut the levels of lipid peroxidation and reduced glutathione. The activity of total superoxide dismutase was significantly inhibited after CIN treatment at the end of incubation, whereas the activities of catalase and glutathione peroxidase were opposite. These results indicated that the inhibitory effect of CIN could attribute to oxidative stress alleviation possibly induced by modifications of cellular structure as well as redox status.

  2. Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

    PubMed Central

    Buchanan, R L; Lewis, D F

    1984-01-01

    Catabolism of carbohydrates has been implicated in the regulation of aflatoxin synthesis. To characterize this effect further, the activities of various enzymes associated with glucose catabolism were determined in Aspergillus parasiticus organisms that were initially cultured in peptone-mineral salts medium and then transferred to glucose-mineral salts and peptone-mineral salts media. After an initial increase in activity, the levels of glucose 6-phosphate dehydrogenase, mannitol dehydrogenase, and malate dehydrogenase were lowered in the presence of glucose. Phosphofructokinase activity was greater in the peptone-grown mycelium, but fructose diphosphatase was largely unaffected by carbon source. Likewise, carbon source had relatively little effect on the activities of pyruvate kinase, malic enzyme, isocitrate-NADP dehydrogenase, and isocitrate-NAD dehydrogenase. The results suggest that glucose may, in part, regulate aflatoxin synthesis via a carbon catabolite repression of NADPH-generating and tricarboxylic acid cycle enzymes. PMID:6091545

  3. Fragmentation of an aflatoxin-like gene cluster in a forest pathogen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Secondary metabolic pathway genes are typically clustered in fungi. An exception to this paradigm is seen for genes required for the production of dothistromin, an aflatoxin-like virulence factor produced by the pine needle pathogen Dothistroma septosporum. In contrast to the tight clustering of gen...

  4. Network analysis of maize RNA transport pathway genes associated with maize resistance to aflatoxin accumulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus is a pathogenic fungus producing alfatoxins that cause significant economic losses in maize production. This study analyzes the differences in expression levels of maize genes in response to A. flavus infection and aflatoxin accumulation. Identification of defense related genes an...

  5. The inhibitory effect of Bacillus megaterium on aflatoxin and cyclopiazonic acid biosynthetic pathway gene expression in Aspergillus flavus.

    PubMed

    Kong, Qing; Chi, Chen; Yu, Jiujiang; Shan, Shihua; Li, Qiyu; Li, Qianting; Guan, Bin; Nierman, William C; Bennett, Joan W

    2014-06-01

    Aspergillus flavus is one of the major moulds that colonize peanut in the field and during storage. The impact to human and animal health, and to the economy in agriculture and commerce, is significant since this mold produces the most potent known natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect in inhibiting aflatoxin formation in A. flavus through down-regulating aflatoxin pathway gene expression as demonstrated by gene chip analysis. Aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98 %) inhibited by co-cultivation with B. megaterium. Growth was also reduced. Using expression studies, we identified the fungal genes down-regulated by co-cultivation with B. megaterium across the entire fungal genome and specifically within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX). Modulating the expression of these genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was significantly down-regulated during co-cultivation. We present a model showing a hypothesis of the regulatory mechanism of aflatoxin production suppression by AflS and AflR through B. megaterium co-cultivation.

  6. Blakeslea trispora Genes for Carotene Biosynthesis

    PubMed Central

    Rodríguez-Sáiz, M.; Paz, B.; de la Fuente, J. L.; López-Nieto, M. J.; Cabri, W.; Barredo, J. L.

    2004-01-01

    We cloned the carB and carRA genes involved in β-carotene biosynthesis from overproducing and wild-type strains of Blakeslea trispora. The carB gene has a length of 1,955 bp, including two introns of 141 and 68 bp, and encodes a protein of 66.4 kDa with phytoene dehydrogenase activity. The carRA gene contains 1,894 bp, with a single intron of 70 bp, and encodes a protein of 69.6 kDa with separate domains for lycopene cyclase and phytoene synthase. The estimated transcript sizes for carB and carRA were 1.8 and 1.9 kb, respectively. CarB from the β-carotene-overproducing strain B. trispora F-744 had an S528R mutation and a TAG instead of a TAA stop codon. The overproducing strain also had a P143S mutation in CarRA. Both B. trispora genes could complement mutations in orthologous genes in Mucor circinelloides and could be used to construct transformed strains of M. circinelloides that produced higher levels of β-carotene than did the nontransformed parent. The results show that these genes are conserved across the zygomycetes and that the B. trispora carB and carRA genes are functional and potentially useable to increase carotenoid production. PMID:15345447

  7. A Biotin Biosynthesis Gene Restricted to Helicobacter.

    PubMed

    Bi, Hongkai; Zhu, Lei; Jia, Jia; Cronan, John E

    2016-02-12

    In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was demonstrated using a reconstituted in vitro desthiobiotin synthesis system. BioV homologues seem the sole pimeloyl-ACP methyl ester esterase present in the Helicobacter species and their occurrence only in H. pylori and close relatives provide a target for development of drugs to specifically treat Helicobacter infections.

  8. A Biotin Biosynthesis Gene Restricted to Helicobacter

    PubMed Central

    Bi, Hongkai; Zhu, Lei; Jia, Jia; Cronan, John E.

    2016-01-01

    In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was demonstrated using a reconstituted in vitro desthiobiotin synthesis system. BioV homologues seem the sole pimeloyl-ACP methyl ester esterase present in the Helicobacter species and their occurrence only in H. pylori and close relatives provide a target for development of drugs to specifically treat Helicobacter infections. PMID:26868423

  9. Gene inactivation in Lactococcus lactis: histidine biosynthesis.

    PubMed Central

    Delorme, C; Godon, J J; Ehrlich, S D; Renault, P

    1993-01-01

    Lactococcus lactis strains from dairy and nondairy sources were tested for the ability to grow in the absence of histidine. Among 60 dairy strains tested, 56 required histidine, whereas only 1 of 11 nondairy strains had this requirement. Moreover, 10 of the 56 auxotrophic strains were able to grow in the presence of histidinol (Hol+), the immediate histidine precursor. This indicates that adaptation to milk often results in histidine auxotrophy. The histidine operon was detected by Southern hybridization in eight dairy auxotrophic strains tested. A large part of the histidine operon (8 kb, containing seven histidine biosynthetic genes and three unrelated open reading frames [ORFs]) was cloned from an auxotroph, which had an inactive hisD gene, as judged by its inability to grow on histidinol. Complementation analysis of three genes, hisA, hisB, and hisG, in Escherichia coli showed that they also were inactive. Sequence analysis of the cloned histidine region, which revealed 98.6% overall homology with that of the previously analyzed prototrophic strain, showed the presence of frameshift mutations in three his genes, hisC, hisG, and hisH, and two genes unrelated to histidine biosynthesis, ORF3 and ORF6. In addition, several mutations were detected in the promoter region of the operon. Northern (RNA) hybridization analysis showed a much lower amount of the his transcript in the auxotrophic strain than in the prototrophic strain. The mutations detected account for the histidine auxotrophy of the analyzed strain. Certain other dairy auxotrophic strains carry a lower number of mutations, since they were able to revert either to a Hol+ phenotype or to histidine prototrophy. Images PMID:7687248

  10. Microbe-Mediated Control of Mycotoxigenic Grain Fungi in Stored Rice with Focus on Aflatoxin Biodegradation and Biosynthesis Inhibition

    PubMed Central

    Mannaa, Mohamed

    2016-01-01

    Rice contaminated with fungal species during storage is not only of poor quality and low economic value, but may also have harmful effects on human and animal health. The predominant fungal species isolated from rice grains during storage belong to the genera Aspergillus and Penicillium. Some of these fungal species produce mycotoxins; they are responsible for adverse health effects in humans and animals, particularly Aspergillus flavus, which produces the extremely carcinogenic aflatoxins. Not surprisingly, there have been numerous attempts to devise safety procedure for the control of such harmful fungi and production of mycotoxins, including aflatoxins. This review provides information about fungal and mycotoxin contamination of stored rice grains, and microbe-based (biological) strategies to control grain fungi and mycotoxins. The latter will include information regarding attempts undertaken for mycotoxin (especially aflatoxin) bio-detoxification and microbial interference with the aflatoxin-biosynthetic pathway in the toxin-producing fungi. PMID:27433116

  11. Effects of Zinc Chelators on Aflatoxin Production in Aspergillus parasiticus

    PubMed Central

    Wee, Josephine; Day, Devin M.; Linz, John E.

    2016-01-01

    Zinc concentrations strongly influence aflatoxin accumulation in laboratory media and in food and feed crops. The presence of zinc stimulates aflatoxin production, and the absence of zinc impedes toxin production. Initial studies that suggested a link between zinc and aflatoxin biosynthesis were presented in the 1970s. In the present study, we utilized two zinc chelators, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and 2,3-dimercapto-1-propanesulfonic acid (DMPS) to explore the effect of zinc limitation on aflatoxin synthesis in Aspergillus parasiticus. TPEN but not DMPS decreased aflatoxin biosynthesis up to six-fold depending on whether A. parasiticus was grown on rich or minimal medium. Although we observed significant inhibition of aflatoxin production by TPEN, no detectable changes were observed in expression levels of the aflatoxin pathway gene ver-1 and the zinc binuclear cluster transcription factor, AflR. Treatment of growing A. parasiticus solid culture with a fluorescent zinc probe demonstrated an increase in intracellular zinc levels assessed by increases in fluorescent intensity of cultures treated with TPEN compared to controls. These data suggest that TPEN binds to cytoplasmic zinc therefore limiting fungal access to zinc. To investigate the efficacy of TPEN on food and feed crops, we found that TPEN effectively decreases aflatoxin accumulation on peanut medium but not in a sunflower seeds-derived medium. From an application perspective, these data provide the basis for biological differences that exist in the efficacy of different zinc chelators in various food and feed crops frequently contaminated by aflatoxin. PMID:27271668

  12. Roles of lignin biosynthesis and regulatory genes in plant development

    PubMed Central

    Yoon, Jinmi; Choi, Heebak

    2015-01-01

    Abstract Lignin is an important factor affecting agricultural traits, biofuel production, and the pulping industry. Most lignin biosynthesis genes and their regulatory genes are expressed mainly in the vascular bundles of stems and leaves, preferentially in tissues undergoing lignification. Other genes are poorly expressed during normal stages of development, but are strongly induced by abiotic or biotic stresses. Some are expressed in non‐lignifying tissues such as the shoot apical meristem. Alterations in lignin levels affect plant development. Suppression of lignin biosynthesis genes causes abnormal phenotypes such as collapsed xylem, bending stems, and growth retardation. The loss of expression by genes that function early in the lignin biosynthesis pathway results in more severe developmental phenotypes when compared with plants that have mutations in later genes. Defective lignin deposition is also associated with phenotypes of seed shattering or brittle culm. MYB and NAC transcriptional factors function as switches, and some homeobox proteins negatively control lignin biosynthesis genes. Ectopic deposition caused by overexpression of lignin biosynthesis genes or master switch genes induces curly leaf formation and dwarfism. PMID:26297385

  13. Roles of lignin biosynthesis and regulatory genes in plant development.

    PubMed

    Yoon, Jinmi; Choi, Heebak; An, Gynheung

    2015-11-01

    Lignin is an important factor affecting agricultural traits, biofuel production, and the pulping industry. Most lignin biosynthesis genes and their regulatory genes are expressed mainly in the vascular bundles of stems and leaves, preferentially in tissues undergoing lignification. Other genes are poorly expressed during normal stages of development, but are strongly induced by abiotic or biotic stresses. Some are expressed in non-lignifying tissues such as the shoot apical meristem. Alterations in lignin levels affect plant development. Suppression of lignin biosynthesis genes causes abnormal phenotypes such as collapsed xylem, bending stems, and growth retardation. The loss of expression by genes that function early in the lignin biosynthesis pathway results in more severe developmental phenotypes when compared with plants that have mutations in later genes. Defective lignin deposition is also associated with phenotypes of seed shattering or brittle culm. MYB and NAC transcriptional factors function as switches, and some homeobox proteins negatively control lignin biosynthesis genes. Ectopic deposition caused by overexpression of lignin biosynthesis genes or master switch genes induces curly leaf formation and dwarfism.

  14. Sesterterpene ophiobolin biosynthesis involving multiple gene clusters in Aspergillus ustus

    PubMed Central

    Chai, Hangzhen; Yin, Ru; Liu, Yongfeng; Meng, Huiying; Zhou, Xianqiang; Zhou, Guolin; Bi, Xupeng; Yang, Xue; Zhu, Tonghan; Zhu, Weiming; Deng, Zixin; Hong, Kui

    2016-01-01

    Terpenoids are the most diverse and abundant natural products among which sesterterpenes account for less than 2%, with very few reports on their biosynthesis. Ophiobolins are tricyclic 5–8–5 ring sesterterpenes with potential pharmaceutical application. Aspergillus ustus 094102 from mangrove rizhosphere produces ophiobolin and other terpenes. We obtained five gene cluster knockout mutants, with altered ophiobolin yield using genome sequencing and in silico analysis, combined with in vivo genetic manipulation. Involvement of the five gene clusters in ophiobolin synthesis was confirmed by investigation of the five key terpene synthesis relevant enzymes in each gene cluster, either by gene deletion and complementation or in vitro verification of protein function. The results demonstrate that ophiobolin skeleton biosynthesis involves five gene clusters, which are responsible for C15, C20, C25, and C30 terpenoid biosynthesis. PMID:27273151

  15. Suppression of spore germination and aflatoxin biosynthesis in Aspergillus parasiticus during and after exposure to high levels of phosphine.

    PubMed

    Antonacci, L; Salvat, A E; Faifer, G C; Godoy, H M

    1999-01-01

    Agar cultures of toxigenic Aspergillus parasiticus NRRL 2999 were exposed to phosphine (PH3), in levels ranging from 0 to 2000 ppm (vol/vol). It was found that with PH3 concentrations of 400 ppm or higher the growth of the fungus was totally arrested. When PH3 was vented and the agar plates were exposed to open air, 100% of the initial CFU developed into fully grown colonies after PH3 levels below 300 ppm, but at higher PH3 concentrations only 50% of the colonies developed. The same strain of A. parasiticus was inoculated into high moisture corn under conditions highly favorable for aflatoxin production, and it was exposed to a range of PH3 levels. After exposure to 500 ppm PH3, both fungal growth and aflatoxin synthesis resumed shortly after elimination of the toxic gas, but after exposure to PH3 levels of 1000 ppm and higher, the physical appearance of the contaminated corn was remarkably changed, showing reduced mycelial growth and almost complete absence of green pigmentation. In addition, aflatoxin synthesis was totally absent for the remainder of the experiment (20 days). These results strongly suggest that exposure to PH3 levels of 1000 ppm or higher could bring about persistent metabolic changes in surviving Aspergillus organisms.

  16. Characterization of the chitinase gene family and the effect on A. flavus and aflatoxin resistance in maize.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize (Zea mays L.) is a crop of global importance, but is prone to contamination by aflatoxins produced by fungi in the genus Aspergillus. The development of resistant germplasm and the identification of genes contributing to resistance would aid in the reduction of the problem with a minimal need ...

  17. Upstream regulation of mycotoxin biosynthesis.

    PubMed

    Alkhayyat, Fahad; Yu, Jae-Hyuk

    2014-01-01

    Mycotoxins are natural contaminants of food and feed products, posing a substantial health risk to humans and animals throughout the world. A plethora of filamentous fungi has been identified as mycotoxin producers and most of these fungal species belong to the genera Aspergillus, Fusarium, and Penicillium. A number of studies have been conducted to better understand the molecular mechanisms of biosynthesis of key mycotoxins and the regulatory cascades controlling toxigenesis. In many cases, the mycotoxin biosynthetic genes are clustered and regulated by one or more pathway-specific transcription factor(s). In addition, as biosynthesis of many secondary metabolites is coordinated with fungal growth and development, there are a number of upstream regulators affecting biosynthesis of mycotoxins in fungi. This review presents a concise summary of the regulation of mycotoxin biosynthesis, focusing on the roles of the upstream regulatory elements governing biosynthesis of aflatoxin and sterigmatocystin in Aspergillus.

  18. Isolation of methyl syringate as a specific aflatoxin production inhibitor from the essential oil of Betula alba and aflatoxin production inhibitory activities of its related compounds.

    PubMed

    Jermnak, Usuma; Yoshinari, Tomoya; Sugiyama, Yasumasa; Tsuyuki, Rie; Nagasawa, Hiromichi; Sakuda, Shohei

    2012-02-15

    Methyl syringate was isolated from the essential oil of Betula alba as an aflatoxin production inhibitor. It inhibited aflatoxin production of Aspergillus parasiticus and Aspergillus flavus with IC(50) values of 0.9 and 0.8 mM, respectively, without significantly inhibiting fungal growth. Methyl syringate reduced mRNA levels of genes (aflR, pksA, and omtB) [corrected] encoding proteins required for aflatoxin biosynthesis. Methyl gallate, methyl 3,4,5-trimethoxybenzoate, and methyl 3-O-methylgallate inhibited both aflatoxin production and fungal growth of A. parasiticus and A. flavus. However, their acids and syringic acid did not inhibit aflatoxin production and growth of A. parasiticus significantly, although gallic acid inhibited aflatoxin production of A. flavus with selectivity. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of methyl syringate was much weaker than that of gallic acid. These results showed that methyl syringate has a unique inhibitory activity toward aflatoxin production with a different mode of action from that of gallic acid.

  19. The major volatile compound 2-phenylethanol from the biocontrol yeast, Pichia anomala, inhibits growth and expression of aflatoxin biosynthetic genes of Aspergillus flavus.

    PubMed

    Hua, Sui Sheng T; Beck, John J; Sarreal, Siov Bouy L; Gee, Wai

    2014-05-01

    Aspergillus flavus is a ubiquitous saprophyte that is able to produce the most potent natural carcinogenic compound known as aflatoxin B1 (AFB1). This toxin frequently contaminates crops including corn, cotton, peanuts, and tree nuts causing substantial economic loss worldwide. Consequently, more than 100 countries have strict regulations limiting AFB1 in foodstuffs and feedstuffs. Plants and microbes are able to produce volatile compounds that act as a defense mechanism against other organisms. Pichia anomala strain WRL-076 is a biocontrol yeast currently being tested to reduce AF contamination of tree nuts in California. We used the SPME-GC/MS analysis and identified the major volatile compound produced by this strain to be 2-phenylethanol (2-PE). It inhibited spore germination and AF production of A. flavus. Inhibition of AF formation by 2-PE was correlated with significant down regulation of clustering AF biosynthesis genes as evidenced by several to greater than 10,000-fold decrease in gene expression. In a time-course analysis we found that 2-PE also altered the expression patterns of chromatin modifying genes, MYST1, MYST2, MYST3, gcn5, hdaA and rpdA. The biocontrol capacity of P. anomala can be attributed to the production of 2-PE, which affects spore germination, growth, toxin production, and gene expression in A. flavus.

  20. Inhibitory effect of eugenol on aflatoxin B1 production in Aspergillus parasiticus by downregulating the expression of major genes in the toxin biosynthetic pathway.

    PubMed

    Jahanshiri, Zahra; Shams-Ghahfarokhi, Masoomeh; Allameh, Abdolamir; Razzaghi-Abyaneh, Mehdi

    2015-07-01

    Aflatoxin contamination of grains and agro-products is a serious food safety issue and a significant economic concern worldwide. In the present study, the effects of eugenol on Aspergillus parasiticus growth and aflatoxin production were studied in relation to the expression of some essential genes involved in aflatoxin biosynthetic pathway. The fungus was cultured in presence of serial two-fold concentrations of eugenol (15.62-500 μg mL(-1)) for 3 days at 28 °C. Mycelia dry weight was determined as an index of fungal growth, while aflatoxin production was assessed by high performance liquid chromatography. The expression of aflatoxin biosynthetic genes including ver-1, nor-1, pksA, omtA and aflR were evaluated by real-time PCR. Eugenol strongly inhibited A. parasiticus growth in the range of 19.16-95.83 % in a dose-dependent manner. Aflatoxin B1 production was also inhibited by the compound in the range of 15.07-98.0 %. The expressions of ver-1, nor-1, pksA, omtA and aflR genes were significantly suppressed by eugenol at concentrations of 62.5 and 125 μg mL(-1). These results indicate that eugenol may be considered as a good candidate to control toxigenic fungal growth and the subsequent contamination of food, feed and agricultural commodities by carcinogenic aflatoxins.

  1. Spiroethers of German chamomile inhibit production of aflatoxin G and trichothecene mycotoxin by inhibiting cytochrome P450 monooxygenases involved in their biosynthesis.

    PubMed

    Yoshinari, Tomoya; Yaguchi, Atsushi; Takahashi-Ando, Naoko; Kimura, Makoto; Takahashi, Haruo; Nakajima, Takashi; Sugita-Konishi, Yoshiko; Nagasawa, Hiromichi; Sakuda, Shohei

    2008-07-01

    The essential oil of German chamomile showed specific inhibition toward aflatoxin G(1) (AFG(1)) production, and (E)- and (Z)-spiroethers were isolated as the active compounds from the oil. The (E)- and (Z)-spiroethers inhibited AFG(1) production of Aspergillus parasiticus with inhibitory concentration 50% (IC(50)) values of 2.8 and 20.8 microM, respectively, without inhibiting fungal growth. Results of an O-methylsterigmatocystin (OMST) conversion study indicated that the spiroethers specifically inhibited the OMST to AFG(1) pathway. A cytochrome P450 monooxygenase, CYPA, is known as an essential enzyme for this pathway. Because CYPA has homology with TRI4, a key enzyme catalyzing early steps in the biosynthesis of trichothecenes, the inhibitory actions of the two spiroethers against TRI4 reactions and 3-acetyldeoxynivalenol (3-ADON) production were tested. (E)- and (Z)-spiroethers inhibited the enzymatic activity of TRI4 dose-dependently and interfered with 3-ADON production by Fusarium graminearum, with IC(50) values of 27.1 and 103 microM, respectively. Our results suggest that the spiroethers inhibited AFG(1) and 3-ADON production by inhibiting CYPA and TRI4, respectively.

  2. Effect of climate change on Aspergillus flavus and aflatoxin B1 production.

    PubMed

    Medina, Angel; Rodriguez, Alicia; Magan, Naresh

    2014-01-01

    This review considers the available information on the potential impact of key environmental factors and their interactions on the molecular ecology, growth and aflatoxin production by Aspergillus flavus in vitro and in maize grain. The recent studies which have been carried out to examine the impact of water activity × temperature on aflatoxin biosynthesis and phenotypic aflatoxin production are examined. These have shown that there is a direct relationship between the relative expression of key regulatory and structural genes under different environmental conditions which correlate directly with aflatoxin B1 production. A model has been developed to integrate the relative expression of 10 biosynthetic genes in the pathway, growth and aflatoxin B1 (AFB1) production which was validated under elevated temperature and water stress conditions. The effect of interacting conditions of aw × temperature × elevated CO2 (2 × and 3 × existing levels) are detailed for the first time. This suggests that while such interacting environmental conditions have little effect on growth they do have a significant impact on aflatoxin biosynthetic gene expression (structural aflD and regulatory aflR genes) and can significantly stimulate the production of AFB1. While the individual factors alone have an impact, it is the combined effect of these three abiotic factors which have an impact on mycotoxin production. This approach provides data which is necessary to help predict the real impacts of climate change on mycotoxigenic fungi.

  3. Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut.

    PubMed

    Power, Imana L; Dang, Phat M; Sobolev, Victor S; Orner, Valerie A; Powell, Joseph L; Lamb, Marshall C; Arias, Renee S

    2017-04-01

    Aflatoxin contamination is a major constraint in food production worldwide. In peanut (Arachis hypogaea L.), these toxic and carcinogenic aflatoxins are mainly produced by Aspergillus flavus Link and A. parasiticus Speare. The use of RNA interference (RNAi) is a promising method to reduce or prevent the accumulation of aflatoxin in peanut seed. In this study, we performed high-throughput sequencing of small RNA populations in a control line and in two transformed peanut lines that expressed an inverted repeat targeting five genes involved in the aflatoxin-biosynthesis pathway and that showed up to 100% less aflatoxin B1 than the controls. The objective was to determine the putative involvement of the small RNA populations in aflatoxin reduction. In total, 41 known microRNA (miRNA) families and many novel miRNAs were identified. Among those, 89 known and 10 novel miRNAs were differentially expressed in the transformed lines. We furthermore found two small interfering RNAs derived from the inverted repeat, and 39 sRNAs that mapped without mismatches to the genome of A. flavus and were present only in the transformed lines. This information will increase our understanding of the effectiveness of RNAi and enable the possible improvement of the RNAi technology for the control of aflatoxins.

  4. Sexuality generates diversity in the aflatoxin gene cluster: evidence on a global scale

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The worldwide costs associated with aflatoxin monitoring and crop losses are in the hundreds of millions of dollars. Aflatoxins also account for considerable health risks, even in countries where food contamination is regulated. Aspergillus flavus and A. parasiticus are the most common agents of af...

  5. Differential expression of genes during aflatoxin B1-induced hepatocarcinogenesis in tree shrews

    PubMed Central

    Li, Yuan; Wan, Da-Fang; Su, Jian-Jia; Cao, Ji; Ou, Chao; Qiu, Xiao-Kun; Ban, Ke-Chen; Yang, Chun; Qin, Liu-Liang; Luo, Dan; Yue, Hui-Fen; Zhang, Li-Sheng; Gu, Jian-Ren

    2004-01-01

    AIM: Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B1 (AFB1), to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism. METHODS: Tree shrews (Tupaia belangeri chinensis) were treated with or without AFB1 for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding non-cancerous liver tissues, 2 biopsied tissues at the early stage (30th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0th week) of the experiment, and another 1 mixed sample of two liver tissues from the untreated control animals biopsied at the 90th week of the experiment. The samples were then tested with the method of AtlasTM cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared. RESULTS: The profiles of differently expressed genes were quite different in different ways of comparison. At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up- or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as “important genes” because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC, genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC. CONCLUSION: A considerable number of genes could

  6. Next Generation Sequencing in Predicting Gene Function in Podophyllotoxin Biosynthesis*

    PubMed Central

    Marques, Joaquim V.; Kim, Kye-Won; Lee, Choonseok; Costa, Michael A.; May, Gregory D.; Crow, John A.; Davin, Laurence B.; Lewis, Norman G.

    2013-01-01

    Podophyllum species are sources of (−)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (−)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (−)-matairesinol into (−)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (−)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways. PMID:23161544

  7. Regulation of aflatoxin biosynthesis and branched-chain amino acids metabolism in Aspergillus flavus by 2-phenylethanol reveal biocontrol mechanism of Pichia anomala

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pichia anomala WRL-076 is a biocontrol yeast which has been shown to inhibit growth and aflatoxin production of A. flavus. Using the SPME-GC/MS analysis we identified that the volatile, 2-phenylethanol (2-PE) produced by this yeast and demonstrated that the compound inhibited aflatoxin production. W...

  8. Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

    PubMed Central

    Delorme, C; Ehrlich, S D; Renault, P

    1992-01-01

    The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases. PMID:1400209

  9. Novel Vibrio cholerae O139 genes involved in lipopolysaccharide biosynthesis.

    PubMed Central

    Stroeher, U H; Parasivam, G; Dredge, B K; Manning, P A

    1997-01-01

    The sequence of part of the rfb region of Vibrio cholerae serogroup O139 and the physical map of a 35-kb region of the O139 chromosome have been determined. The O139 rfb region presented contains a number of open reading frames which show similarities to other rfb and capsular biosynthesis genes found in members of the Enterobacteriaceae family and in V. cholerae O1. The cloned and sequenced region can complement the defects in O139 antigen biosynthesis in transposon insertions within the O139 rfb cluster. Linkage is demonstrated among IS1358 of V. cholerae O139, the rfb region, and the recently reported otnA and otnB genes (E. M. Bik, A. E. Bunschoten, R. D. Gouw, and F. R. Mooi, EMBO J. 14:209-216, 1995). In addition, the whole of this region has been linked to the rfaD gene. Furthermore, determination of the sequence flanking IS1358 has revealed homology to other rfb-like genes. The exact site of insertion with respect to rfaD is defined for the novel DNAs of both the Bengal and the Argentinian O139 isolates. PMID:9098074

  10. The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis.

    PubMed

    Dekkers, Katherine L; You, Bang-Jau; Gowda, Vivek S; Liao, Hui-Ling; Lee, Miin-Huey; Bau, Huey-Jiunn; Ueng, Peter P; Chung, Kuang-Ren

    2007-05-01

    Cercosporin, a photo-activated, non-host-selective phytotoxin produced by many species of the plant pathogenic fungus Cercospora, causes peroxidation of plant cell membranes by generating reactive oxygen species and is an important virulence determinant. Here we report a new gene, CTB3 that is involved in cercosporin biosynthesis in Cercospora nicotianae. CTB3 is adjacent to a previously identified CTB1 encoding a polyketide synthase which is also required for cercosporin production. CTB3 contains a putative O-methyltransferase domain in the N-terminus and a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase domain in the C-terminus. The N-terminal amino acid sequence also is similar to that of the transcription enhancer AFLS (formerly AFLJ) involved in aflatoxin biosynthesis. Expression of CTB3 was differentially regulated by light, medium, nitrogen and carbon sources and pH. Disruption of the N- or C-terminus of CTB3 yielded mutants that failed to accumulate the CTB3 transcript and cercosporin. The Deltactb3 disruptants produced a yellow pigment that is not toxic to tobacco suspension cells. Production of cercosporin in a Deltactb3 null mutant was fully restored when transformed with a functional CTB3 clone or when paired with a Deltactb1-null mutant (defective in polyketide synthase) by cross feeding of the biosynthetic intermediates. Pathogenicity assays using detached tobacco leaves revealed that the Deltactb3 disruptants drastically reduced lesion formation.

  11. Diversity of aflatoxin-producing fungi and their impact on food safety in sub-Saharan Africa.

    PubMed

    Probst, C; Bandyopadhyay, R; Cotty, P J

    2014-03-17

    Crops frequently contaminated by aflatoxins are important sources of revenue and daily nourishment in many portions of sub-Saharan Africa. In recent years, reports have associated aflatoxins with diminished human health and export opportunities in many African Nations. Aflatoxins are highly carcinogenic metabolites mainly produced by members of Aspergillus sect. Flavi. The current study examined aflatoxin-producing fungi associated with maize grain intended for human consumption in 18 sub-Saharan African countries. 4469 Aspergillus sect. Flavi isolates were obtained from 339 samples. The majority (75%) of isolates belonged to the L strain morphotype of A. flavus. Minor percentages were A. tamarii (6%), A. parasiticus (1%), and isolates with S strain morphology (3%). No A. bombycis or A. nomius isolates were detected. Phylogenetic analyses of partial sequences of the nitrate reductase gene (niaD, 1.3kb) and the aflatoxin pathway transcription factor gene (aflR, 1.7kb) were used to verify isolate assignments into species and lineages. Phylogenetics resolved S strain isolates producing only B aflatoxins into two lineages fully supported by sizes of deletions in the gene region spanning the aflatoxin biosynthesis genes cypA (aflU) and norB (aflF). One lineage was the A. flavus S strain with either 0.9 or 1.5kb deletions. The second lineage, recently described from Kenya, has a 2.2kb deletion. Taxa with S strain morphology differed in distribution with strain SBG limited to West Africa and both A. minisclerotigenes and the new lineage from Kenya in Central and East Africa. African A. flavus L strain isolates formed a single clade with L strain isolates from other continents. The sampled maize frequently tested positive for aflatoxins (65%), fumonisins (81%), and deoxynivalenol (40%) indicating the presence of fungi capable of producing the respective toxins. Percentage of samples exceeding US limits for total aflatoxins (regulatory limit), fumonisins (advisory limit

  12. Swainsonine Biosynthesis Genes in Diverse Symbiotic and Pathogenic Fungi.

    PubMed

    Cook, Daniel; Donzelli, Bruno G G; Creamer, Rebecca; Baucom, Deana L; Gardner, Dale R; Pan, Juan; Moore, Neil; Jaromczyk, Jerzy W; Schardl, Christopher L

    2017-04-04

    Swainsonine, a cytotoxic fungal alkaloid and a potential cancer therapy drug, is produced by the insect pathogen and plant symbiont, Metarhizium robertsii, the clover pathogen Slafractonia leguminicola, locoweed symbionts belonging to Alternaria sect. Undifilum, and a recently discovered morning glory symbiont belonging to order Chaetothyriales. Genome sequence analyses revealed that these fungi shared orthologous gene clusters, designated "SWN," which included a multifunctional swnK gene comprising predicted adenylylation and acyltransferase domains with their associated thiolation domains, a β-ketoacyl synthase domain, and two reductase domains. The role of swnK was demonstrated by inactivating it in M. robertsii through homologous gene replacement to give a ∆swnK mutant that produced no detectable swainsonine, then complementing the mutant with the wild-type gene to restore swainsonine biosynthesis. Other SWN cluster genes were predicted to encode two putative hydroxylases and two reductases, as expected to complete biosynthesis of swainsonine from the predicted SwnK product. SWN gene clusters were identified in six out of seven sequenced genomes of Metarhzium species, and in all 15 sequenced genomes of Arthrodermataceae, a family of fungi that cause athlete's foot and ringworm diseases in humans and other mammals. Representative isolates of all of these species were cultured, and all Metarhizium spp. with SWN clusters, as well as all but one of the Arthrodermataceae, produced swainsonine. These results suggested a new biosynthetic hypothesis for this alkaloid, extended the known taxonomic breadth of swainsonine producers to five orders of Ascomycota, and suggested that swainsonine has roles in mutualistic symbioses and diseases of plants and animals.

  13. Effect of the combined probiotics with aflatoxin B₁-degrading enzyme on aflatoxin detoxification, broiler production performance and hepatic enzyme gene expression.

    PubMed

    Zuo, Rui-yu; Chang, Juan; Yin, Qing-qiang; Wang, Ping; Yang, Yu-rong; Wang, Xiao; Wang, Guo-qiang; Zheng, Qiu-hong

    2013-09-01

    In order to degrade aflatoxin B₁ (AFB₁), AFB₁-degrading microbes (probiotics) such as Lactobacillus casei, Bacillus subtilis and Pichia anomala, and the AFB₁-degrading enzyme from Aspergillus oryzae were selected and combined to make feed additive. Seventy-five 43-day-old male Arbor Acres broilers were randomly divided into 5 groups, 15 broilers for each group. The broilers were given with 5 kinds of diets such as the basal diet, 400 μg/kg AFB₁ supplement without feed additive, and 200, 400, 800 μg/kg AFB₁ supplement with 0.15% feed additive. The feeding experimental period was 30 d, which was used to determine production performance of broilers. In addition, serum, liver and chest muscle were selected for measuring AFB₁ residues, gene expressions, microscopic and antioxidant analyses. The results showed that adding 0.15% feed additive in broiler diets could significantly relieve the negative effect of AFB₁ on chicken's production performance and nutrient metabolic rates (P<0.05). It could also improve AFB₁ metabolism, hepatic cell structure, antioxidant activity, and many hepatic enzyme gene expressions involved in oxidoreductase, apoptosis, cell growth, immune system and metabolic process (P<0.05). It could be concluded that the feed additive was able to degrade AFB₁ and improve animal production.

  14. Testing an aflatoxin B1 gene signature in rat archival tissues.

    PubMed

    Merrick, B Alex; Auerbach, Scott S; Stockton, Patricia S; Foley, Julie F; Malarkey, David E; Sills, Robert C; Irwin, Richard D; Tice, Raymond R

    2012-05-21

    Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced

  15. Genes and enzymes of ectoine biosynthesis in halotolerant methanotrophs.

    PubMed

    Reshetnikov, Alexander S; Khmelenina, Valentina N; Mustakhimov, Ildar I; Trotsenko, Yuri A

    2011-01-01

    Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) is a widely distributed compatible solute accumulated by halophilic and halotolerant microorganisms to prevent osmotic stress in highly saline environments. Ectoine as a highly water keeping compound stabilizing biomolecules and whole cells can be used in scientific work, cosmetics, and medicine. Detailed understanding of the organization/regulation of the ectoine biosynthetic pathway in various producers is an active area of research. Here we review current knowledge on some genetic and enzymatic aspects of ectoine biosynthesis in halophilic and halotolerant methanotrophs. By using PCR methodology, the genes coding for the specific enzymes of ectoine biosynthesis, diaminobutyric acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA), and ectoine synthase (EctC), were identified in several methanotrophic species. Organization of these genes in either ectABC or ectABC-ask operons, the latter additionally encoding aspartate kinase isozyme (Ask), correlated well with methanotroph halotolerance and intracellular ectoine level. A new gene, ectR1 encoding the MarR-like transcriptional regulatory protein EctR1, negatively controlling transcription of ectoine biosynthetic genes was found upstream of ectABC-ask operon in Methylomicrobium alcaliphilum 20Z. The ectR-like genes were also found in halotolerant methanol utilizers Methylophaga alcalica and Methylophaga thalassica as well as in several genomes of nonmethylotrophic species. The His(6)-tagged DABA acetyltransferases from Mm. alcaliphilum, M. alcalica, and M. thalassica were purified and the enzyme properties were found to correlate with the ecophysiologies of these bacteria. All these discoveries should be very helpful for better understanding the biosynthetic mechanism of this important natural compound, and for the targeted metabolic engineering of its producers.

  16. Alkane Biosynthesis Genes in Cyanobacteria and Their Transcriptional Organization

    PubMed Central

    Klähn, Stephan; Baumgartner, Desirée; Pfreundt, Ulrike; Voigt, Karsten; Schön, Verena; Steglich, Claudia; Hess, Wolfgang R.

    2014-01-01

    In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl–acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria. PMID

  17. Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods.

    PubMed

    Rodríguez, Alicia; Rodríguez, Mar; Luque, M Isabel; Martín, Alberto; Córdoba, Juan J

    2012-08-01

    Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products.

  18. A public platform for the verification of the phenotypic effect of candidate genes for resistance to aflatoxin accumulation and Aspergillus flavus infection in maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A public candidate gene testing pipeline for resistance to aflatoxin accumulation or Aspergillus flavus infection in maize is presented here. The pipeline consists of steps for identifying, testing, and verifying the association of any maize gene sequence with resistance under field conditions. Reso...

  19. Engineering of glucosinolate biosynthesis: candidate gene identification and validation.

    PubMed

    Møldrup, Morten E; Salomonsen, Bo; Halkier, Barbara A

    2012-01-01

    The diverse biological roles of glucosinolates as plant defense metabolites and anticancer compounds have spurred a strong interest in their biosynthetic pathways. Since the completion of the Arabidopsis genome, functional genomics approaches have enabled significant progress on the elucidation of glucosinolate biosynthesis, although in planta validation of candidate gene function often is hampered by time-consuming generation of knockout and overexpression lines in Arabidopsis. To better exploit the increasing amount of data available from genomic sequencing, microarray database and RNAseq, time-efficient methods for identification and validation of candidate genes are needed. This chapter covers the methodology we are using for gene discovery in glucosinolate engineering, namely, guilt-by-association-based in silico methods and fast proof-of-function screens by transient expression in Nicotiana benthamiana. Moreover, the lessons learned in the rapid, transient tobacco system are readily translated to our robust, versatile yeast expression platform, where additional genes critical for large-scale microbial production of glucosinolates can be identified. We anticipate that the methodology presented here will be beneficial to elucidate and engineer other plant biosynthetic pathways.

  20. The inhibitory effect of Bacillus megaterium on aflatoxin biosynthetic pathway gene expression in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus is one of the major fungal mold that colonize peanut in the field and during storage. The impacts to human and animal health and to economy in agriculture and commerce are significant since this mould produces the most potent natural toxins, aflatoxins, which are carcinogenic, mut...

  1. RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

    PubMed

    Merrick, B Alex; Phadke, Dhiral P; Auerbach, Scott S; Mav, Deepak; Stiegelmeyer, Suzy M; Shah, Ruchir R; Tice, Raymond R

    2013-01-01

    Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the

  2. Cloning and characterization of the polyether salinomycin biosynthesis gene cluster of Streptomyces albus XM211.

    PubMed

    Jiang, Chunyan; Wang, Hougen; Kang, Qianjin; Liu, Jing; Bai, Linquan

    2012-02-01

    Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity.

  3. [Gene cloning and bioinformatics analysis of new gene for chlorogenic acid biosynthesis of Lonicera hypoglauca].

    PubMed

    Yu, Shu-lin; Huang, Lu-qi; Yuan, Yuan; Qi, Lin-jie; Liu, Da-hui

    2015-03-01

    To obtain the key genes for chlorogenic acid biosynthesis of Lonicera hypoglauca, four new genes ware obtained from the our dataset of L. hypoglauca. And we also predicted the structure and function of LHPAL4, LHHCT1 , LHHCT2 and LHHCT3 proteins. The phylogenetic tree showed that LHPAL4 was closely related with LHPAL1, LHHCT1 was closely related with LHHCT3, LHHCT2 clustered into a single group. By Real-time PCR to detect the gene expressed level in different organs of L. hypoglauca, we found that the transcripted level of LHPAL4, LHHCT1 and LHHCT3 was the highest in defeat flowers, and the transcripted level of LHHCT2 was the highest in leaves. These result provided a basis to further analysis the mechanism of active ingredients in different organs, as well as the element for in vitro biosynthesis of active ingredients.

  4. Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom

    PubMed Central

    2009-01-01

    Background As a major component of plant cell wall, lignin plays important roles in mechanical support, water transport, and stress responses. As the main cause for the recalcitrance of plant cell wall, lignin modification has been a major task for bioenergy feedstock improvement. The study of the evolution and function of lignin biosynthesis genes thus has two-fold implications. First, the lignin biosynthesis pathway provides an excellent model to study the coordinative evolution of a biochemical pathway in plants. Second, understanding the function and evolution of lignin biosynthesis genes will guide us to develop better strategies for bioenergy feedstock improvement. Results We analyzed lignin biosynthesis genes from fourteen plant species and one symbiotic fungal species. Comprehensive comparative genome analysis was carried out to study the distribution, relatedness, and family expansion of the lignin biosynthesis genes across the plant kingdom. In addition, we also analyzed the comparative synteny map between rice and sorghum to study the evolution of lignin biosynthesis genes within the Poaceae family and the chromosome evolution between the two species. Comprehensive lignin biosynthesis gene expression analysis was performed in rice, poplar and Arabidopsis. The representative data from rice indicates that different fates of gene duplications exist for lignin biosynthesis genes. In addition, we also carried out the biomass composition analysis of nine Arabidopsis mutants with both MBMS analysis and traditional wet chemistry methods. The results were analyzed together with the genomics analysis. Conclusion The research revealed that, among the species analyzed, the complete lignin biosynthesis pathway first appeared in moss; the pathway is absent in green algae. The expansion of lignin biosynthesis gene families correlates with substrate diversity. In addition, we found that the expansion of the gene families mostly occurred after the divergence of monocots

  5. Two new aflatoxin producing species, and an overview of Aspergillus section Flavi

    PubMed Central

    Varga, J.; Frisvad, J.C.; Samson, R.A.

    2011-01-01

    Aspergillus subgenus Circumdati section Flavi includes species with usually biseriate conidial heads, in shades of yellow-green to brown, and dark sclerotia. Several species assigned to this section are either important mycotoxin producers including aflatoxins, cyclopiazonic acid, ochratoxins and kojic acid, or are used in oriental food fermentation processes and as hosts for heterologous gene expression. A polyphasic approach was applied using morphological characters, extrolite data and partial calmodulin, β-tubulin and ITS sequences to examine the evolutionary relationships within this section. The data indicate that Aspergillus section Flavi involves 22 species, which can be grouped into seven clades. Two new species, A. pseudocaelatus sp. nov. and A. pseudonomius sp. nov. have been discovered, and can be distinguished from other species in this section based on sequence data and extrolite profiles. Aspergillus pseudocaelatus is represented by a single isolate collected from Arachis burkartii leaf in Argentina, is closely related to the non-aflatoxin producing A. caelatus, and produces aflatoxins B & G, cyclopiazonic acid and kojic acid, while A. pseudonomius was isolated from insects and soil in the USA. This species is related to A. nomius, and produces aflatoxin B1 (but not G-type aflatoxins), chrysogine and kojic acid. In order to prove the aflatoxin producing abilities of the isolates, phylogenetic analysis of three genes taking part in aflatoxin biosynthesis, including the transcriptional regulator aflR, norsolonic acid reductase and O-methyltransferase were also carried out. A detailed overview of the species accepted in Aspergillus section Flavi is presented. PMID:21892243

  6. Genetic Analysis of the Aspergillus flavus Vegetative Compatibility Group to Which a Biological Control Agent That Limits Aflatoxin Contamination in U.S. Crops Belongs.

    PubMed

    Grubisha, Lisa C; Cotty, Peter J

    2015-09-01

    Some filamentous fungi in Aspergillus section Flavi produce carcinogenic secondary compounds called aflatoxins. Aflatoxin contamination is routinely managed in commercial agriculture with strains of Aspergillus flavus that do not produce aflatoxins. These non-aflatoxin-producing strains competitively exclude aflatoxin producers and reshape fungal communities so that strains with the aflatoxin-producing phenotype are less frequent. This study evaluated the genetic variation within naturally occurring atoxigenic A. flavus strains from the endemic vegetative compatibility group (VCG) YV36. AF36 is a strain of VCG YV36 and was the first fungus used in agriculture for aflatoxin management. Genetic analyses based on mating-type loci, 21 microsatellite loci, and a single nucleotide polymorphism (SNP) in the aflC gene were applied to a set of 237 YV36 isolates collected from 1990 through 2005 from desert legumes and untreated fields and from fields previously treated with AF36 across the southern United States. One haplotype dominated across time and space. No recombination with strains belonging to VCGs other than YV36 was detected. All YV36 isolates carried the SNP in aflC that prevents aflatoxin biosynthesis and the mat1-2 idiomorph at the mating-type locus. These results suggest that VCG YV36 has a clonal population structure maintained across both time and space. These results demonstrate the genetic stability of atoxigenic strains belonging to a broadly distributed endemic VCG in both untreated populations and populations where the short-term frequency of VCG YV36 has increased due to applications of a strain used to competitively exclude aflatoxin producers. This work supports the hypothesis that strains of this VCG are not involved in routine genetic exchange with aflatoxin-producing strains.

  7. Genetic Analysis of the Aspergillus flavus Vegetative Compatibility Group to Which a Biological Control Agent That Limits Aflatoxin Contamination in U.S. Crops Belongs

    PubMed Central

    Cotty, Peter J.

    2015-01-01

    Some filamentous fungi in Aspergillus section Flavi produce carcinogenic secondary compounds called aflatoxins. Aflatoxin contamination is routinely managed in commercial agriculture with strains of Aspergillus flavus that do not produce aflatoxins. These non-aflatoxin-producing strains competitively exclude aflatoxin producers and reshape fungal communities so that strains with the aflatoxin-producing phenotype are less frequent. This study evaluated the genetic variation within naturally occurring atoxigenic A. flavus strains from the endemic vegetative compatibility group (VCG) YV36. AF36 is a strain of VCG YV36 and was the first fungus used in agriculture for aflatoxin management. Genetic analyses based on mating-type loci, 21 microsatellite loci, and a single nucleotide polymorphism (SNP) in the aflC gene were applied to a set of 237 YV36 isolates collected from 1990 through 2005 from desert legumes and untreated fields and from fields previously treated with AF36 across the southern United States. One haplotype dominated across time and space. No recombination with strains belonging to VCGs other than YV36 was detected. All YV36 isolates carried the SNP in aflC that prevents aflatoxin biosynthesis and the mat1-2 idiomorph at the mating-type locus. These results suggest that VCG YV36 has a clonal population structure maintained across both time and space. These results demonstrate the genetic stability of atoxigenic strains belonging to a broadly distributed endemic VCG in both untreated populations and populations where the short-term frequency of VCG YV36 has increased due to applications of a strain used to competitively exclude aflatoxin producers. This work supports the hypothesis that strains of this VCG are not involved in routine genetic exchange with aflatoxin-producing strains. PMID:26092465

  8. A regulatory gene (ECO-orf4) required for ECO-0501 biosynthesis in Amycolatopsis orientalis.

    PubMed

    Shen, Yang; Huang, He; Zhu, Li; Luo, Minyu; Chen, Daijie

    2014-02-01

    ECO-0501 is a novel linear polyene antibiotic, which was discovered from Amycolatopsis orientalis. Recent study of ECO-0501 biosynthesis pathway revealed the presence of regulatory gene: ECO-orf4. The A. orientalis ECO-orf4 gene from the ECO-0501 biosynthesis cluster was analyzed, and its deduced protein (ECO-orf4) was found to have amino acid sequence homology with large ATP-binding regulators of the LuxR (LAL) family regulators. Database comparison revealed two hypothetical domains, a LuxR-type helix-turn-helix (HTH) DNA binding motif near the C-terminal and an N-terminal nucleotide triphosphate (NTP) binding motif included. Deletion of the corresponding gene (ECO-orf4) resulted in complete loss of ECO-0501 production. Complementation by one copy of intact ECO-orf4 restored the polyene biosynthesis demonstrating that ECO-orf4 is required for ECO-0501 biosynthesis. The results of overexpression ECO-orf4 on ECO-0501 production indicated that it is a positive regulatory gene. Gene expression analysis by reverse transcription PCR of the ECO-0501 gene cluster showed that the transcription of ECO-orf4 correlates with that of genes involved in polyketide biosynthesis. These results demonstrated that ECO-orf4 is a pathway-specific positive regulatory gene that is essential for ECO-0501 biosynthesis.

  9. Discovery of genes essential for heme biosynthesis through large-scale gene expression analysis

    PubMed Central

    Nilsson, Roland; Schultz, Iman J.; Pierce, Eric L.; Soltis, Kathleen A.; Naranuntarat, Amornrat; Ward, Diane M.; Baughman, Joshua; Paradkar, Prasad N.; Kingsley, Paul D.; Culotta, Valeria C.; Kaplan, Jerry; Palis, James; Paw, Barry H.; Mootha, Vamsi K.

    2009-01-01

    Summary Heme biosynthesis consists of a series of eight enzymatic reactions that originate in mitochondria and continue in the cytosol before returning to mitochondria. Although these core enzymes are well studied, additional mitochondrial transporters and regulatory factors are predicted to be required. To discover such unknown components, we utilized a large-scale computational screen to identify mitochondrial proteins whose transcripts consistently co-express with the core machinery of heme biosynthesis. We identified SLC25A39, SLC22A4 and TMEM14C, which are putative mitochondrial transporters, as well as C1orf69 and ISCA1, which are iron-sulfur cluster proteins. Targeted knockdowns of all five genes in zebrafish resulted in profound anemia without impacting erythroid lineage specification. Moreover, silencing of Slc25a39 in murine erythroleukemia cells impaired iron incorporation into protoporphyrin IX, and vertebrate Slc25a39 complemented an iron homeostasis defect in the orthologous yeast mtm1Δ deletion mutant. Our results advance the molecular understanding of heme biosynthesis and offer promising candidate genes for inherited anemias. PMID:19656490

  10. Exopolysaccharide biosynthesis genes required for social motility in Myxococcus xanthus.

    PubMed

    Lu, Ann; Cho, Kyunyung; Black, Wesley P; Duan, Xue-Yan; Lux, Renate; Yang, Zhaomin; Kaplan, Heidi B; Zusman, David R; Shi, Wenyuan

    2005-01-01

    Social (S)-motility in Myxococcus xanthus is a flagellum-independent gliding motility system that allows bacteria to move in groups on solid surfaces. S-motility has been shown to require type IV pili (TFP), exopolysaccharide (EPS; a component of fibrils) and lipopolysaccharide (LPS). Previously, information concerning EPS biogenesis in M. xanthus was lacking. In this study, we screened 5000 randomly mutagenized colonies for defects in S-motility and EPS and identified two genetic regions essential for EPS biogenesis: the EPS synthesis (eps) region and the EPS-associated (eas) region. Mutants with insertions in the eps and eas regions were defective in S-motility and fruiting body formation. These mutants failed to bind the dye calcofluor white, indicating that they lacked EPS; however, they retained normal TFP and LPS. Analysis of the eps locus showed several open reading frames (ORFs) that encode homologues to glycosyltransferases, glucanases and EPS transporters as well as regulatory proteins; the eas locus contains two ORFs: one exhibits homology to hypothetical proteins with a conserved domain of unknown function and the other displays no apparent homology to other proteins in the database. Further genetic mutagenesis analysis indicates that the whole eps region is involved in the biosynthesis of fibrils and fibril EPS. The operon at the proximal end of the eps region was analysed by generating in-frame deletion mutations. These mutants showed varying degrees of defects in the bacterium's ability to produce EPS or perform EPS-related functions, confirming the involvement of these genes in M. xanthus EPS biogenesis.

  11. Foreign gene recruitment to the fatty acid biosynthesis pathway in diatoms.

    PubMed

    Chan, Cheong Xin; Baglivi, Francesca L; Jenkins, Christina E; Bhattacharya, Debashish

    2013-09-01

    Diatoms are highly successful marine and freshwater algae that contribute up to 20% of global carbon fixation. These species are leading candidates for biofuel production owing to ease of culturing and high fatty acid content. To assist in strain improvement and downstream applications for potential use as a biofuel, it is important to understand the evolution of lipid biosynthesis in diatoms. The evolutionary history of diatoms is however complicated by likely multiple endosymbioses involving the capture of foreign cells and horizontal gene transfer into the host genome. Using a phylogenomic approach, we assessed the evolutionary history of 12 diatom genes putatively encoding functions related to lipid biosynthesis. We found evidence of gene transfer likely from a green algal source for seven of these genes, with the remaining showing either vertical inheritance or evolutionary histories too complicated to interpret given current genome data. The functions of horizontally transferred genes encompass all aspects of lipid biosynthesis (initiation, biosynthesis, and desaturation of fatty acids) as well as fatty acid elongation, and are not restricted to plastid-targeted proteins. Our findings demonstrate that the transfer, duplication, and subfunctionalization of genes were key steps in the evolution of lipid biosynthesis in diatoms and other photosynthetic eukaryotes. This target pathway for biofuel research is highly chimeric and surprisingly, our results suggest that research done on related genes in green algae may have application to diatom models.

  12. Genome-wide transcriptome analysis of cotton (Gossypium hirsutum L.) identifies candidate gene signatures in response to aflatoxin producing fungus Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aflatoxins are toxic metabolites and potent carcinogen produced from asexual fungi Aspergillus flavus and A. parasiticus. Aflatoxins can contaminate cottonseed under conducive preharvest and postharvest conditions. U.S. federal regulations restrict the use of aflatoxin contaminated cottonseed at >20...

  13. Evaluation of the expression genes associated with resistance to Aspergillus flavus colonization and aflatoxin production in different maize lines.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aflatoxins are carcinogenic toxic compounds produced by Aspergillus flavus during infection of crops including maize (Zea mays L.). Contamination of maize with aflatoxin is exacerbated by late season drought stress. Previous studies have implicated numerous resistance-associated proteins (RAPs) that...

  14. Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple

    PubMed Central

    Liao, Liao; Vimolmangkang, Sornkanok; Wei, Guochao; Zhou, Hui; Korban, Schuyler S.; Han, Yuepeng

    2015-01-01

    Proanthocyanidins (PAs) are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis. PMID:25914714

  15. Genes Involved in the Biosynthesis and Transport of Acinetobactin in Acinetobacter baumannii

    PubMed Central

    Hasan, Tarik; Choi, Chul Hee

    2015-01-01

    Pathogenic bacteria survive in iron-limited host environments by using several iron acquisition mechanisms. Acinetobacter baumannii, causing serious infections in compromised patients, produces an iron-chelating molecule, called acinetobactin, which is composed of equimolar quantities of 2,3-dihydroxybenzoic acid (DHBA), L-threonine, and N-hydroxyhistamine, to compete with host cells for iron. Genes that are involved in the production and transport of acinetobactin are clustered within the genome of A. baumannii. A recent study showed that entA, located outside of the acinetobactin gene cluster, plays important roles in the biosynthesis of the acinetobactin precursor DHBA and in bacterial pathogenesis. Therefore, understanding the genes that are associated with the biosynthesis and transport of acinetobactin in the bacterial genome is required. This review is intended to provide a general overview of the genes in the genome of A. baumannii that are required for acinetobactin biosynthesis and transport. PMID:25873846

  16. Deep sequencing of the Camellia chekiangoleosa transcriptome revealed candidate genes for anthocyanin biosynthesis.

    PubMed

    Wang, Zhong-Wei; Jiang, Cong; Wen, Qiang; Wang, Na; Tao, Yuan-Yuan; Xu, Li-An

    2014-03-15

    Camellia chekiangoleosa is an important species of genus Camellia. It provides high-quality edible oil and has great ornamental value. The flowers are big and red which bloom between February and March. Flower pigmentation is closely related to the accumulation of anthocyanin. Although anthocyanin biosynthesis has been studied extensively in herbaceous plants, little molecular information on the anthocyanin biosynthesis pathway of C. chekiangoleosa is yet known. In the present study, a cDNA library was constructed to obtain detailed and general data from the flowers of C. chekiangoleosa. To explore the transcriptome of C. chekiangoleosa and investigate genes involved in anthocyanin biosynthesis, a 454 GS FLX Titanium platform was used to generate an EST dataset. About 46,279 sequences were obtained, and 24,593 (53.1%) were annotated. Using Blast search against the AGRIS, 1740 unigenes were found homologous to 599 Arabidopsis transcription factor genes. Based on the transcriptome dataset, nine anthocyanin biosynthesis pathway genes (PAL, CHS1, CHS2, CHS3, CHI, F3H, DFR, ANS, and UFGT) were identified and cloned. The spatio-temporal expression patterns of these genes were also analyzed using quantitative real-time polymerase chain reaction. The study results not only enrich the gene resource but also provide valuable information for further studies concerning anthocyanin biosynthesis.

  17. Altered biochemical profile and gene expression in aflatoxin B-1-transformed C3H10T1/2 cells.

    PubMed

    Nadadur, S; Lisciandro, K; Mudipalli, A; Maccubbin, A; Faletto, M; Gurtoo, H

    1997-06-01

    A transformed cell line 7SA, obtained by transformation of C3H10T1/2 cells with irt vitro activated aflatoxin B-1 (AFB(1)), was used to investigate biochemical and molecular alterations associated with transformation by AFB(1). 7SA cells demonstrate an altered biochemical phenotype characterized by alterations in phase I and phase II enzymes in a manner that would allow these cells to survive in a hostile chemical environment. Investigations of the molecular basis of transformation revealed no mutations in codons 12/13 and 61 of ras genes (Ha-, Ki- and N-ras) and in exons 5, 6, 7 and 8 of p53 tumor suppressor gene. However, subtractive hybridization led to the isolation of seven novel cDNA clones that demonstrated 2 to 10-fold overexpression of the mRNAs corresponding to the five cDNAs (SK1, SK2, SK3, SK4 and SK5) and >400 fold overexpression of the mRNAs corresponding to the other two cDNAs (SK67 and SK153). In addition, part of the sequence of the cDNA clone SK5 demonstrated >88% identity with L1-like mobile genetic element and Southern analysis of the DNA with SK5 cDNA as a probe revealed gene rearrangement in 7SA DNA, compared to DNA from C3H10T1/2 cells.

  18. Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

  19. A hypothesis to explain how laeA specifically regulates certain secondary metabolite biosynthesis gene clusters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biosynthesis of mycotoxins involves transcriptional co-regulation of sets of clustered genes. We hypothesize that specific control of transcription of genes in these clusters by LaeA, a global regulator of secondary metabolite production and development in aspergilli and other filamentous fungi, re...

  20. Developmental and feedforward control of the expression of folate biosynthesis genes in tomato fruit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about how plants regulate their folate content, including whether the expression of folate biosynthesis genes is orchestrated during development or modulated by folate levels. Nor is much known about how folate levels impact the expression of other genes. These points were addressed ...

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

    PubMed Central

    Guleria, Praveen; Yadav, Sudesh Kumar

    2013-01-01

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

  2. Cloning and characterization of the Streptomyces peucetius dnrQS genes encoding a daunosamine biosynthesis enzyme and a glycosyl transferase involved in daunorubicin biosynthesis.

    PubMed Central

    Otten, S L; Liu, X; Ferguson, J; Hutchinson, C R

    1995-01-01

    The dnrQS genes from the daunorubicin producer Streptomyces peucetius were characterized by DNA sequencing, complementation analysis, and gene disruption. The dnrQ gene is required for daunosamine biosynthesis, and dnrS appears to encode a glycosyltransferase for the addition of the 2,3,6-trideoxy-3-aminohexose, daunosamine, to epsilon-rhodomycinone. PMID:7592454

  3. RNA Sequencing of Contaminated Seeds Reveals the State of the Seed Permissive for Pre-Harvest Aflatoxin Contamination and Points to a Potential Susceptibility Factor

    PubMed Central

    Clevenger, Josh; Marasigan, Kathleen; Liakos, Vasileios; Sobolev, Victor; Vellidis, George; Holbrook, Corley; Ozias-Akins, Peggy

    2016-01-01

    Pre-harvest aflatoxin contamination (PAC) is a major problem facing peanut production worldwide. Produced by the ubiquitous soil fungus, Aspergillus flavus, aflatoxin is the most naturally occurring known carcinogen. The interaction between fungus and host resulting in PAC is complex, and breeding for PAC resistance has been slow. It has been shown that aflatoxin production can be induced by applying drought stress as peanut seeds mature. We have implemented an automated rainout shelter that controls temperature and moisture in the root and peg zone to induce aflatoxin production. Using polymerase chain reaction (PCR) and high performance liquid chromatography (HPLC), seeds meeting the following conditions were selected: infected with Aspergillus flavus and contaminated with aflatoxin; and not contaminated with aflatoxin. RNA sequencing analysis revealed groups of genes that describe the transcriptional state of contaminated vs. uncontaminated seed. These data suggest that fatty acid biosynthesis and abscisic acid (ABA) signaling are altered in contaminated seeds and point to a potential susceptibility factor, ABR1, as a repressor of ABA signaling that may play a role in permitting PAC. PMID:27827875

  4. Comparative Transcriptomic Analyses of Differentially Expressed Genes in Transgenic Melatonin Biosynthesis Ovine HIOMT Gene in Switchgrass

    PubMed Central

    Yuan, Shan; Guan, Cong; Liu, Sijia; Huang, Yanhua; Tian, Danyang; Cui, Xin; Zhang, Yunwei; Yang, Fuyu

    2016-01-01

    Melatonin serves pleiotropic functions in prompting plant growth and resistance to various stresses. The accurate biosynthetic pathway of melatonin remains elusive in plant species, while the N-acetyltransferase and O-methyltransferase were considered to be the last two key enzymes during its biosynthesis. To investigate the biosynthesis and metabolic pathway of melatonin in plants, the RNA-seq profile of overexpression of the ovine HIOMT was analyzed and compared with the previous transcriptome of transgenic oAANAT gene in switchgrass, a model plant for cellulosic ethanol production. A total of 946, 405, and 807 differentially expressed unigenes were observed in AANAT vs. control, HIOMT vs. control, and AANAT vs. HIOMT, respectively. Two hundred and seventy-five upregulated and 130 downregulated unigenes were detected in transgenic oHIOMT line comparing with control, including the significantly upregulated (F-box/kelch-repeat protein, zinc finger BED domain-containing protein-3) genes, which were potentially correlated with enhanced phenotypes of shoot, stem and root growth in transgenic oHIOMT switchgrass. Several stress resistant related genes (SPX domain-containing membrane protein, copper transporter 1, late blight resistance protein homolog R1A-6 OS etc.) were specifically and significantly upregulated in transgenic oHIOMT only, while metabolism-related genes (phenylalanine-4-hydroxylase, tyrosine decarboxylase 1, protein disulfide-isomerase and galactinol synthase 2 etc.) were significantly upregulated in transgenic oAANAT only. These results provide new sights into the biosynthetic and physiological functional networks of melatonin in plants. PMID:27877177

  5. Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesis genes required for aklanonic acid biosynthesis.

    PubMed Central

    Rajgarhia, V B; Strohl, W R

    1997-01-01

    The structure of the Streptomyces sp. strain C5 daunorubicin type II polyketide synthase (PKS) gene region is different from that of other known type II PKS gene clusters. Directly downstream of the genes encoding ketoacylsynthase alpha and beta (KS alpha, KS beta) are two genes (dpsC, dpsD) encoding proteins of unproven function, both absent from other type II PKS gene clusters. Also in contrast to other type II PKS clusters, the gene encoding the acyl carrier protein (ACP), dpsG, is located about 6.8 kbp upstream of the genes encoding the daunorubicin KS alpha and KS beta. In this work, we demonstrate that the minimal genes required to produce aklanonic acid in heterologous hosts are dpsG (ACP), dauI (regulatory activator), dpsA (KS alpha), dpsB (KS beta), dpsF (aromatase), dpsE (polyketide reductase), and dauG (putative deoxyaklanonic acid oxygenase). The two unusual open reading frames, dpsC (KASIII homolog lacking a known active site) and dpsD (acyltransferase homolog), are not required to synthesize aklanonic acid. Additionally, replacement of dpsD or dpsCD in Streptomyces sp. strain C5 with a neomycin resistance gene (aphI) results in mutant strains that still produced anthracyclines. PMID:9098068

  6. Biosynthesis and Toxicological Effects of Patulin

    PubMed Central

    Puel, Olivier; Galtier, Pierre; Oswald, Isabelle P.

    2010-01-01

    Patulin is a toxic chemical contaminant produced by several species of mold, especially within Aspergillus, Penicillium and Byssochlamys. It is the most common mycotoxin found in apples and apple-derived products such as juice, cider, compotes and other food intended for young children. Exposure to this mycotoxin is associated with immunological, neurological and gastrointestinal outcomes. Assessment of the health risks due to patulin consumption by humans has led many countries to regulate the quantity in food. A full understanding of the molecular genetics of patulin biosynthesis is incomplete, unlike other regulated mycotoxins (aflatoxins, trichothecenes and fumonisins), although the chemical structures of patulin precursors are now known. The biosynthetic pathway consists of approximately 10 steps, as suggested by biochemical studies. Recently, a cluster of 15 genes involved in patulin biosynthesis was reported, containing characterized enzymes, a regulation factor and transporter genes. This review includes information on the current understanding of the mechanisms of patulin toxinogenesis and summarizes its toxicological effects. PMID:22069602

  7. Cloning of a Vibrio cholerae vibriobactin gene cluster: identification of genes required for early steps in siderophore biosynthesis.

    PubMed Central

    Wyckoff, E E; Stoebner, J A; Reed, K E; Payne, S M

    1997-01-01

    Vibrio cholerae secretes the catechol siderophore vibriobactin in response to iron limitation. Vibriobactin is structurally similar to enterobactin, the siderophore produced by Escherichia coli, and both organisms produce 2,3-dihydroxybenzoic acid (DHBA) as an intermediate in siderophore biosynthesis. To isolate and characterize V. cholerae genes involved in vibriobactin biosynthesis, we constructed a genomic cosmid bank of V. cholerae DNA and isolated clones that complemented mutations in E. coli enterobactin biosynthesis genes. V. cholerae homologs of entA, entB, entC, entD, and entE were identified on overlapping cosmid clones. Our data indicate that the vibriobactin genes are clustered, like the E. coli enterobactin genes, but the organization of the genes within these clusters is different. In this paper, we present the organization and sequences of genes involved in the synthesis and activation of DHBA. In addition, a V. cholerae strain with a chromosomal mutation in vibA was constructed by marker exchange. This strain was unable to produce vibriobactin or DHBA, confirming that in V. cholerae VibA catalyzes an early step in vibriobactin biosynthesis. PMID:9371453

  8. Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus).

    PubMed

    Fedorov, Vadim B; Goropashnaya, Anna V; Tøien, Oivind; Stewart, Nathan C; Gracey, Andrew Y; Chang, Celia; Qin, Shizhen; Pertea, Geo; Quackenbush, John; Showe, Louise C; Showe, Michael K; Boyer, Bert B; Barnes, Brian M

    2009-04-10

    We conducted a large-scale gene expression screen using the 3,200 cDNA probe microarray developed specifically for Ursus americanus to detect expression differences in liver and skeletal muscle that occur during winter hibernation compared with animals sampled during summer. The expression of 12 genes, including RNA binding protein motif 3 (Rbm3), that are mostly involved in protein biosynthesis, was induced during hibernation in both liver and muscle. The Gene Ontology and Gene Set Enrichment analysis consistently showed a highly significant enrichment of the protein biosynthesis category by overexpressed genes in both liver and skeletal muscle during hibernation. Coordinated induction in transcriptional level of genes involved in protein biosynthesis is a distinctive feature of the transcriptome in hibernating black bears. This finding implies induction of translation and suggests an adaptive mechanism that contributes to a unique ability to reduce muscle atrophy over prolonged periods of immobility during hibernation. Comparing expression profiles in bears to small mammalian hibernators shows a general trend during hibernation of transcriptional changes that include induction of genes involved in lipid metabolism and carbohydrate synthesis as well as depression of genes involved in the urea cycle and detoxification function in liver.

  9. Organization of genes required for gellan polysaccharide biosynthesis in Sphingomonas elodea ATCC 31461.

    PubMed

    Harding, Nancy E; Patel, Yamini N; Coleman, Russell J

    2004-02-01

    Sphingomonas elodea ATCC 31461 produces gellan, a capsular polysaccharide that is useful as a gelling agent for food and microbiological media. Complementation of nonmucoid S. elodea mutants with a gene library resulted in identification of genes essential for gellan biosynthesis. A cluster of 18 genes spanning 21 kb was isolated. These 18 genes are homologous to genes for synthesis of sphingan polysaccharide S-88 from Sphingomonas sp. ATCC 31554, with predicted amino acid identities varying from 61% to 98%. Both polysaccharides have the same tetrasaccharide repeat unit, comprised of [-->4)-alpha- l-rhamnose-(1-->3)-beta- d-glucose-(1-->4)-beta- d-glucuronic acid-(1-->4)-beta- d-glucose-(1-->]. Polysaccharide S-88, however, has mannose or rhamnose in the fourth position and has a rhamnosyl side chain, while gellan has no sugar side chain but is modified by glyceryl and acetyl substituents. Genes for synthesis of the precursor dTDP- l-rhamnose were highly conserved. The least conserved genes in this cluster encode putative glycosyl transferases III and IV and a gene of unknown function, gelF. Three genes ( gelI, gelM, and gelN) affected the amount and rheology of gellan produced. Four additional genes present in the S-88 sphingan biosynthetic gene cluster did not have homologs in the gene cluster for gellan biosynthesis. Three of these gene homologs, gelR, gelS, and gelG, were found in an operon unlinked to the main gellan biosynthetic gene cluster. In a third region, a gene possibly involved in positive regulation of gellan biosynthesis was identified.

  10. Identification of solanapyrone biosynthesis genes and generation of solanapyrone-deficient mutants in Ascochyta rabiei

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ascochyta rabiei, the causal agent of Ascochyta blight of chickpea, produces solanapyrone toxins which are toxic to chickpea. However, very little is known about the genetics of toxin production and the role of the toxins in pathogenesis. In the present study, solanapyrone biosynthesis genes in A. ...

  11. Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

    PubMed Central

    Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

    2016-01-01

    Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides. PMID:27625674

  12. Co-regulation of brassinosteroid biosynthesis-related genes during xylem cell differentiation.

    PubMed

    Yamamoto, Ryo; Fujioka, Shozo; Iwamoto, Kuninori; Demura, Taku; Takatsuto, Suguru; Yoshida, Shigeo; Fukuda, Hiroo

    2007-01-01

    To understand the regulatory mechanisms of brassinosteroid (BR) biosynthesis in specific plant developmental processes, we first investigated the accumulation profiles of BRs and sterols in xylem differentiation in a Zinnia culture. The amounts of many substances in the late C28 sterol biosynthetic pathway to campesterol (CR), such as episterol and 24-methylenecholesterol, as well as those in the BR-specific biosynthetic pathway from CR to brassinolide (BL), were elevated in close association with tracheary element differentiation. Among them, 6-deoxotyphasterol (6-deoxoTY) accumulated to unusually high levels within cells cultured in tracheary element-inductive medium, while castasterone (CS) was not elevated either within or outside cells. To identify the molecular basis of this co-up-regulation of BRs and C28 sterols, we isolated Zinnia genes for the key enzymes of BR biosynthesis, ZeSTE1, ZeDIM, ZeDWF4, ZeCPD1 and ZeCPD2. RNA gel blot analysis of these genes indicated a coordinated increase in transcripts for ZeSTE1, ZeDIM, ZeDWF4 and ZeCPD1, and a tracheary element differentiation-specific increase in transcripts for ZeDWF4 and ZeCPD1. In situ hybridization experiments of ZeDWF4 and ZeCPD1 mRNAs revealed their preferential accumulation in procambium cells, immature xylem cells and xylem parenchyma cells. These results suggest that BR biosynthesis during tracheary element differentiation may be regulated by the coordinated regulation of broad sterol biosynthesis and specific regulation of BR biosynthesis, which occurs in part by elevated transcript levels of genes encoding BR biosynthetic enzymes, specifically ZeDWF4 and ZeCPD1. These data provide new insights into the regulation of BR biosynthesis and BR signaling during plant development.

  13. Identification of the Lomofungin Biosynthesis Gene Cluster and Associated Flavin-Dependent Monooxygenase Gene in Streptomyces lomondensis S015

    PubMed Central

    Zhang, Chunxiao; Sheng, Chaolan; Wang, Wei; Hu, Hongbo; Peng, Huasong; Zhang, Xuehong

    2015-01-01

    Streptomyces lomondensis S015 synthesizes the broad-spectrum phenazine antibiotic lomofungin. Whole genome sequencing of this strain revealed a genomic locus consisting of 23 open reading frames that includes the core phenazine biosynthesis gene cluster lphzGFEDCB. lomo10, encoding a putative flavin-dependent monooxygenase, was also identified in this locus. Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin. This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces. PMID:26305803

  14. Clade classification of monolignol biosynthesis gene family members reveals target genes to decrease lignin in Lolium perenne.

    PubMed

    van Parijs, F R D; Ruttink, T; Boerjan, W; Haesaert, G; Byrne, S L; Asp, T; Roldán-Ruiz, I; Muylle, H

    2015-07-01

    In monocots, lignin content has a strong impact on the digestibility of the cell wall fraction. Engineering lignin biosynthesis requires a profound knowledge of the role of paralogues in the multigene families that constitute the monolignol biosynthesis pathway. We applied a bioinformatics approach for genome-wide identification of candidate genes in Lolium perenne that are likely to be involved in the biosynthesis of monolignols. More specifically, we performed functional subtyping of phylogenetic clades in four multigene families: 4CL, COMT, CAD and CCR. Essential residues were considered for functional clade delineation within these families. This classification was complemented with previously published experimental evidence on gene expression, gene function and enzymatic activity in closely related crops and model species. This allowed us to assign functions to novel identified L. perenne genes, and to assess functional redundancy among paralogues. We found that two 4CL paralogues, two COMT paralogues, three CCR paralogues and one CAD gene are prime targets for genetic studies to engineer developmentally regulated lignin in this species. Based on the delineation of sequence conservation between paralogues and a first analysis of allelic diversity, we discuss possibilities to further study the roles of these paralogues in lignin biosynthesis, including expression analysis, reverse genetics and forward genetics, such as association mapping. We propose criteria to prioritise paralogues within multigene families and certain SNPs within these genes for developing genotyping assays or increasing power in association mapping studies. Although L. perenne was the target of the analyses presented here, this functional subtyping of phylogenetic clades represents a valuable tool for studies investigating monolignol biosynthesis genes in other monocot species.

  15. Role of EctR as transcriptional regulator of ectoine biosynthesis genes in Methylophaga thalassica.

    PubMed

    Mustakhimov, I I; Reshetnikov, A S; Fedorov, D N; Khmelenina, V N; Trotsenko, Y A

    2012-08-01

    In the halophilic aerobic methylotrophic bacterium Methylophaga thalassica, the genes encoding the enzymes for biosynthesis of the osmoprotectant ectoine were shown to be located in operon ectABC-ask. Transcription of the ect-operon was started from the two promoters homologous to the σ(70)-dependent promoter of Escherichia coli and regulated by protein EctR, whose encoding gene, ectR, is transcribed from three promoters. Genes homologous to ectR of methylotrophs were found in clusters of ectoine biosynthesis genes in some non-methylotrophic halophilic bacteria. EctR proteins of methylotrophic and heterotrophic halophiles belong to the MarR-family of transcriptional regulators but form a separate branch on the phylogenetic tree of the MarR proteins.

  16. Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68.

    PubMed

    Samadlouie, Hamid-Reza; Hamidi-Esfahani, Zohreh; Alavi, Seyed-Mehdi; Varastegani, Boshra

    2014-01-01

    The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.

  17. Regulation of the Flavonoid Biosynthesis Pathway Genes in Purple and Black Grains of Hordeum vulgare

    PubMed Central

    Mock, Hans-Peter; Kukoeva, Tatjana V.; Börner, Andreas; Khlestkina, Elena K.

    2016-01-01

    Barley grain at maturity can have yellow, purple, blue, and black pigmentations which are suggested to play a protective role under stress conditions. The first three types of the colors are caused by phenolic compounds flavonoids; the last one is caused by phytomelanins, oxidized and polymerized phenolic compounds. Although the genetic basis of the flavonoid biosynthesis pathway in barley has been thoroughly studied, there is no data yet on its regulation in purple and black barley grains. In the current study, genetic model of Hordeum vulgare ‘Bowman’ near-isogenic lines (NILs) was used to investigate the regulation of the flavonoid biosynthesis in white, purple, and black barley grains. Microsatellite genotyping revealed donor segments in the purple- and black-grained lines on chromosomes 2H (in region of the Ant2 gene determining purple color of grains) and 1H (in region of the Blp gene determining black lemma and pericarp), respectively. The isolated dominant Ant2 allele of the purple-grained line has high level of sequence similarity with the recessive Bowman’s ant2 in coding region, whereas an insertion of 179 bp was detected in promoter region of ant2. This structural divergence between Ant2 and ant2 alleles may underlie their different expression in grain pericarp: Bowman’s Ant2 is not transcribed, whereas it was up-regulated in the purple-grained line with coordinately co-expressed flavonoid biosynthesis structural genes (Chs, Chi, F3h, F3’h, Dfr, Ans). This led to total anthocyain content increase in purple-grained line identified by ultra-performance liquid chromatography (HPLC). Collectively, these results proved the regulatory function of the Ant2 gene in anthocyanin biosynthesis in barley grain pericarp. In the black-grained line, the specific transcriptional regulation of the flavonoid biosynthesis pathway genes was not detected, suggesting that flavonoid pigments are not involved in development of black lemma and pericarp trait. PMID

  18. Transcriptome Sequencing and Expression Analysis of Terpenoid Biosynthesis Genes in Litsea cubeba

    PubMed Central

    Han, Xiao-Jiao; Wang, Yang-Dong; Chen, Yi-Cun; Lin, Li-Yuan; Wu, Qing-Ke

    2013-01-01

    Background Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour.) Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes. Results Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56%) unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions. Conclusion RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The results may help

  19. Transcriptomic Analysis Reveals Key Genes Related to Betalain Biosynthesis in Pulp Coloration of Hylocereus polyrhizus

    PubMed Central

    Qingzhu, Hua; Chengjie, Chen; Zhe, Chen; Pengkun, Chen; Yuewen, Ma; Jingyu, Wu; Jian, Zheng; Guibing, Hu; Jietang, Zhao; Yonghua, Qin

    2016-01-01

    Betalains have high nutritional value and bioactivities. Red pulp pitaya (Hylocereus polyrhizus) is the only fruit containing abundant betalains for consumer. However, no information is available about genes involved in betalain biosynthesis in H. polyrhizus. Herein, two cDNA libraries of pitaya pulps with two different coloration stages (white and red pulp stages) of Guanhuahong (H. polyrhizus) were constructed. A total of about 12 Gb raw RNA-Seq data was generated and was de novo assembled into 122,677 transcripts with an average length of 1183 bp and an N50 value of 2008. Approximately 99.99% of all transcripts were annotated based on seven public databases. A total of 8871 transcripts were significantly regulated. Thirty-three candidate transcripts related to betalain biosynthesis were obtained from the transcriptome data. Transcripts encoding enzymes involved in betalain biosynthesis were analyzed using RT-qPCR at the whole pulp coloration stages of H. polyrhizus (7-1) and H. undatus (132-4). Nine key transcripts of betalain biosynthesis were identified. They were assigned to four kinds of genes in betalain biosynthetic pathway, including tyrosinase, 4, 5-DOPA dioxygenase extradiol, cytochrome P450 and glucosyltransferase. Ultimately, a preliminary betalain biosynthetic pathway for pitaya was proposed based on betalain analyses, gene expression profiles and published documents. PMID:26779215

  20. Transcriptomic Analysis Reveals Key Genes Related to Betalain Biosynthesis in Pulp Coloration of Hylocereus polyrhizus.

    PubMed

    Qingzhu, Hua; Chengjie, Chen; Zhe, Chen; Pengkun, Chen; Yuewen, Ma; Jingyu, Wu; Jian, Zheng; Guibing, Hu; Jietang, Zhao; Yonghua, Qin

    2015-01-01

    Betalains have high nutritional value and bioactivities. Red pulp pitaya (Hylocereus polyrhizus) is the only fruit containing abundant betalains for consumer. However, no information is available about genes involved in betalain biosynthesis in H. polyrhizus. Herein, two cDNA libraries of pitaya pulps with two different coloration stages (white and red pulp stages) of Guanhuahong (H. polyrhizus) were constructed. A total of about 12 Gb raw RNA-Seq data was generated and was de novo assembled into 122,677 transcripts with an average length of 1183 bp and an N50 value of 2008. Approximately 99.99% of all transcripts were annotated based on seven public databases. A total of 8871 transcripts were significantly regulated. Thirty-three candidate transcripts related to betalain biosynthesis were obtained from the transcriptome data. Transcripts encoding enzymes involved in betalain biosynthesis were analyzed using RT-qPCR at the whole pulp coloration stages of H. polyrhizus (7-1) and H. undatus (132-4). Nine key transcripts of betalain biosynthesis were identified. They were assigned to four kinds of genes in betalain biosynthetic pathway, including tyrosinase, 4, 5-DOPA dioxygenase extradiol, cytochrome P450 and glucosyltransferase. Ultimately, a preliminary betalain biosynthetic pathway for pitaya was proposed based on betalain analyses, gene expression profiles and published documents.

  1. Gene transfers shaped the evolution of de novo NAD+ biosynthesis in eukaryotes.

    PubMed

    Ternes, Chad M; Schönknecht, Gerald

    2014-09-01

    NAD(+) is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD(+) biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD(+) biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD(+) biosynthesis in eukaryotes was shaped by numerous gene transfers.

  2. Genes associated with 2-methylisoborneol biosynthesis in cyanobacteria: isolation, characterization, and expression in response to light.

    PubMed

    Wang, Zhongjie; Xu, Yao; Shao, Jihai; Wang, Jie; Li, Renhui

    2011-04-07

    The volatile microbial metabolite 2-methylisoborneol (2-MIB) is a root cause of taste and odor issues in freshwater. Although current evidence suggests that 2-MIB is not toxic, this compound degrades water quality and presents problems for water treatment. To address these issues, cyanobacteria and actinomycetes, the major producers of 2-MIB, have been investigated extensively. In this study, two 2-MIB producing strains, coded as Pseudanabaena sp. and Planktothricoids raciborskii, were used in order to elucidate the genetic background, light regulation, and biochemical mechanisms of 2-MIB biosynthesis in cyanobacteria. Genome walking and PCR methods revealed that two adjacent genes, SAM-dependent methyltransferanse gene and monoterpene cyclase gene, are responsible for GPP methylation and subsequent cyclization to 2-MIB in cyanobacteria. These two genes are located in between two homologous cyclic nucleotide-binding protein genes that may be members of the Crp-Fnr regulator family. Together, this sequence of genes forms a putative operon. The synthesis of 2-MIB is similar in cyanobacteria and actinomycetes. Comparison of the gene arrangement and functional sites between cyanobacteria and other organisms revealed that gene recombination and gene transfer probably occurred during the evolution of 2-MIB-associated genes. All the microorganisms examined have a common origin of 2-MIB biosynthesis capacity, but cyanobacteria represent a unique evolutionary lineage. Gene expression analysis suggested that light is a crucial, but not the only, active regulatory factor for the transcription of 2-MIB synthesis genes. This light-regulated process is immediate and transient. This study is the first to identify the genetic background and evolution of 2-MIB biosynthesis in cyanobacteria, thus enhancing current knowledge on 2-MIB contamination of freshwater.

  3. Host-Induced Gene Silencing (HIGS) of aflatoxin synthesis genes in peanut and maize: use of RNA interference and genetic diversity of Aspergillus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Approximately 4.5 billion people are chronically exposed to aflatoxins, these are powerful carcinogens produced by Aspergillus flavus and A. parasiticus. High levels of aflatoxins in crops result in approximately 100 million metric tons of cereals, ¬nuts, root crops and other agricultural products ...

  4. The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts.

    PubMed

    Kowalska, Ewa; Kozik, Andrzej

    2008-01-01

    Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)-4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge.

  5. Genome-Wide Survey of Flavonoid Biosynthesis Genes and Gene Expression Analysis between Black- and Yellow-Seeded Brassica napus

    PubMed Central

    Qu, Cunmin; Zhao, Huiyan; Fu, Fuyou; Wang, Zhen; Zhang, Kai; Zhou, Yan; Wang, Xin; Wang, Rui; Xu, Xinfu; Tang, Zhanglin; Lu, Kun; Li, Jia-Na

    2016-01-01

    Flavonoids, the compounds that impart color to fruits, flowers, and seeds, are the most widespread secondary metabolites in plants. However, a systematic analysis of these loci has not been performed in Brassicaceae. In this study, we isolated 649 nucleotide sequences related to flavonoid biosynthesis, i.e., the Transparent Testa (TT) genes, and their associated amino acid sequences in 17 Brassicaceae species, grouped into Arabidopsis or Brassicaceae subgroups. Moreover, 36 copies of 21 genes of the flavonoid biosynthesis pathway were identified in Arabidopsis thaliana, 53 were identified in Brassica rapa, 50 in Brassica oleracea, and 95 in B. napus, followed the genomic distribution, collinearity analysis and genes triplication of them among Brassicaceae species. The results showed that the extensive gene loss, whole genome triplication, and diploidization that occurred after divergence from the common ancestor. Using qRT-PCR methods, we analyzed the expression of 18 flavonoid biosynthesis genes in 6 yellow- and black-seeded B. napus inbred lines with different genetic background, found that 12 of which were preferentially expressed during seed development, whereas the remaining genes were expressed in all B. napus tissues examined. Moreover, 14 of these genes showed significant differences in expression level during seed development, and all but four of these (i.e., BnTT5, BnTT7, BnTT10, and BnTTG1) had similar expression patterns among the yellow- and black-seeded B. napus. Results showed that the structural genes (BnTT3, BnTT18, and BnBAN), regulatory genes (BnTTG2 and BnTT16) and three encoding transfer proteins (BnTT12, BnTT19, and BnAHA10) might play an crucial roles in the formation of different seed coat colors in B. napus. These data will be helpful for illustrating the molecular mechanisms of flavonoid biosynthesis in Brassicaceae species. PMID:27999578

  6. Expression and mapping of anthocyanin biosynthesis genes in carrot

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anthocyanin gene expression has been extensively studied in leaves, fruits and flowers of numerous plants. Little, however, is known about anthocyanin accumulation in roots, or in carrots or other Apiaceae. We quantified expression of six anthocyanin biosynthetic genes (phenylalanine ammonia-lyase (...

  7. Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) in Streptomyces chartreusis NRRL 3882▿

    PubMed Central

    Wu, Qiulin; Liang, Jingdan; Lin, Shuangjun; Zhou, Xiufen; Bai, Linquan; Deng, Zixin; Wang, Zhijun

    2011-01-01

    The pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis. PMID:21173184

  8. Comparative and Genetic Analyses of the Putative Vibrio cholerae Lipopolysaccharide Core Oligosaccharide Biosynthesis (wav) Gene Cluster

    PubMed Central

    Nesper, Jutta; Kraiß, Anita; Schild, Stefan; Blaβ, Julia; Klose, Karl E.; Bockemühl, Jochen; Reidl, Joachim

    2002-01-01

    We identified five different putative wav gene cluster types, which are responsible for the synthesis of the core oligosaccharide (OS) region of Vibrio cholerae lipopolysaccharide. Preliminary evidence that the genes encoded by this cluster are involved in core OS biosynthesis came from analysis of the recently released O1 El Tor V. cholerae genome sequence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of O1 El Tor mutant strains defective in three genes (waaF, waaL, and wavB). Investigations of 38 different V. cholerae strains by Southern blotting, PCR, and sequencing analyses showed that the O1 El Tor wav gene cluster type is prevalent among clinical isolates of different serogroups associated with cholera and environmental O1 strains. In contrast, we found differences in the wav gene contents of 19 unrelated non-O1, non-O139 environmental and human isolates not associated with cholera. These strains contained four new wav gene cluster types that differ from each other in distinct gene loci, providing evidence for horizontal transfer of wav genes and for limited structural diversity of the core OS among V. cholerae isolates. Our results show genetic diversity in the core OS biosynthesis gene cluster and predominance of the type 1 wav gene locus in strains associated with clinical cholera, suggesting that a specific core OS structure could contribute to V. cholerae virulence. PMID:11953379

  9. Silver nanoparticles mediated altered gene expression of melanin biosynthesis genes in Bipolaris sorokiniana.

    PubMed

    Mishra, Sandhya; Singh, H B

    2015-03-01

    Melanin production in many fungal phytopathogens has been investigated to play direct or indirect role in pathogenesis. However, in Bipolaris sorokiniana, the spot blotch pathogen of wheat, much less is known about the role melanin play in pathogenesis. As an extension of our previous report, the present study aims to investigate the plausible association between melanin production and virulence factor in B. sorokiniana. In the previous study, we carried out analysis on the antifungal efficacy of biosynthesized silver nanoparticles (AgNPs) against B. sorokiniana. The present investigation revealed the gene expression analysis of melanin biosynthesis genes viz. polyketide synthase (PKS1) and scytalone dehydratase (SCD1) under the influence of AgNPs. The 0.05mg/ml concentration of AgNPs yielded noticeable inhibition of B. sorokiniana growth, while 0.1mg/ml concentration of AgNPs accounted for complete inhibition of pathogen growth. In addition, the semiquantitative RT-PCR analysis exhibited reduced expression of PKS1 and SCD1 under the influence of AgNPs treatment. Furthermore, the qRT-PCR demonstrated 6.47 and 1.808 fold significant decrease in the expression pattern of PKS1 and SCD1, respectively, in B. sorokiniana treated with AgNPs. The present study provides probable understanding of molecular events underlying the antifungal role of AgNPs against B. sorokiniana.

  10. The rubber tree genome shows expansion of gene family associated with rubber biosynthesis.

    PubMed

    Lau, Nyok-Sean; Makita, Yuko; Kawashima, Mika; Taylor, Todd D; Kondo, Shinji; Othman, Ahmad Sofiman; Shu-Chien, Alexander Chong; Matsui, Minami

    2016-06-24

    Hevea brasiliensis Muell. Arg, a member of the family Euphorbiaceae, is the sole natural resource exploited for commercial production of high-quality natural rubber. The properties of natural rubber latex are almost irreplaceable by synthetic counterparts for many industrial applications. A paucity of knowledge on the molecular mechanisms of rubber biosynthesis in high yield traits still persists. Here we report the comprehensive genome-wide analysis of the widely planted H. brasiliensis clone, RRIM 600. The genome was assembled based on ~155-fold combined coverage with Illumina and PacBio sequence data and has a total length of 1.55 Gb with 72.5% comprising repetitive DNA sequences. A total of 84,440 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed strong synteny between H. brasiliensis and other Euphorbiaceae genomes. Our data suggest that H. brasiliensis's capacity to produce high levels of latex can be attributed to the expansion of rubber biosynthesis-related genes in its genome and the high expression of these genes in latex. Using cap analysis gene expression data, we illustrate the tissue-specific transcription profiles of rubber biosynthesis-related genes, revealing alternative means of transcriptional regulation. Our study adds to the understanding of H. brasiliensis biology and provides valuable genomic resources for future agronomic-related improvement of the rubber tree.

  11. The rubber tree genome shows expansion of gene family associated with rubber biosynthesis

    PubMed Central

    Lau, Nyok-Sean; Makita, Yuko; Kawashima, Mika; Taylor, Todd D.; Kondo, Shinji; Othman, Ahmad Sofiman; Shu-Chien, Alexander Chong; Matsui, Minami

    2016-01-01

    Hevea brasiliensis Muell. Arg, a member of the family Euphorbiaceae, is the sole natural resource exploited for commercial production of high-quality natural rubber. The properties of natural rubber latex are almost irreplaceable by synthetic counterparts for many industrial applications. A paucity of knowledge on the molecular mechanisms of rubber biosynthesis in high yield traits still persists. Here we report the comprehensive genome-wide analysis of the widely planted H. brasiliensis clone, RRIM 600. The genome was assembled based on ~155-fold combined coverage with Illumina and PacBio sequence data and has a total length of 1.55 Gb with 72.5% comprising repetitive DNA sequences. A total of 84,440 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed strong synteny between H. brasiliensis and other Euphorbiaceae genomes. Our data suggest that H. brasiliensis’s capacity to produce high levels of latex can be attributed to the expansion of rubber biosynthesis-related genes in its genome and the high expression of these genes in latex. Using cap analysis gene expression data, we illustrate the tissue-specific transcription profiles of rubber biosynthesis-related genes, revealing alternative means of transcriptional regulation. Our study adds to the understanding of H. brasiliensis biology and provides valuable genomic resources for future agronomic-related improvement of the rubber tree. PMID:27339202

  12. Transcriptome Sequencing of Codonopsis pilosula and Identification of Candidate Genes Involved in Polysaccharide Biosynthesis

    PubMed Central

    Gao, Jian Ping; Wang, Dong; Cao, Ling Ya; Sun, Hai Feng

    2015-01-01

    Background Codonopsis pilosula (Franch.) Nannf. is one of the most widely used medicinal plants. Although chemical and pharmacological studies have shown that codonopsis polysaccharides (CPPs) are bioactive compounds and that their composition is variable, their biosynthetic pathways remain largely unknown. Next-generation sequencing is an efficient and high-throughput technique that allows the identification of candidate genes involved in secondary metabolism. Principal Findings To identify the components involved in CPP biosynthesis, a transcriptome library, prepared using root and other tissues, was assembled with the help of Illumina sequencing. A total of 9.2 Gb of clean nucleotides was obtained comprising 91,175,044 clean reads, 102,125 contigs, and 45,511 unigenes. After aligning the sequences to the public protein databases, 76.1% of the unigenes were annotated. Among these annotated unigenes, 26,189 were assigned to Gene Ontology categories, 11,415 to Clusters of Orthologous Groups, and 18,848 to Kyoto Encyclopedia of Genes and Genomes pathways. Analysis of abundance of transcripts in the library showed that genes, including those encoding metallothionein, aquaporin, and cysteine protease that are related to stress responses, were in the top list. Among genes involved in the biosynthesis of CPP, those responsible for the synthesis of UDP-L-arabinose and UDP-xylose were highly expressed. Significance To our knowledge, this is the first study to provide a public transcriptome dataset prepared from C. pilosula and an outline of the biosynthetic pathway of polysaccharides in a medicinal plant. Identified candidate genes involved in CPP biosynthesis provide understanding of the biosynthesis and regulation of CPP at the molecular level. PMID:25719364

  13. Identification and Expression Profiles of Sex Pheromone Biosynthesis and Transport Related Genes in Spodoptera litura

    PubMed Central

    Zhang, Ya-Nan; Zhu, Xiu-Yun; Fang, Li-Ping; He, Peng; Wang, Zhi-Qiang; Chen, Geng; Sun, Liang; Ye, Zhan-Feng; Deng, Dao-Gui; Li, Jin-Bu

    2015-01-01

    Although the general pathway of sex pheromone synthesis in moth species has been established, the molecular mechanisms remain poorly understood. The common cutworm Spodoptera litura is an important agricultural pest worldwide and causes huge economic losses annually. The female sex pheromone of S. litura comprises Z9,E11-14:OAc, Z9,E12-14:OAc, Z9-14:OAc, and E11-14:OAc. By sequencing and analyzing the transcriptomic data of the sex pheromone glands, we identified 94 candidate genes related to pheromone biosynthesis (55 genes) or chemoreception (39 genes). Gene expression patterns and phylogenetic analysis revealed that two desaturase genes (SlitDes5 and SlitDes11) and one fatty acyl reductase gene (SlitFAR3) showed pheromone gland (PG) biased or specific expression, and clustered with genes known to be involved in pheromone synthesis in other moth species. Furthermore, 4 chemoreception related genes (SlitOBP6, SlitOBP11, SlitCSP3, and SlitCSP14) also showed higher expression in the PG, and could be additional candidate genes involved in sex pheromone transport. This study provides the first solid background information that should facilitate further elucidation of sex pheromone biosynthesis and transport, and indicates potential targets to disrupt sexual communication in S. litura for a novel pest management strategy. PMID:26445454

  14. Identification and Expression Profiles of Sex Pheromone Biosynthesis and Transport Related Genes in Spodoptera litura.

    PubMed

    Zhang, Ya-Nan; Zhu, Xiu-Yun; Fang, Li-Ping; He, Peng; Wang, Zhi-Qiang; Chen, Geng; Sun, Liang; Ye, Zhan-Feng; Deng, Dao-Gui; Li, Jin-Bu

    2015-01-01

    Although the general pathway of sex pheromone synthesis in moth species has been established, the molecular mechanisms remain poorly understood. The common cutworm Spodoptera litura is an important agricultural pest worldwide and causes huge economic losses annually. The female sex pheromone of S. litura comprises Z9,E11-14:OAc, Z9,E12-14:OAc, Z9-14:OAc, and E11-14:OAc. By sequencing and analyzing the transcriptomic data of the sex pheromone glands, we identified 94 candidate genes related to pheromone biosynthesis (55 genes) or chemoreception (39 genes). Gene expression patterns and phylogenetic analysis revealed that two desaturase genes (SlitDes5 and SlitDes11) and one fatty acyl reductase gene (SlitFAR3) showed pheromone gland (PG) biased or specific expression, and clustered with genes known to be involved in pheromone synthesis in other moth species. Furthermore, 4 chemoreception related genes (SlitOBP6, SlitOBP11, SlitCSP3, and SlitCSP14) also showed higher expression in the PG, and could be additional candidate genes involved in sex pheromone transport. This study provides the first solid background information that should facilitate further elucidation of sex pheromone biosynthesis and transport, and indicates potential targets to disrupt sexual communication in S. litura for a novel pest management strategy.

  15. Contents of therapeutic metabolites in Swertia chirayita correlate with the expression profiles of multiple genes in corresponding biosynthesis pathways.

    PubMed

    Padhan, Jibesh Kumar; Kumar, Varun; Sood, Hemant; Singh, Tiratha Raj; Chauhan, Rajinder S

    2015-08-01

    Swertia chirayita, an endangered medicinal herb, contains three major secondary metabolites swertiamarin, amarogentin and mangiferin, exhibiting valuable therapeutic traits. No information exists as of today on the biosynthesis of these metabolites in S. chirayita. The current study reports the expression profiling of swertiamarin, amarogentin and mangiferin biosynthesis pathway genes and their correlation with the respective metabolites content in different tissues of S. chirayita. Root tissues of greenhouse grown plants contained the maximum amount of secoiridoids (swertiamarin, 2.8% of fr. wt and amarogentin, 0.1% of fr. wt), whereas maximum accumulation of mangiferin (1.0% of fr. wt) was observed in floral organs. Differential gene expression analysis and their subsequent principal component analysis unveiled ten genes (encoding HMGR, PMK, MVK, ISPD, ISPE, GES, G10H, 8HGO, IS and 7DLGT) of the secoiridoids biosynthesis pathway and five genes (encoding EPSPS, PAL, ADT, CM and CS) of mangiferin biosynthesis with elevated transcript amounts in relation to corresponding metabolite contents. Three genes of the secoiridoids biosynthesis pathway (encoding PMK, ISPD and IS) showed elevated levels (∼57-104 fold increase in roots), and EPSPS of mangiferin biosynthesis showed an about 117 fold increase in transcripts in leaf tissues of the greenhouse grown plants. The study does provide leads on potential candidate genes correlating with the metabolites biosynthesis in S. chirayita as an initiative towards its genetic improvement.

  16. Exploring the distribution of citrinin biosynthesis related genes among Monascus species.

    PubMed

    Chen, Yi-Pei; Tseng, Ching-Ping; Chien, I-Ling; Wang, Wei-Yi; Liaw, Li-Ling; Yuan, Gwo-Fang

    2008-12-24

    Citrinin, a hepato-nephrotoxic compound to humans, can be produced by the food fermentation microorganisms Monascus spp. In this study, we investigated the distribution of mycotoxin citrinin biosynthesis genes in 18 Monascus strains. The results show that the acyl-transferase and keto-synthase domains of the pksCT gene encoding citrinin polyketide synthase were found in Monascus purpureus, Monascus kaoliang, and Monascus sanguineus. Furthermore, the ctnA gene, a major activator for citrinin biosynthesis, was found in M. purpureus and M. kaoliang, but was absent in M. sanguineus. The orf3 gene encoding oxygenase, located between pksCT and ctnA, was also present in M. purpureus and M. kaoliang. The pksCT gene was highly conserved in M. purpureus, M. kaoliang, and M. sanguineus, while the ctnA and orf3 genes were shown to be highly homologous in M. purpureus and M. kaoliang. In contrast, the PCR and Southern blot analyses suggest that pksCT, ctnA, and orf3 were absent or significantly different in Monascus pilosus, Monascus ruber, Monascus barkeri, Monascus floridanus, Monascus lunisporas, and Monascus pallens. A citrinin-producing phenotype was detected only in M. purpureus and M. kaoliang using high performance liquid chromatography (HPLC). These results clearly indicate that the highly conserved citrinin gene cluster in M. purpureus and M. kaoliang carry out citrinin biosynthesis. In addition, according to the phylogenetic subgroups established with the beta-tubulin gene, the citrinin gene cluster can group the species of Monascus.

  17. SNP in starch biosynthesis genes associated with nutritional and functional properties of rice

    PubMed Central

    Kharabian-Masouleh, Ardashir; Waters, Daniel L. E.; Reinke, Russell F.; Ward, Rachelle; Henry, Robert J.

    2012-01-01

    Starch is a major component of human diets. The relative contribution of variation in the genes of starch biosynthesis to the nutritional and functional properties of the rice was evaluated in a rice breeding population. Sequencing 18 genes involved in starch synthesis in a population of 233 rice breeding lines discovered 66 functional SNPs in exonic regions. Five genes, AGPS2b, Isoamylase1, SPHOL, SSIIb and SSIVb showed no polymorphism. Association analysis found 31 of the SNP were associated with differences in pasting and cooking quality properties of the rice lines. Two genes appear to be the major loci controlling traits under human selection in rice, GBSSI (waxy gene) and SSIIa. GBSSI influenced amylose content and retrogradation. Other genes contributing to retrogradation were GPT1, SSI, BEI and SSIIIa. SSIIa explained much of the variation in cooking characteristics. Other genes had relatively small effects. PMID:22870386

  18. Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation.

    PubMed

    Piraee, Mahmood; White, Robert L; Vining, Leo C

    2004-01-01

    Five ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13-15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmlS) and conjugal transfer of the insertionally inactivated genes to S. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. 1H-NMR established that the principal metabolite in the disrupted strains was the alpha-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH2-dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.

  19. Conversion of 11-hydroxy-O-methylsterigmatocystin to aflatoxin G1 in Aspergillus parasiticus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In aflatoxin biosynthesis, aflatoxins G1 (AFG1) and B1 (AFB1) are independently produced from a common precursor, O-methylsterigmatocystin (OMST). Recently, 11-hydroxy-O-methylsterigmatocystin (HOMST) was identified as a later precursor involved in the conversion of OMST to AFB1. However, the invo...

  20. Genes involved in long-chain alkene biosynthesis in Micrococcus luteus

    SciTech Connect

    Beller, Harry R.; Goh, Ee-Been; Keasling, Jay D.

    2010-01-07

    Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which four decades ago was reported to biosynthesize iso- and anteiso branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty-acid overproducing E. coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-CoA produced the same C27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or -ACP) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (?-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen condensation during

  1. Identification of maize genes associated with host plant resistance and susceptibility to Aspergillus flavus infection and aflatoxin accumulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus infection and aflatoxin contamination of maize pose negative impacts in agriculture and health. Commercial maize hybrids are generally susceptible to this fungus. Significant levels of host plant resistance have been observed in certain maize inbred lines. This study was conducted...

  2. Correlation Index-Based Responsible-Enzyme Gene Screening (CIRES), a Novel DNA Microarray-Based Method for Enzyme Gene Involved in Glycan Biosynthesis

    PubMed Central

    Yamamoto, Harumi; Takematsu, Hiromu; Fujinawa, Reiko; Naito, Yuko; Okuno, Yasushi; Tsujimoto, Gozoh; Suzuki, Akemi; Kozutsumi, Yasunori

    2007-01-01

    Background Glycan biosynthesis occurs though a multi-step process that requires a variety of enzymes ranging from glycosyltransferases to those involved in cytosolic sugar metabolism. In many cases, glycan biosynthesis follows a glycan-specific, linear pathway. As glycosyltransferases are generally regulated at the level of transcription, assessing the overall transcriptional profile for glycan biosynthesis genes seems warranted. However, a systematic approach for assessing the correlation between glycan expression and glycan-related gene expression has not been reported previously. Methodology To facilitate genetic analysis of glycan biosynthesis, we sought to correlate the expression of genes involved in cell-surface glycan formation with the expression of the glycans, as detected by glycan-recognizing probes. We performed cross-sample comparisons of gene expression profiles using a newly developed, glycan-focused cDNA microarray. Cell-surface glycan expression profiles were obtained using flow cytometry of cells stained with plant lectins. Pearson's correlation coefficients were calculated for these profiles and were used to identify enzyme genes correlated with glycan biosynthesis. Conclusions This method, designated correlation index-based responsible-enzyme gene screening (CIRES), successfully identified genes already known to be involved in the biosynthesis of certain glycans. Our evaluation of CIRES indicates that it is useful for identifying genes involved in the biosynthesis of glycan chains that can be probed with lectins using flow cytometry. PMID:18043739

  3. Aflatoxins as a cause of hepatocellular carcinoma.

    PubMed

    Kew, Michael C

    2013-09-01

    Aflatoxins, metabolites of the fungi Aspergillus flavus and Aspergillus parasiticus, are frequent contaminants of a number of staple foods, particularly maize and ground nuts, in subsistence farming communities in tropical and sub-tropical climates in sub-Saharan Africa, Eastern Asia and parts of South America. Contamination of foods occurs during growth and as a result of storage in deficient or inappropriate facilities. These toxins pose serious public health hazards, including the causation of hepatocellular carcinoma by aflatoxin B1. Exposure begins in utero and is life-long. The innocuous parent molecule of the fungus is converted by members of the cytochrome p450 family into mutagenic and carcinogenic intermediates. Aflatoxin-B1 is converted into aflatoxin B1-8,9 exo-epoxide, which is in turn converted into 8,9-dihydroxy-8-(N7) guanyl-9-hydroxy aflatoxin B1 adduct. This adduct is metabolized into aflatoxin B1 formaminopyrimidine adduct. These adducts are mutagenic and carcinogenic. In addition, an arginine to serine mutation at codon 249 of the p53 tumor suppressor gene is produced, abrogating the function of the tumor suppressor gene, and contributing to hepatocarcinogenesis. Aflatoxin B1 acts synergistically with hepatitis B virus in causing hepatocellular carcinoma. A number of interactions between the two carcinogens may be responsible for this action, including integration of hepatitis B virus x gene and its consequences, as well as interference with nucleotide excision repair, activation of p21waf1/cip1, generation of DNA mutations, and altered methylation of genes. But much remains to be learnt about the precise pathogenetic mechanisms responsible for aflatoxin B1-induced hepatocellular carcinoma as well as the interaction between the toxin and hepatitis B virus in causing the tumor.

  4. Gene-to-metabolite network for biosynthesis of lignans in MeJA-elicited Isatis indigotica hairy root cultures

    PubMed Central

    Chen, Ruibing; Li, Qing; Tan, Hexin; Chen, Junfeng; Xiao, Ying; Ma, Ruifang; Gao, Shouhong; Zerbe, Philipp; Chen, Wansheng; Zhang, Lei

    2015-01-01

    Root and leaf tissue of Isatis indigotica shows notable anti-viral efficacy, and are widely used as “Banlangen” and “Daqingye” in traditional Chinese medicine. The plants' pharmacological activity is attributed to phenylpropanoids, especially a group of lignan metabolites. However, the biosynthesis of lignans in I. indigotica remains opaque. This study describes the discovery and analysis of biosynthetic genes and AP2/ERF-type transcription factors involved in lignan biosynthesis in I. indigotica. MeJA treatment revealed differential expression of three genes involved in phenylpropanoid backbone biosynthesis (IiPAL, IiC4H, Ii4CL), five genes involved in lignan biosynthesis (IiCAD, IiC3H, IiCCR, IiDIR, and IiPLR), and 112 putative AP2/ERF transcription factors. In addition, four intermediates of lariciresinol biosynthesis were found to be induced. Based on these results, a canonical correlation analysis using Pearson's correlation coefficient was performed to construct gene-to-metabolite networks and identify putative key genes and rate-limiting reactions in lignan biosynthesis. Over-expression of IiC3H, identified as a key pathway gene, was used for metabolic engineering of I. indigotica hairy roots, and resulted in an increase in lariciresinol production. These findings illustrate the utility of canonical correlation analysis for the discovery and metabolic engineering of key metabolic genes in plants. PMID:26579184

  5. Coordinated gene expression for pheromone biosynthesis in the pine engraver beetle, Ips pini (Coleoptera: Scolytidae)

    NASA Astrophysics Data System (ADS)

    Keeling, Christopher I.; Blomquist, Gary J.; Tittiger, Claus

    In several pine bark beetle species, phloem feeding induces aggregation pheromone production to coordinate a mass attack on the host tree. Male pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), produce the monoterpenoid pheromone component ipsdienol de novo via the mevalonate pathway in the anterior midgut upon feeding. To understand how pheromone production is regulated in this tissue, we used quantitative real-time PCR to examine feeding-induced changes in gene expression of seven mevalonate pathway genes: acetoacetyl-coenzyme A thiolase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate 5-diphosphate decarboxylase, isopentenyl-diphosphate isomerase, geranyl-diphosphate synthase (GPPS), and farnesyl-diphosphate synthase (FPPS). In males, expression of all these genes significantly increased upon feeding. In females, the expression of the early mevalonate pathway genes (up to and including the isomerase) increased significantly, but the expression of the later genes (GPPS and FPPS) was unaffected or decreased upon feeding. Thus, feeding coordinately regulates expression of the mevalonate pathway genes necessary for pheromone biosynthesis in male, but not female, midguts. Furthermore, basal mRNA levels were 5- to 41-fold more abundant in male midguts compared to female midguts. This is the first report of coordinated regulation of mevalonate pathway genes in an invertebrate model consistent with their sex-specific role in de novo pheromone biosynthesis.

  6. Carotenoids in unexpected places: gall midges, lateral gene transfer, and carotenoid biosynthesis in animals.

    PubMed

    Cobbs, Cassidy; Heath, Jeremy; Stireman, John O; Abbot, Patrick

    2013-08-01

    Carotenoids are conjugated isoprenoid molecules with many important physiological functions in organisms, including roles in photosynthesis, oxidative stress reduction, vision, diapause, photoperiodism, and immunity. Until recently, it was believed that only plants, microorganisms, and fungi were capable of synthesizing carotenoids and that animals acquired them from their diet, but recent studies have demonstrated that two arthropods (pea aphid and spider mite) possess a pair of genes homologous to those required for the first step of carotenoid biosynthesis. Absent in all other known animal genomes, these genes appear to have been acquired by aphids and spider mites in one or several lateral gene transfer events from a fungal donor. We report the third case of fungal carotenoid biosynthesis gene homologs in an arthropod: flies from the family Cecidomyiidae, commonly known as gall midges. Using phylogenetic analyses we show that it is unlikely that lycopene cyclase/phytoene synthase and phytoene desaturase homologs were transferred singly to an ancient arthropod ancestor; instead we propose that genes were transferred independently from related fungal donors after divergence of the major arthropod lineages. We also examine variation in intron placement and copy number of the carotenoid genes that may underlie function in the midges. This trans-kingdom transfer of carotenoid genes may represent a key innovation, underlying the evolution of phytophagy and plant-galling in gall midges and facilitating their extensive diversification across plant lineages.

  7. A Photoperiod-Regulating Gene CONSTANS Is Correlated to Lipid Biosynthesis in Chlamydomonas reinhardtii

    PubMed Central

    Deng, Xiaodong; Fan, Xinzhao; Li, Ping; Fei, Xiaowen

    2015-01-01

    Background. The regulation of lipid biosynthesis is essential in photosynthetic eukaryotic cells. Thus far, no regulatory genes have been reported in the lipid metabolism pathway. Plant CONSTANS (CO) gene regulates blooming by participating in photoperiod and biological clock. Apart from regulating photoperiod, the Chlamydomonas CO gene also regulates starch content. Results. In this study, the results showed that, under HSM-S condition, cells accumulated more lipids at short-day conditions than at long-day conditions. The silencing of the CrCO gene via RNA interference resulted in an increase in lipid content and an increase in triacylglyceride (TAG) level by 24.5%. CrCO RNAi strains accumulated more lipids at short-day conditions than at long-day conditions. The decrease in CrCO expression resulted in the increased expression of TAG biosynthesis-related genes, such as DGAT2, PAP2, and PDAT3, whereas CIS and FBP1 genes showed a decrease in their mRNA when the CrCO expression was suppressed. On the other hand, the overexpression of CrCO resulted in the decrease in lipid content and TAG level. Conclusions. The results of this study revealed a relationship between CrCO gene and lipid metabolism in Chlamydomonas, suggesting that increasing oil by suppressing CrCO expression in microalgae is feasible. PMID:25654119

  8. A potato skin SSH library yields new candidate genes for suberin biosynthesis and periderm formation.

    PubMed

    Soler, Marçal; Serra, Olga; Fluch, Silvia; Molinas, Marisa; Figueras, Mercè

    2011-05-01

    Potato (Solanum tuberosum) tubers are underground storage organs covered by the skin or periderm, a suberized layer that protects inner flesh from dehydration and pathogens. Understanding the molecular processes associated with periderm formation is of great importance for a better knowledge of this protective tissue and for improving the storage life of tubers. Here, to isolate new candidate genes for potato periderm, a suppression subtractive hybridization library from potato skin was performed. This library yielded a comprehensive list of 108 candidate genes that were manually sorted in functional categories according to the main cellular and metabolic processes in periderm. As expected, the list contains Suberin and wax genes, including some genes with a demonstrated role in the biosynthesis of these cell wall aliphatic compounds. Moreover, Regulation and Stress and defence genes are highly abundant in the library in general agreement with previous potato skin proteomic studies. The putative function of the genes in periderm is discussed.

  9. Yeast HXK2 gene reverts glucose regulation mutation of penicillin biosynthesis in P. chrysogenum.

    PubMed

    Pérez, Edmundo A; Fernández, Francisco J; Fierro, Francisco; Mejía, Armando; Marcos, Ana T; Martín, Juan F; Barrios-González, Javier

    2014-01-01

    The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and β-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of β-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and β-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent.

  10. Yeast HXK2 gene reverts glucose regulation mutation of penicillin biosynthesis in P. chrysogenum

    PubMed Central

    Pérez, Edmundo A.; Fernández, Francisco J.; Fierro, Francisco; Mejía, Armando; Marcos, Ana T.; Martín, Juan F.; Barrios-González, Javier

    2014-01-01

    The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and β-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of β-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and β-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent. PMID:25477921

  11. AtTHIC, a gene involved in thiamine biosynthesis in Arabidopsis thaliana.

    PubMed

    Kong, Danyu; Zhu, Yuxing; Wu, Huilan; Cheng, Xudong; Liang, Hui; Ling, Hong-Qing

    2008-05-01

    Thiamine (vitamin B(1)) is an essential compound for organisms. It contains a pyrimidine ring structure and a thiazole ring structure. These two moieties of thiamine are synthesized independently and then coupled together. Here we report the molecular characterization of AtTHIC, which is involved in thiamine biosynthesis in Arabidopsis. AtTHIC is similar to Escherichia coli ThiC, which is involved in pyrimidine biosynthesis in prokaryotes. Heterologous expression of AtTHIC could functionally complement the thiC knock-out mutant of E. coli. Downregulation of AtTHIC expression by T-DNA insertion at its promoter region resulted in a drastic reduction of thiamine content in plants and the knock-down mutant thic1 showed albino (white leaves) and lethal phenotypes under the normal culture conditions. The thic1 mutant could be rescued by supplementation of thiamine and its defect functions could be complemented by expression of AtTHIC cDNA. Transient expression analysis revealed that the AtTHIC protein targets plastids and chloroplasts. AtTHIC was strongly expressed in leaves, flowers and siliques and the transcription of AtTHIC was downregulated by extrinsic thiamine. In conclusion, AtTHIC is a gene involved in pyrimidine synthesis in the thiamine biosynthesis pathway of Arabidopsis, and our results provide some new clues for elucidating the pathway of thiamine biosynthesis in plants.

  12. The role of enoyl reductase genes in phloridzin biosynthesis in apple.

    PubMed

    Dare, Andrew P; Tomes, Sumathi; Cooney, Janine M; Greenwood, David R; Hellens, Roger P

    2013-11-01

    Phloridzin is the predominant polyphenol in apple (Malus × domestica Borkh.) where it accumulates to high concentrations in many tissues including the leaves, bark, roots and fruit. Despite its relative abundance in apple the biosynthesis of phloridzin and other related dihydrochalcones remains only partially understood. The key unidentified enzyme in phloridzin biosynthesis is a putative carbon double bond reductase which is thought to act on p-coumaroyl-CoA to produce the dihydro-p-coumaroyl-CoA precursor. A functional screen of six apple enoyl reductase-like (ENRL) genes was carried out using transient infiltration into tobacco and gene silencing by RNA interference (RNAi) in order to determine carbon double bond reductase activity and contribution to foliar phloridzin concentrations. The ENRL-3 gene caused a significant increase in phloridzin concentration when infiltrated into tobacco leaves whilst a second protein ENRL-5, with over 98% amino acid sequence similarity to ENRL-3, showed p-coumaroyl-CoA reductase activity in enzyme assays. Finally, an RNAi study showed that reducing the transcript levels of ENRL-3 in transgenic 'Royal Gala' led to a 66% decrease in the concentration of dihydrochalcones in the leaves in the one available silenced line. Overall these results suggest that ENRL-3, and its close homolog ENRL-5, may contribute to the biosynthesis of phloridzin in apple.

  13. The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis.

    PubMed Central

    MacCabe, A P; Riach, M B; Unkles, S E; Kinghorn, J R

    1990-01-01

    Clones of Aspergillus nidulans genomic DNA spanning 20 kb have been isolated and shown by a combination of classical and molecular genetic means to represent the npeA locus, previously found to be one of four loci (npeA, npeB, npeC and npeD) involved in the synthesis of penicillin. As well as containing the gene encoding the second enzyme for penicillin biosynthesis, namely isopenicillin N synthetase (IPNS) (designated ipnA), our results show that these clones (pSTA200, pSTA201 and pSTA207) contain two more genes to form a cluster of three contiguous penicillin biosynthetic genes. Our evidence suggests that these genes encode delta (L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and acyl transferase (ACYT) (designated acvA and acyA respectively), the first and third enzymes required for penicillin biosynthesis, with the gene order being acvA-ipnA-acyA. Transcripts have been identified for the three genes and their approximate sizes determined--acvA 9.5 kb, ipnA 1.4 kb and acyA 1.6 kb. All three mRNA species are observed in cells grown in fermentation medium but not in cells grown in minimal medium, suggesting that the control of penicillin biosynthesis is, in part, at the level of mRNA accumulation. Finally our results show that acvA and ipnA genes are divergently transcribed, whilst acyA is transcribed in the same orientation as ipnA. Images Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:2403928

  14. Identification and Expression Analysis of Candidate Genes Involved in Carotenoid Biosynthesis in Chickpea Seeds

    PubMed Central

    Rezaei, Mohammad K.; Deokar, Amit; Tar'an, Bunyamin

    2016-01-01

    Plant carotenoids have a key role in preventing various diseases in human because of their antioxidant and provitamin A properties. Chickpea is a good source of carotenoid among legumes and its diverse germplasm and genome accessibility makes it a good model for carotenogenesis studies. The structure, location, and copy numbers of genes involved in carotenoid biosynthesis were retrieved from the chickpea genome. The majority of the single nucleotide polymorphism (SNPs) within these genes across five diverse chickpea cultivars was synonymous mutation. We examined the expression of the carotenogenesis genes and their association with carotenoid concentration at different seed development stages of five chickpea cultivars. Total carotenoid concentration ranged from 22 μg g−1 in yellow cotyledon kabuli to 44 μg g−1 in green cotyledon desi at 32 days post anthesis (DPA). The majority of carotenoids in chickpea seeds consists of lutein and zeaxanthin. The expression of the selected 19 genes involved in carotenoid biosynthesis pathway showed common pattern across five cultivars with higher expression at 8 and/or 16 DPA then dropped considerably at 24 and 32 DPA. Almost all genes were up-regulated in CDC Jade cultivar. Correlation analysis between gene expression and carotenoid concentration showed that the genes involved in the primary step of carotenoid biosynthesis pathway including carotenoid desaturase and isomerase positively correlated with various carotenoid components in chickpea seeds. A negative correlation was found between hydroxylation activity and provitamin A concentration in the seeds. The highest provitamin A concentration including β-carotene and β-cryptoxanthin were found in green cotyledon chickpea cultivars. PMID:28018400

  15. Molecular evolution and functional characterisation of haplotypes of an important rubber biosynthesis gene in Hevea brasiliensis.

    PubMed

    Uthup, T K; Rajamani, A; Ravindran, M; Saha, T

    2016-07-01

    Hydroxy-methylglutaryl coenzyme-A synthase (HMGS) is a rate-limiting enzyme in the cytoplasmic isoprenoid biosynthesis pathway leading to natural rubber production in Hevea brasiliensis (rubber). Analysis of the structural variants of this gene is imperative to understand their functional significance in rubber biosynthesis so that they can be properly utilised for ongoing crop improvement programmes in Hevea. We report here allele richness and diversity of the HMGS gene in selected popular rubber clones. Haplotypes consisting of single nucleotide polymorphisms (SNPs) from the coding and non-coding regions with a high degree of heterozygosity were identified. Segregation and linkage disequilibrium analysis confirmed that recombination is the major contributor to the generation of allelic diversity, rather than point mutations. The evolutionarily conserved nature of some SNPs was identified by comparative DNA sequence analysis of HMGS orthologues from diverse taxa, demonstrating the molecular evolution of rubber biosynthesis genes in general. In silico three-dimensional structural studies highlighting the structural positioning of non-synonymous SNPs from different HMGS haplotypes revealed that the ligand-binding site on the enzyme remains impervious to the reported sequence variations. In contrast, gene expression results indicated the possibility of association between specific haplotypes and HMGS expression in Hevea clones, which may have a downstream impact up to the level of rubber production. Moreover, haplotype diversity of the HMGS gene and its putative association with gene expression can be the basis for further genetic association studies in rubber. Furthermore, the data also show the role of SNPs in the evolution of candidate genes coding for functional traits in plants.

  16. Digital Gene Expression Profiling to Explore Differentially Expressed Genes Associated with Terpenoid Biosynthesis during Fruit Development in Litsea cubeba.

    PubMed

    Gao, Ming; Lin, Liyuan; Chen, Yicun; Wang, Yangdong

    2016-09-20

    Mountain pepper (Litseacubeba (Lour.) Pers.) (Lauraceae) is an important industrial crop as an ingredient in cosmetics, pesticides, food additives and potential biofuels. These properties are attributed to monoterpenes and sesquiterpenes. However, there is still no integrated model describing differentially expressed genes (DEGs) involved in terpenoid biosynthesis during the fruit development of L. cubeba. Here, we performed digital gene expression (DGE) using the Illumina NGS platform to evaluated changes in gene expression during fruit development in L. cubeba. DGE generated expression data for approximately 19354 genes. Fruit at 60 days after flowering (DAF) served as the control, and a total of 415, 1255, 449 and 811 up-regulated genes and 505, 1351, 1823 and 1850 down-regulated genes were identified at 75, 90, 105 and 135 DAF, respectively. Pathway analysis revealed 26 genes involved in terpenoid biosynthesis pathways. Three DEGs had continued increasing or declining trends during the fruit development. The quantitative real-time PCR (qRT-PCR) results of five differentially expressed genes were consistent with those obtained from Illumina sequencing. These results provide a comprehensive molecular biology background for research on fruit development, and information that should aid in metabolic engineering to increase the yields of L. cubeba essential oil.

  17. Detection and quantification of Aspergillus section Flavi spp. in stored peanuts by real-time PCR of nor-1 gene, and effects of storage conditions on aflatoxin production.

    PubMed

    Passone, María Alejandra; Rosso, Laura Cristina; Ciancio, Aurelio; Etcheverry, Miriam

    2010-04-15

    Aspergillus flavus and A. parasiticus are the main species from section Flavi responsible for aflatoxin accumulation in stored peanuts. A real-time PCR (RT-PCR) system directed against the nor-1 gene of the aflatoxin biosynthetic pathway as target sequence was applied to monitor and quantify Aspergillus section Flavi population in peanuts. Kernels were conditioned at four water activity (a(W)) levels and stored during a 4-month period. The quantification of fungal genomic DNA in naturally contaminated peanut samples was performed using TaqMan fluorescent probe technology. Sensitivity tests demonstrated that DNA amounts accounting for a single conidium of A. parasiticus RCP08300 can be detected. A standard curve relating nor-1 copy numbers to colony forming units (cfu) was constructed. Counts of species of Aspergillus section Flavi from unknown samples obtained by molecular and conventional count (CC) methodologies were compared. A correlation between cfu data obtained by RT-PCR and CC methods was observed (r=0.613; p<0.0001); and the former always showed values higher by 0.5-1 log units. A decrease of fungal density was observed throughout the storage period, regardless of the quantification methodology applied. Total aflatoxin levels ranging from 1.1 to 200.4 ng/g were registered in peanuts conditioned at the higher a(W) values (0.94-0.84 a(W)). The RT-PCR assay developed appears to be a promising tool in the prediction of potential aflatoxigenic risk in stored peanuts, even in case of low-level infections, and suitable for rapid, automated and high throughput analysis.

  18. Transcriptional analysis of exopolysaccharides biosynthesis gene clusters in Lactobacillus plantarum.

    PubMed

    Vastano, Valeria; Perrone, Filomena; Marasco, Rosangela; Sacco, Margherita; Muscariello, Lidia

    2016-04-01

    Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and find applications also in non-dairy foods and in therapeutics. Recently, four clusters of genes (cps) associated with surface polysaccharide production have been identified in Lactobacillus plantarum WCFS1, a probiotic and food-associated lactobacillus. These clusters are involved in cell surface architecture and probably in release and/or exposure of immunomodulating bacterial molecules. Here we show a transcriptional analysis of these clusters. Indeed, RT-PCR experiments revealed that the cps loci are organized in five operons. Moreover, by reverse transcription-qPCR analysis performed on L. plantarum WCFS1 (wild type) and WCFS1-2 (ΔccpA), we demonstrated that expression of three cps clusters is under the control of the global regulator CcpA. These results, together with the identification of putative CcpA target sequences (catabolite responsive element CRE) in the regulatory region of four out of five transcriptional units, strongly suggest for the first time a role of the master regulator CcpA in EPS gene transcription among lactobacilli.

  19. Isolation and identification of fungi from a meju contaminated with aflatoxins.

    PubMed

    Jung, Yu Jung; Chung, Soo Hyun; Lee, Hyo Ku; Chun, Hyang Sook; Hong, Seung Beom

    2012-12-01

    A home-made meju sample contaminated naturally with aflatoxins was used for isolation of fungal strains. Overall, 230 fungal isolates were obtained on dichloran rosebengal chloramphenicol (DRBC) and dichloran 18% glycerol (DG18) agar plates. Morphological characteristics and molecular analysis of a partial beta-tubulin gene and the internal transcribed spacer (ITS) of rDNA were used for the identification of the isolates. The fungal isolates were divided into 7 genera: Aspergillus, Eurotium, Penicillium, Eupenicillium, Mucor, Lichtheimia, and Curvularia. Three strains from 56 isolates of the A. oryzae/flavus group were found to be aflatoxigenic A. flavus, by the presence of the aflatoxin biosynthesis genes and confirmatory aflatoxin production by high-performance liquid chromatography (HPLC). The predominant isolate from DRBC plates was A. oryzae (42 strains, 36.2%), whereas that from DG18 was A. candidus (61 strains, 53.5%). Out of the 230 isolates, the most common species was A. candidus (34.3%) followed by A. oryzae (22.2%), Mucor circinelloides (13.0%), P. polonicum (10.0%), A. tubingensis (4.8%), and L. ramosa (3.5%). A. flavus and E. chevalieri presented occurrence levels of 2.2%, respectively. The remaining isolates of A. unguis, P. oxalicum, Eupenicillium cinnamopurpureum, A. acidus, E. rubrum, P. chrysogenum, M. racemosus, and C. inaequalis had lower occurrence levels of < 2.0%.

  20. Functional Characterization of 4'OMT and 7OMT Genes in BIA Biosynthesis.

    PubMed

    Gurkok, Tugba; Ozhuner, Esma; Parmaksiz, Iskender; Özcan, Sebahattin; Turktas, Mine; İpek, Arif; Demirtas, Ibrahim; Okay, Sezer; Unver, Turgay

    2016-01-01

    Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L.), the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA) including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes remain to be revealed. In this study, expressions of 3-hydroxy-N-methylcoclaurine 4'-methyltransferase (4'OMT) and reticuline 7-O-methyltransferase (7OMT) genes were subjected to manipulation to functionally characterize their roles in BIA biosynthesis. Measurements of alkaloid accumulation were performed in leaf, stem, and capsule tissues accordingly. Suppression of 4'OMT expression caused reduction in the total alkaloid content in stem tissue whereas total alkaloid content was significantly induced in the capsule. Silencing of the 7OMT gene also caused repression in total alkaloid content in the stem. On the other hand, over-expression of 4'OMT and 7OMT resulted in higher morphine accumulation in the stem but suppressed amount in the capsule. Moreover, differential expression in several BIA synthesis genes (CNMT, TYDC, 6OMT, SAT, COR, 4'OMT, and 7OMT) were observed upon manipulation of 4'OMT and 7OMT expression. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4'OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues.

  1. Transcriptome Analysis of Medicinal Plant Salvia miltiorrhiza and Identification of Genes Related to Tanshinone Biosynthesis

    PubMed Central

    Yang, Lei; Ding, Guohui; Lin, Haiyan; Cheng, Haining; Kong, Yu; Wei, Yukun; Fang, Xin; Liu, Renyi; Wang, Lingiian; Chen, Xiaoya; Yang, Changqing

    2013-01-01

    Salvia miltiorrhiza Bunge, a perennial plant of Lamiaceae, accumulates abietane-type diterpenoids of tanshinones in root, which have been used as traditional Chinese medicine to treat neuroasthenic insomnia and cardiovascular diseases. However, to date the biosynthetic pathway of tanshinones is only partially elucidated and the mechanism for their root-specific accumulation remains unknown. To identify enzymes and transcriptional regulators involved in the biosynthesis of tanshinones, we conducted transcriptome profiling of S. miltiorrhiza root and leaf tissues using the 454 GS-FLX pyrosequencing platform, which generated 550,546 and 525,292 reads, respectively. RNA sequencing reads were assembled and clustered into 64,139 unigenes (29,883 isotigs and 34,256 singletons). NCBI non-redundant protein databases (NR) and Swiss-Prot database searches anchored 32,096 unigenes (50%) with functional annotations based on sequence similarities. Further assignments with Gene Ontology (GO) terms and KEGG biochemical pathways identified 168 unigenes referring to the terpenoid backbone biosynthesis (including 144 MEP and MVA pathway genes and 24 terpene synthases). Comparative analysis of the transcriptomes identified 2,863 unigenes that were highly expressed in roots, including those encoding enzymes of early steps of tanshinone biosynthetic pathway, such as copalyl diphosphate synthase (SmCPS), kaurene synthase-like (SmKSL) and CYP76AH1. Other differentially expressed unigenes predicted to be related to tanshinone biosynthesis fall into cytochrome P450 monooxygenases, dehydrogenases and reductases, as well as regulatory factors. In addition, 21 P450 genes were selectively confirmed by real-time PCR. Thus we have generated a large unigene dataset which provides a valuable resource for further investigation of the radix development and biosynthesis of tanshinones. PMID:24260395

  2. Enzymology of aminoglycoside biosynthesis-deduction from gene clusters.

    PubMed

    Wehmeier, Udo F; Piepersberg, Wolfgang

    2009-01-01

    The classical aminoglycosides are, with very few exceptions, typically actinobacterial secondary metabolites with antimicrobial activities all mediated by inhibiting translation on the 30S subunit of the bacterial ribosome. Some chemically related natural products inhibit glucosidases by mimicking oligo-alpha-1,4-glucosides. The biochemistry of the aminoglycoside biosynthetic pathways is still a developing field since none of the pathways has been analyzed to completeness as yet. In this chapter we treat the enzymology of aminoglycoside biosyntheses as far as it becomes apparent from recent investigations based on the availability of DNA sequence data of biosynthetic gene clusters for all major structural classes of these bacterial metabolites. We give a more general overview of the field, including descriptions of some key enzymes in various aminoglycoside pathways, whereas in Chapter 20 provides a detailed account of the better-studied enzymology thus far known for the neomycin and butirosin pathways.

  3. Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds

    SciTech Connect

    Josse, Rozenn; Dumont, Julie; Fautrel, Alain; Robin, Marie-Anne; Guillouzo, André

    2012-01-15

    Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24 h exposure of these hepatocyte models to 0.05 and 0.25 μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cell cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds. -- Highlights: ► Gene expression profile changes induced by aflatoxin B1 in human hepatocytes. ► AFB1 modulates various genes including tumor suppressor genes and proto-oncogenes. ► Important inter-individual variations in the response to AFB1. ► Some genes also altered by other

  4. Coregulated expression of loline alkaloid-biosynthesis genes in Neotyphodium uncinatum cultures.

    PubMed

    Zhang, Dong-Xiu; Stromberg, Arnold J; Spiering, Martin J; Schardl, Christopher L

    2009-08-01

    Epichloë endophytes (holomorphic Epichloë spp. and anamorphic Neotyphodium spp.) are systemic, often heritable symbionts of cool-season grasses (subfamily Pooideae). Many epichloae provide protection to their hosts by producing anti-insect compounds. Among these are the loline alkaloids (LA), which are toxic and deterrent to a broad range of herbivorous insects but not to mammalian herbivores. LOL, a gene cluster containing nine genes, is associated with LA biosynthesis. We investigated coordinate regulation between LOL-gene expression and LA production in minimal medium (MM) cultures of Neotyphodium uncinatum. Expression of all LOL genes significantly fit temporal quadratic patterns during LA production. LOL-gene expression started before LA were detectable, and increased while LA accumulated. The highest gene expression level was reached at close to the time of most rapid LA accumulation, and gene expression declined to a very low level as amounts of LA plateaued. Temporal expression profiles of the nine LOL genes were tightly correlated with each other, but not as tightly correlated with proC and metE (genes for biosynthesis of precursor amino acids). Furthermore, the start days and peak days of expression significantly correlated with the order of the LOL-cluster genes in the genome. Hierarchical cluster analysis indicated three pairs of genes-lolA and lolC, lolO and lolD, and lolT and lolE-expression of which was especially tightly correlated. Of these, lolA and lolC tended to be expressed early, and lolT and lolE tended to be expressed late, in keeping with the putative roles of the respective gene products in the LA-biosynthesis pathway. Several common transcriptional binding sites were discovered in the LOL upstream regions. However, low expression of P(lolC2)uidA and P(lolA2)uidA in N. uncinatum transformants suggested induced expression of LOL genes might be subject to position effect at the LOL locus.

  5. Identification and Characterization of Multiple Intermediate Alleles of the Key Genes Regulating Brassinosteroid Biosynthesis Pathways

    PubMed Central

    Du, Junbo; Zhao, Baolin; Sun, Xin; Sun, Mengyuan; Zhang, Dongzhi; Zhang, Shasha; Yang, Wenyu

    2017-01-01

    Most of the early identified brassinosteroid signaling and biosynthetic mutants are null mutants, exhibiting extremely dwarfed phenotypes and male sterility. These null mutants are usually unable to be directly transformed via a routinely used Agrobacterium-mediated gene transformation system and therefore are less useful for genetic characterization of the brassinosteroid (BR)-related pathways. Identification of intermediate signaling mutants such as bri1–5 and bri1–9 has contributed drastically to the elucidation of BR signaling pathway using both genetic and biochemical approaches. However, intermediate mutants of key genes regulating BR biosynthesis have seldom been reported. Here we report identification of several intermediate BR biosynthesis mutants mainly resulted from leaky transcriptions due to the insertions of T-DNAs in the introns. These mutants are semi-dwarfed and fertile and capable to be transformed. These intermediate mutants could be useful tools for future discovery and analyses of novel components regulating BR biosynthesis and catabolism via genetic modifier screen. PMID:28138331

  6. Identification and Characterization of a Novel Biotin Biosynthesis Gene in Saccharomyces cerevisiae

    PubMed Central

    Wu, Hong; Ito, Kiyoshi; Shimoi, Hitoshi

    2005-01-01

    Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae. PMID:16269718

  7. Complex Patterns of Gene Fission in the Eukaryotic Folate Biosynthesis Pathway

    PubMed Central

    Maguire, Finlay; Henriquez, Fiona L.; Leonard, Guy; Dacks, Joel B.; Brown, Matthew W.; Richards, Thomas A.

    2014-01-01

    Shared derived genomic characters can be useful for polarizing phylogenetic relationships, for example, gene fusions have been used to identify deep-branching relationships in the eukaryotes. Here, we report the evolutionary analysis of a three-gene fusion of folB, folK, and folP, which encode enzymes that catalyze consecutive steps in de novo folate biosynthesis. The folK-folP fusion was found across the eukaryotes and a sparse collection of prokaryotes. This suggests an ancient derivation with a number of gene losses in the eukaryotes potentially as a consequence of adaptation to heterotrophic lifestyles. In contrast, the folB-folK-folP gene is specific to a mosaic collection of Amorphea taxa (a group encompassing: Amoebozoa, Apusomonadida, Breviatea, and Opisthokonta). Next, we investigated the stability of this character. We identified numerous gene losses and a total of nine gene fission events, either by break up of an open reading frame (four events identified) or loss of a component domain (five events identified). This indicates that this three gene fusion is highly labile. These data are consistent with a growing body of data indicating gene fission events occur at high relative rates. Accounting for these sources of homoplasy, our data suggest that the folB-folK-folP gene fusion was present in the last common ancestor of Amoebozoa and Opisthokonta but absent in the Metazoa including the human genome. Comparative genomic data of these genes provides an important resource for designing therapeutic strategies targeting the de novo folate biosynthesis pathway of a variety of eukaryotic pathogens such as Acanthamoeba castellanii. PMID:25252772

  8. Comparative analysis of transcription factor gene families from Papaver somniferum: identification of regulatory factors involved in benzylisoquinoline alkaloid biosynthesis.

    PubMed

    Agarwal, Parul; Pathak, Sumya; Lakhwani, Deepika; Gupta, Parul; Asif, Mehar Hasan; Trivedi, Prabodh Kumar

    2016-05-01

    Opium poppy (Papaver somniferum L.), known for biosynthesis of several therapeutically important benzylisoquinoline alkaloids (BIAs), has emerged as the premier organism to study plant alkaloid metabolism. The most prominent molecules produced in opium poppy include narcotic analgesic morphine, the cough suppressant codeine, the muscle relaxant papaverine and the anti-microbial agent sanguinarine and berberine. Despite several health benefits, biosynthesis of some of these molecules is very low due to tight temporal and spatial regulation of the genes committed to their biosynthesis. Transcription factors, one of the prime regulators of secondary plant product biosynthesis, might be involved in controlled biosynthesis of BIAs in P. somniferum. In this study, identification of members of different transcription factor gene families using transcriptome datasets of 10 cultivars of P. somniferum with distinct chemoprofile has been carried out. Analysis suggests that most represented transcription factor gene family in all the poppy cultivars is WRKY. Comparative transcriptome analysis revealed differential expression pattern of the members of a set of transcription factor gene families among 10 cultivars. Through analysis, two members of WRKY and one member of C3H gene family were identified as potential candidates which might regulate thebaine and papaverine biosynthesis, respectively, in poppy.

  9. RNA-seq Analysis of Overexpressing Ovine AANAT Gene of Melatonin Biosynthesis in Switchgrass

    PubMed Central

    Yuan, Shan; Huang, Yanhua; Liu, Sijia; Guan, Cong; Cui, Xin; Tian, Danyang; Zhang, Yunwei; Yang, Fuyu

    2016-01-01

    Melatonin serves important functions in the promotion of growth and anti-stress regulation by efficient radical scavenging and regulation of antioxidant enzyme activity in various plants. To investigate its regulatory roles and metabolism pathways, the transcriptomic profile of overexpressing the ovine arylalkylamine N-acetyltransferase (oAANAT) gene, encoding the penultimate enzyme in melatonin biosynthesis, was compared with empty vector control using RNA-seq in switchgrass, a model plant of cellulosic ethanol conversion. The 85.22 million high quality reads that were assembled into 135,684 unigenes were generated by Illumina sequencing for transgenic oAANAT switchgrass with an average sequence length of 716 bp. A total of 946 differentially expression genes in transgenic line comparing to control switchgrass, including 737 up-regulated and 209 down-regulated genes, were mainly enriched with two main functional patterns of melatonin identifying by gene ontology analysis: the growth regulator and stress tolerance. Furthermore, KEGG maps indicated that the biosynthetic pathways of secondary metabolite (phenylpropanoids, flavonoids, steroids, stilbenoid, diarylheptanoid, and gingerol) and signaling pathways (MAPK signaling pathway, estrogen signaling pathway) were involved in melatonin metabolism. This study substantially expands the transcriptome information for switchgrass and provides valuable clues for identifying candidate genes involved in melatonin biosynthesis and elucidating the mechanism of melatonin metabolism. PMID:27656186

  10. Characterization of the Promoter Region of Biosynthetic Enzyme Genes Involved in Berberine Biosynthesis in Coptis japonica

    PubMed Central

    Yamada, Yasuyuki; Yoshimoto, Tadashi; Yoshida, Sayumi T.; Sato, Fumihiko

    2016-01-01

    The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs), a plant-specific WRKY-type TF, CjWRKY1, and a basic helix-loop-helix TF, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4′OMT and CYP719A1) were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC) reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay and by a chromatin immunoprecipitation assay. In addition, CjbHLH1 also activated transcription from truncated 4′OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. japonica are discussed. PMID:27642289

  11. Variations in critical morphine biosynthesis genes and their potential to influence human health.

    PubMed

    Mantione, Kirk; Kream, Richard M; Stefano, George B

    2010-01-01

    Endogenous morphine has been detected in human tissues from the vascular, immune and nervous systems. The genes/enzymes (CYP2D6, COMT and PNMT) that are involved in the biosynthesis of morphine have variations that affect their functionality. Some of these variations are the result of single nucleotide polymorphisms of DNA sequences. This review highlights some of the functional differences in the critical enzymes required for the biosynthesis of morphine that may affect human health. These variations have been shown to change the way animals react to stressors, perceive pain and behave. The presence of morphine signaling in almost all organ systems suggests that it is most likely playing a role in maintaining the health and promoting the normal functioning of these physiological systems.

  12. Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium

    PubMed Central

    Lohße, Anna; Kolinko, Isabel; Raschdorf, Oliver; Uebe, René; Borg, Sarah; Brachmann, Andreas; Plitzko, Jürgen M.; Müller, Rolf; Zhang, Youming

    2016-01-01

    ABSTRACT Magnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear chain. The number and size of magnetosome particles have to be critically controlled to build a sensor sufficiently strong to ensure the efficient alignment of cells within Earth's weak magnetic field while at the same time minimizing the metabolic costs imposed by excessive magnetosome biosynthesis. Apart from their biological function, bacterial magnetosomes have gained considerable interest since they provide a highly useful model for prokaryotic organelle formation and represent biogenic magnetic nanoparticles with exceptional properties. However, potential applications have been hampered by the difficult cultivation of these fastidious bacteria and their poor yields of magnetosomes. In this study, we found that the size and number of magnetosomes within the cell are controlled by many different Mam and Mms proteins. We present a strategy for the overexpression of magnetosome biosynthesis genes in the alphaproteobacterium Magnetospirillum gryphiswaldense by chromosomal multiplication of individual and multiple magnetosome gene clusters via transposition. While stepwise amplification of the mms6 operon resulted in the formation of increasingly larger crystals (increase of ∼35%), the duplication of all major magnetosome operons (mamGFDC, mamAB, mms6, and mamXY, comprising 29 genes in total) yielded an overproducing strain in which magnetosome numbers were 2.2-fold increased. We demonstrate that the tuned expression of the mam and mms clusters provides a powerful strategy for the control of magnetosome size and number, thereby setting the stage for high-yield production of tailored magnetic nanoparticles by synthetic biology approaches. IMPORTANCE Before our study, it had remained unknown how the upper sizes and numbers of magnetosomes are genetically regulated, and overproduction of

  13. Diversity and phylogeny of the ectoine biosynthesis genes in aerobic, moderately halophilic methylotrophic bacteria.

    PubMed

    Reshetnikov, Alexander S; Khmelenina, Valentina N; Mustakhimov, Ildar I; Kalyuzhnaya, Marina; Lidstrom, Mary; Trotsenko, Yuri A

    2011-11-01

    The genes of ectoine biosynthesis pathway were identified in six species of aerobic, slightly halophilic bacteria utilizing methane, methanol or methylamine. Two types of ectoine gene cluster organization were revealed in the methylotrophs. The gene cluster ectABC coding for diaminobutyric acid (DABA) acetyltransferase (EctA), DABA aminotransferase (EctB) and ectoine synthase (EctC) was found in methanotrophs Methylobacter marinus 7C and Methylomicrobium kenyense AMO1(T). In methanotroph Methylomicrobium alcaliphilum ML1, methanol-utilizers Methylophaga thalassica 33146(T) , Methylophaga alcalica M8 and methylamine-utilizer Methylarcula marina h1(T), the genes forming the ectABC-ask operon are preceded by ectR, encoding a putative transcriptional regulatory protein EctR. Phylogenetic relationships of the Ect proteins do not correlate with phylogenetic affiliation of the strains, thus implying that the ability of methylotrophs to produce ectoine is most likely the result of a horizontal transfer event.

  14. Functional characterization of human COQ4, a gene required for Coenzyme Q{sub 10} biosynthesis

    SciTech Connect

    Casarin, Alberto; Trevisson, Eva; Pertegato, Vanessa; Doimo, Mara; Ferrero-Gomez, Maria Lara; Abbadi, Sara; Quinzii, Catarina; Hirano, Michio; Basso, Giuseppe; Salviati, Leonardo

    2008-07-18

    Defects in genes involved in coenzyme Q (CoQ) biosynthesis cause primary CoQ deficiency, a severe multisystem disorders presenting as progressive encephalomyopathy and nephropathy. The COQ4 gene encodes an essential factor for biosynthesis in Saccharomyces cerevisiae. We have identified and cloned its human ortholog, COQ4, which is located on chromosome 9q34.13, and is transcribed into a 795 base-pair open reading frame, encoding a 265 amino acid (aa) protein (Isoform 1) with a predicted N-terminal mitochondrial targeting sequence. It shares 39% identity and 55% similarity with the yeast protein. Coq4 protein has no known enzymatic function, but may be a core component of multisubunit complex required for CoQ biosynthesis. The human transcript is detected in Northern blots as a {approx}1.4 kb single band and is expressed ubiquitously, but at high levels in liver, lung, and pancreas. Transcription initiates at multiple sites, located 333-23 nucleotides upstream of the ATG. A second group of transcripts originating inside intron 1 of the gene encodes a 241 aa protein, which lacks the mitochondrial targeting sequence (isoform 2). Expression of GFP-fusion proteins in HeLa cells confirmed that only isoform 1 is targeted to mitochondria. The functional significance of the second isoform is unknown. Human COQ4 isoform 1, expressed from a multicopy plasmid, efficiently restores both growth in glycerol, and CoQ content in COQ4{sup null} yeast strains. Human COQ4 is an interesting candidate gene for patients with isolated CoQ{sub 10} deficiency.

  15. Identification of a gene essential for the first committed step in the biosynthesis of bacteriochlorophyll c.

    PubMed

    Liu, Zhenfeng; Bryant, Donald A

    2011-06-24

    Bacteriochlorophylls (BChls) c, d, and e are the major chlorophylls in chlorosomes, which are the largest and one of the most efficient antennae produced by chlorophototrophic organisms. In the biosynthesis of these three BChls, a C-13(2)-methylcarboxyl group found in all other chlorophylls (Chls) must be removed. This reaction is postulated to be the first committed step in the synthesis of these BChls. Analyses of gene neighborhoods of (B)Chl biosynthesis genes and distribution patterns in organisms producing chlorosomes helped to identify a gene (bciC) that appeared to be a good candidate to produce the enzyme involved in this biochemical reaction. To confirm that this was the case, a deletion mutant of an open reading frame orthologous to bciC, CT1077, was constructed in Chlorobaculum tepidum, a genetically tractible green sulfur bacterium. The CT1077 deletion mutant was unable to synthesize BChl c but still synthesized BChl a and Chl a. The deletion mutant accumulated large amounts of various (bacterio)pheophorbides, all of which still had C-13(2)-methylcarboxyl groups. A C. tepidum strain in which CT1077 was replaced by an orthologous gene, Cabther_B0081 [corrected] from "Candidatus Chloracidobacterium thermophilum" was constructed. Although the product of Cabther_B0081 [corrected] was only 28% identical to the product of CT1077, this strain synthesized BChl c, BChl a, and Chl a in amounts similar to wild-type C. tepidum cells. To indicate their roles in the first committed step of BChl c, d, and e biosynthesis, open reading frames CT1077 and Cabther_B0081 [corrected] have been redesignated bciC. The potential mechanism by which BciC removes the C-13(2)-methylcarboxyl moiety of chlorophyllide a is discussed.

  16. A single cluster of coregulated genes encodes the biosynthesis of the mycotoxins roquefortine C and meleagrin in Penicillium chrysogenum.

    PubMed

    García-Estrada, Carlos; Ullán, Ricardo V; Albillos, Silvia M; Fernández-Bodega, María Ángeles; Durek, Pawel; von Döhren, Hans; Martín, Juan F

    2011-11-23

    A single gene cluster of Penicillium chrysogenum contains genes involved in the biosynthesis and secretion of the mycotoxins roquefortine C and meleagrin. Five of these genes have been silenced by RNAi. Pc21g15480 (rds) encodes a nonribosomal cyclodipeptide synthetase for the biosynthesis of both roquefortine C and meleagrin. Pc21g15430 (rpt) encodes a prenyltransferase also required for the biosynthesis of both mycotoxins. Silencing of Pc21g15460 or Pc21g15470 led to a decrease in roquefortine C and meleagrin, whereas silencing of the methyltransferase gene (Pc21g15440; gmt) resulted in accumulation of glandicolin B, indicating that this enzyme catalyzes the conversion of glandicolin B to meleagrin. All these genes are transcriptionally coregulated. Our results prove that roquefortine C and meleagrin derive from a single pathway.

  17. The SMUL_1544 Gene Product Governs Norcobamide Biosynthesis in the Tetrachloroethene-Respiring Bacterium Sulfurospirillum multivorans

    PubMed Central

    Keller, Sebastian; Treder, Aaron; von Reuss, Stephan H.; Escalante-Semerena, Jorge C.

    2016-01-01

    ABSTRACT The tetrachloroethene (PCE)-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which, in comparison to other cobamides, e.g., cobalamin and pseudo-B12, lacks the methyl group in the linker moiety of the nucleotide loop. In this study, the protein SMUL_1544 was shown to be responsible for the formation of the unusual linker moiety, which is most probably derived from ethanolamine-phosphate (EA-P) as the precursor. The product of the SMUL_1544 gene successfully complemented a Salmonella enterica ΔcobD mutant. The cobD gene encodes an l-threonine-O-3-phosphate (l-Thr-P) decarboxylase responsible for the synthesis of (R)-1-aminopropan-2-ol O-2-phosphate (AP-P), required specifically for cobamide biosynthesis. When SMUL_1544 was produced in the heterologous host lacking CobD, norpseudo-B12 was formed, which pointed toward the formation of EA-P rather than AP-P. Guided cobamide biosynthesis experiments with minimal medium supplemented with l-Thr-P supported cobamide biosynthesis in S. enterica producing SMUL_1544 or S. multivorans. Under these conditions, both microorganisms synthesized pseudo-B12. This observation indicated a flexibility in the SMUL_1544 substrate spectrum. From the formation of catalytically active PCE reductive dehalogenase (PceA) in S. multivorans cells producing pseudo-B12, a compatibility of the respiratory enzyme with the cofactor was deduced. This result might indicate a structural flexibility of PceA in cobamide binding. Feeding of l-[3-13C]serine to cultures of S. multivorans resulted in isotope labeling of the norpseudo-B12 linker moiety, which strongly supports the hypothesis of EA-P formation from l-serine-O-phosphate (l-Ser-P) in this organism. IMPORTANCE The identification of the gene product SMUL_1544 as a putative l-Ser-P decarboxylase involved in norcobamide biosynthesis in S. multivorans adds a novel module to the assembly line of cobamides (complete corrinoids) in prokaryotes

  18. A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection

    PubMed Central

    Almousally, Ibrahem; Shaban, Mouhnad; Blee, Elizabeth

    2015-01-01

    Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease. PMID:26116672

  19. Homeodomain Protein Scr Regulates the Transcription of Genes Involved in Juvenile Hormone Biosynthesis in the Silkworm.

    PubMed

    Meng, Meng; Liu, Chun; Peng, Jian; Qian, Wenliang; Qian, Heying; Tian, Ling; Li, Jiarui; Dai, Dandan; Xu, Anying; Li, Sheng; Xia, Qingyou; Cheng, Daojun

    2015-11-02

    The silkworm Dominant trimolting (Moltinism, M³) mutant undergoes three larval molts and exhibits precocious metamorphosis. In this study, we found that compared with the wild-type (WT) that undergoes four larval molts, both the juvenile hormone (JH) concentration and the expression of the JH-responsive gene Krüppel homolog 1 (Kr-h1) began to be greater in the second instar of the M³ mutant. A positional cloning analysis revealed that only the homeodomain transcription factor gene Sex combs reduced (Scr) is located in the genomic region that is tightly linked to the M³ locus. The expression level of the Scr gene in the brain-corpora cardiaca-corpora allata (Br-CC-CA) complex, which controls the synthesis of JH, was very low in the final larval instar of both the M³ and WT larvae, and exhibited a positive correlation with JH titer changes. Importantly, luciferase reporter analysis and electrophoretic mobility shift assay (EMSA) demonstrated that the Scr protein could promote the transcription of genes involved in JH biosynthesis by directly binding to the cis-regulatory elements (CREs) of homeodomain protein on their promoters. These results conclude that the homeodomain protein Scr is transcriptionally involved in the regulation of JH biosynthesis in the silkworm.

  20. Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph "Methylomicrobium alcaliphilum 20Z".

    PubMed

    Reshetnikov, Alexander S; Khmelenina, Valentina N; Trotsenko, Yuri A

    2006-01-01

    The genes involved in biosynthesis of the major compatible solute ectoine (1,4,5,6-tetrahydro-2-methylpyrimidine carboxylic acid) in halotolerant obligate methanotroph "Methylomicrobium alcaliphilum 20Z" were studied. The complete nucleotide sequences of the structural genes encoding L: -aspartokinase (Ask), L-2,4-diaminobutyric acid transaminase (EctB), L-2,4-diaminobutyric acid acetyltransferase (EctA), and L-ectoine synthase (EctC) were defined and shown to be transcribed as a single operon ectABCask. Phylogenetic analysis revealed high sequence identities (34-63%) of the Ect proteins to those from halophilic heterotrophs with the highest amino acid identities being to Vibrio cholerae enzymes. The chromosomal DNA fragment from "M. alcaliphilum 20Z" containing ectABC genes and putative promoter region was expressed in Escherichia coli. Recombinant cells could grow in the presence of 4% NaCl and synthesize ectoine. The data obtained suggested that despite the ectoine biosynthesis pathway being evolutionary well conserved with respect to the genes and enzymes involved, some differences in their organization and regulation could occur in various halophilic bacteria.

  1. Altered Expression of Genes Implicated in Xylan Biosynthesis Affects Penetration Resistance against Powdery Mildew

    PubMed Central

    Chowdhury, Jamil; Lück, Stefanie; Rajaraman, Jeyaraman; Douchkov, Dimitar; Shirley, Neil J.; Schwerdt, Julian G.; Schweizer, Patrick; Fincher, Geoffrey B.; Burton, Rachel A.; Little, Alan

    2017-01-01

    Heteroxylan has recently been identified as an important component of papillae, which are formed during powdery mildew infection of barley leaves. Deposition of heteroxylan near the sites of attempted fungal penetration in the epidermal cell wall is believed to enhance the physical resistance to the fungal penetration peg and hence to improve pre-invasion resistance. Several glycosyltransferase (GT) families are implicated in the assembly of heteroxylan in the plant cell wall, and are likely to work together in a multi-enzyme complex. Members of key GT families reported to be involved in heteroxylan biosynthesis are up-regulated in the epidermal layer of barley leaves during powdery mildew infection. Modulation of their expression leads to altered susceptibility levels, suggesting that these genes are important for penetration resistance. The highest level of resistance was achieved when a GT43 gene was co-expressed with a GT47 candidate gene, both of which have been predicted to be involved in xylan backbone biosynthesis. Altering the expression level of several candidate heteroxylan synthesis genes can significantly alter disease susceptibility. This is predicted to occur through changes in the amount and structure of heteroxylan in barley papillae.

  2. Banana ethylene response factors are involved in fruit ripening through their interactions with ethylene biosynthesis genes

    PubMed Central

    Xiao, Yun-yi; Chen, Jian-ye; Kuang, Jiang-fei; Shan, Wei; Xie, Hui; Jiang, Yue-ming; Lu, Wang-jin

    2013-01-01

    The involvement of ethylene response factor (ERF) transcription factor (TF) in the transcriptional regulation of ethylene biosynthesis genes during fruit ripening remains largely unclear. In this study, 15 ERF genes, designated as MaERF1–MaERF15, were isolated and characterized from banana fruit. These MaERFs were classified into seven of the 12 known ERF families. Subcellular localization showed that MaERF proteins of five different subfamilies preferentially localized to the nucleus. The 15 MaERF genes displayed differential expression patterns and levels in peel and pulp of banana fruit, in association with four different ripening treatments caused by natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and combined 1-MCP and ethylene treatments. MaERF9 was upregulated while MaERF11 was downregulated in peel and pulp of banana fruit during ripening or after treatment with ethylene. Furthermore, yeast-one hybrid (Y1H) and transient expression assays showed that the potential repressor MaERF11 bound to MaACS1 and MaACO1 promoters to suppress their activities and that MaERF9 activated MaACO1 promoter activity. Interestingly, protein–protein interaction analysis revealed that MaERF9 and -11 physically interacted with MaACO1. Taken together, these results suggest that MaERFs are involved in banana fruit ripening via transcriptional regulation of or interaction with ethylene biosynthesis genes. PMID:23599278

  3. Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation

    PubMed Central

    Vitreschak, Alexey G.; Rodionov, Dmitry A.; Mironov, Andrey A.; Gelfand, Mikhail S.

    2002-01-01

    The riboflavin biosynthesis in bacteria was analyzed using comparative analysis of genes, operons and regulatory elements. A model for regulation based on formation of alternative RNA structures involving the RFN elements is suggested. In Gram-positive bacteria including actinomycetes, Thermotoga, Thermus and Deinococcus, the riboflavin metabolism and transport genes are predicted to be regulated by transcriptional attenuation, whereas in most Gram-negative bacteria, the riboflavin biosynthesis genes seem to be regulated on the level of translation initiation. Several new candidate riboflavin transporters were identified (impX in Desulfitobacterium halfniense and Fusobacterium nucleatum; pnuX in several actinomycetes, including some Corynebacterium species and Strepto myces coelicolor; rfnT in Rhizobiaceae). Traces of a number of likely horizontal transfer events were found: the complete riboflavin operon with the upstream regulatory element was transferred to Haemophilus influenzae and Actinobacillus pleuropneumoniae from some Gram-positive bacterium; non-regulated riboflavin operon in Pyrococcus furiousus was likely transferred from Thermotoga; and the RFN element was inserted into the riboflavin operon of Pseudomonas aeruginosa from some other Pseudomonas species, where it had regulated the ribH2 gene. PMID:12136096

  4. Homeodomain Protein Scr Regulates the Transcription of Genes Involved in Juvenile Hormone Biosynthesis in the Silkworm

    PubMed Central

    Meng, Meng; Liu, Chun; Peng, Jian; Qian, Wenliang; Qian, Heying; Tian, Ling; Li, Jiarui; Dai, Dandan; Xu, Anying; Li, Sheng; Xia, Qingyou; Cheng, Daojun

    2015-01-01

    The silkworm Dominant trimolting (Moltinism, M3) mutant undergoes three larval molts and exhibits precocious metamorphosis. In this study, we found that compared with the wild-type (WT) that undergoes four larval molts, both the juvenile hormone (JH) concentration and the expression of the JH-responsive gene Krüppel homolog 1 (Kr-h1) began to be greater in the second instar of the M3 mutant. A positional cloning analysis revealed that only the homeodomain transcription factor gene Sex combs reduced (Scr) is located in the genomic region that is tightly linked to the M3 locus. The expression level of the Scr gene in the brain-corpora cardiaca-corpora allata (Br-CC-CA) complex, which controls the synthesis of JH, was very low in the final larval instar of both the M3 and WT larvae, and exhibited a positive correlation with JH titer changes. Importantly, luciferase reporter analysis and electrophoretic mobility shift assay (EMSA) demonstrated that the Scr protein could promote the transcription of genes involved in JH biosynthesis by directly binding to the cis-regulatory elements (CREs) of homeodomain protein on their promoters. These results conclude that the homeodomain protein Scr is transcriptionally involved in the regulation of JH biosynthesis in the silkworm. PMID:26540044

  5. A comparison of genes involved in sphingan biosynthesis brought up to date.

    PubMed

    Schmid, Jochen; Sperl, Nadine; Sieber, Volker

    2014-09-01

    Microbial polysaccharides have a wide range of functional properties and show high relevance in industrial applications. The possibility to create tailor-made polysaccharides by genetic engineering will further enhance the product portfolio and may open new fields of application. Here, we have examined in detail the recently sequenced genome of the welan-producing strain Sphingomonas sp. ATCC 31555 to identify the complete welan cluster and further genes involved in EPS production. The corresponding genes were compared on the nucleotide and amino acid sequence level to the EPS clusters of the described gellan-producing Sphingomonas elodea ATCC 31461, diutan-producing Sphingomonas sp. ATCC 53159, and the S-88-producing Sphingomonas sp. ATCC 31554 strains. We also compared the previously mentioned strains to each other and included the genes upstream of the main cluster in gellan and welan cluster. The cluster organization of Sphingomonas strain S-7 was also compared based on previous hybridization experiments, without nucleotide sequences. We have found that the occurrence of genes in all biosynthesis clusters is connected to the structures of the various produced sphingans. Along these lines, homologous genes responsible for the assembly of the identical repeating unit generally show high sequence identity, whereas genes for putative side chain attachment urf31, urf31.4, and urf34 vary more in distinct areas. Moreover, gene clusters for biosynthesis of diutan, welan, gellan, and S-88 as well as S-7 are similar in general organization but differ in location and arrangement of some genes. Finally, we summarized genetic and mutational engineering approaches toward modified sphingan variants as described in literature.

  6. Target genes of the Streptomyces tsukubaensis FkbN regulator include most of the tacrolimus biosynthesis genes, a phosphopantetheinyl transferase and other PKS genes.

    PubMed

    Ordóñez-Robles, María; Rodríguez-García, Antonio; Martín, Juan F

    2016-09-01

    Tacrolimus (FK506) is a 23-membered macrolide immunosuppressant used in current clinics. Understanding how the tacrolimus biosynthetic gene cluster is regulated is important to increase its industrial production. Here, we analysed the effect of the disruption of fkbN (encoding a LAL-type positive transcriptional regulator) on the whole transcriptome of the tacrolimus producer Streptomyces tsukubaensis using microarray technology. Transcription of fkbN in the wild type strain increases from 70 h of cultivation reaching a maximum at 89 h, prior to the onset of tacrolimus biosynthesis. Disruption of fkbN in S. tsukubaensis does not affect growth but prevents tacrolimus biosynthesis. Inactivation of fkbN reduces the transcription of most of the fkb cluster genes, including some all (for allylmalonyl-CoA biosynthesis) genes but does not affect expression of allMNPOS or fkbR (encoding a LysR-type regulator). Disruption of fkbN does not suppress transcription of the cistron tcs6-fkbQ-fkbN; thus, FkbN self-regulates only weakly its own expression. Interestingly, inactivation of FkbN downregulates the transcription of a 4'-phosphopantetheinyl transferase coding gene, which product is involved in tacrolimus biosynthesis, and upregulates the transcription of a gene cluster containing a cpkA orthologous gene, which encodes a PKS involved in coelimycin P1 biosynthesis in Streptomyces coelicolor. We propose an information theory-based model for FkbN binding sequences. The consensus FkbN binding sequence consists of 14 nucleotides with dyad symmetry containing two conserved inverted repeats of 7 nt each. This FkbN target sequence is present in the promoters of FkbN-regulated genes.

  7. Sexual Reproduction in Aflatoxin-Producing Aspergillus nomius

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aflatoxins are fungal secondary metabolites that exhibit carcinogenic, hepatotoxic and immunosuppressive properties. Aspergillus nomius is a potent producer of aflatoxins and was formerly considered to be strictly asexual in reproduction. In this research, mating-type genes MAT1-1 and MAT1-2 were ...

  8. Digital Gene Expression Analysis Provides Insight into the Transcript Profile of the Genes Involved in Aporphine Alkaloid Biosynthesis in Lotus (Nelumbo nucifera)

    PubMed Central

    Yang, Mei; Zhu, Lingping; Li, Ling; Li, Juanjuan; Xu, Liming; Feng, Ji; Liu, Yanling

    2017-01-01

    The predominant alkaloids in lotus leaves are aporphine alkaloids. These are the most important active components and have many pharmacological properties, but little is known about their biosynthesis. We used digital gene expression (DGE) technology to identify differentially-expressed genes (DEGs) between two lotus cultivars with different alkaloid contents at four leaf development stages. We also predicted potential genes involved in aporphine alkaloid biosynthesis by weighted gene co-expression network analysis (WGCNA). Approximately 335 billion nucleotides were generated; and 94% of which were aligned against the reference genome. Of 22 thousand expressed genes, 19,000 were differentially expressed between the two cultivars at the four stages. Gene Ontology (GO) enrichment analysis revealed that catalytic activity and oxidoreductase activity were enriched significantly in most pairwise comparisons. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, dozens of DEGs were assigned to the categories of biosynthesis of secondary metabolites, isoquinoline alkaloid biosynthesis, and flavonoid biosynthesis. The genes encoding norcoclaurine synthase (NCS), norcoclaurine 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3′-hydroxylase (NMCH), and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) in the common pathways of benzylisoquinoline alkaloid biosynthesis and the ones encoding corytuberine synthase (CTS) in aporphine alkaloid biosynthetic pathway, which have been characterized in other plants, were identified in lotus. These genes had positive effects on alkaloid content, albeit with phenotypic lag. The WGCNA of DEGs revealed that one network module was associated with the dynamic change of alkaloid content. Eleven genes encoding proteins with methyltransferase, oxidoreductase and CYP450 activities were identified. These were surmised to be genes involved in aporphine alkaloid biosynthesis. This

  9. Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus.

    PubMed Central

    Woloshuk, C P; Seip, E R; Payne, G A; Adkins, C R

    1989-01-01

    A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin. Images PMID:2495764

  10. A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni).

    PubMed

    Kumar, Hitesh; Kaul, Kiran; Bajpai-Gupta, Suphla; Kaul, Vijay Kumar; Kumar, Sanjay

    2012-01-15

    Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA(3)) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.

  11. Genes involved in long-chain alkene biosynthesis in Micrococcus luteus.

    PubMed

    Beller, Harry R; Goh, Ee-Been; Keasling, Jay D

    2010-02-01

    Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which 4 decades ago was reported to biosynthesize iso- and anteiso-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C(27) monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (beta-ketoacyl-ACP synthase III), which

  12. Genes, Gene Clusters, and Biosynthesis of Trichothecenes and Fumonisins in Fusarium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Trichothecenes and fumonisins are mycotoxins produced by Fusarium, a filamentous fungus that can cause disease on some crop plants, including corn, rice, and wheat. Research on the genetics and biochemistry of trichothecene and fumonisin biosynthesis has provided important insights into the genetic...

  13. Transcriptional analysis of the Streptomyces glaucescens tetracenomycin C biosynthesis gene cluster.

    PubMed Central

    Decker, H; Hutchinson, C R

    1993-01-01

    A 12.6-kb DNA fragment from Streptomyces glaucescens GLA.0 containing the 12 genes for tetracenomycin (TCM) C biosynthesis and resistance enabled Streptomyces lividans to produce TCM C. Transcriptional analysis of the tcmPG intergenic region in this cluster established the presence of two divergent promoters. The tcmIc mutation, a T-to-G transversion in the -10 region of the tcmG promoter, decreased promoter activity drastically at the stationary growth stage and time of maximum TCM C accumulation. This promoter may direct the transcription of a tcmGHIJKLMNO operon, while the other promoter is for tcmP. Images PMID:8509340

  14. Regulation of lysine biosynthesis and transport genes in bacteria: yet another RNA riboswitch?

    PubMed

    Rodionov, Dmitry A; Vitreschak, Alexey G; Mironov, Andrey A; Gelfand, Mikhail S

    2003-12-01

    Comparative analysis of genes, operons and regulatory elements was applied to the lysine biosynthetic pathway in available bacterial genomes. We report identification of a lysine-specific RNA element, named the LYS element, in the regulatory regions of bacterial genes involved in biosynthesis and transport of lysine. Similarly to the previously described RNA regulatory elements for three vitamins (riboflavin, thiamin and cobalamin), purine and methionine regulons, this regulatory RNA structure is highly conserved on the sequence and structural levels. The LYS element includes regions of lysine-constitutive mutations previously identified in Escherichia coli and Bacillus subtilis. A possible mechanism of the lysine-specific riboswitch is similar to the previously defined mechanisms for the other metabolite-specific riboswitches and involves either transcriptional or translational attenuation in various groups of bacteria. Identification of LYS elements in Gram-negative gamma-proteobacteria, Gram-positive bacteria from the Bacillus/Clostridium group, and Thermotogales resulted in description of the previously uncharacterized lysine regulon in these bacterial species. Positional analysis of LYS elements led to identification of a number of new candidate lysine transporters, namely LysW, YvsH and LysXY. Finally, the most likely candidates for genes of lysine biosynthesis missing in Gram- positive bacteria were identified using the genome context analysis.

  15. Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes

    PubMed Central

    Scott, Andrew; Noga, Marek J.; de Graaf, Paul; Westerlaken, Ilja; Yildirim, Esengul; Danelon, Christophe

    2016-01-01

    The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes. Acyl-coenzyme A and glycerol-3-phosphate were used as canonical precursors to generate a variety of important bacterial lipids. Moreover, this study demonstrates that two-step acyl transfer can occur from enzymes synthesized inside vesicles. Besides clear implications for growth and potentially division of a synthetic cell, we postulate that gene-based lipid biosynthesis can become instrumental for ex vivo and protein purification-free production of natural and non-natural lipids. PMID:27711229

  16. Molecular classification of commercial Spirulina strains and identification of their sulfolipid biosynthesis genes.

    PubMed

    Kwei, Chee Kuan; Lewis, David; King, Keith; Donohue, William; Neilan, Brett

    2011-04-01

    Cyanobacterial strains of the genus Spirulina have recently been identified as an excellent source of sulfolipids, some of which possess anti-HIV properties. Thus, to investigate the distribution of sufolipid biosynthesis pathways in Spirulina, a genetic screening/phylogentic study was performed. Five different strains of Spirulina [Spirulina (Jiangmen), Spirulina sp., S. platensis, S. maxima, and Spirulina seawater] sourced from different locations were initially classified via 16S rDNA sequencing, and then screened for the presence of the sulfolipid biosynthesis genes sqdB and sqdX via a PCR. To assess the suitability of these strains for human consumption and safe therapeutic use, the strains were also screened for the presence of genes encoding nonribosomal peptide synthases (NRPSs) and polyketide synthases (PKSs), which are often associated with toxin pathways in cyanobacteria. The results of the 16S rDNA analysis and phylogenetic study indicated that Spirulina sp. is closely related to Halospirulina, whereas the other four Spirulina strains are closely related to Arthrospira. Homologs of sqdB and sqdX were identified in Spirulina (Jiangmen), Spirulina sp., S. platensis, and the Spirulina seawater. None of the Spirulina strains screened in this study tested positive for NRPS or PKS genes, suggesting that these strains do not produce NRP or PK toxins.

  17. Cloning and expression of daunorubicin biosynthesis genes from Streptomyces peucetius and S. peucetius subsp. caesius.

    PubMed Central

    Otten, S L; Stutzman-Engwall, K J; Hutchinson, C R

    1990-01-01

    Genes for the biosynthesis of daunorubicin (daunomycin) and doxorubicin (adriamycin), important antitumor drugs, were cloned from Streptomyces peucetius (the daunorubicin producer) and S. peucetius subsp. caesius (the doxorubicin producer) by use of the actI/tcmIa and actIII polyketide synthase gene probes. Restriction mapping and Southern analysis of the DNA cloned in a cosmid vector established that the DNA represented three nonoverlapping regions of the S. peucetius subsp. caesius genome. These three regions plus an additional one that hybridized to the same probes are present in the S. peucetius genome, as reported previously (K. J. Stutzman-Engwall and C. R. Hutchinson, Proc. Natl. Acad. Sci. USA 86:3135-3139, 1989). Functional analysis of representative clones from some of these regions in S. lividans, S. peucetius ATCC 29050, S. peucetius subsp. caesius ATCC 27952, and two of its blocked mutants (strains H6101 and H6125) showed that many of the antibiotic production genes reside in the region of DNA represented by the group IV clones. This conclusion is based on the production of epsilon-rhodomycinone, a key intermediate of the daunorubicin pathway, in certain S. lividans transformants and on the apparent complementation of mutations that block daunorubicin biosynthesis in strains H6101 and H6125. Some of the transformants of strains 29050, 27952, and H6125 exhibited substantial overproduction of epsilon-rhodomycinone and daunorubicin. PMID:2345153

  18. Lipopolysaccharide biosynthesis genes discriminate between Rubus- and Spiraeoideae-infective genotypes of Erwinia amylovora.

    PubMed

    Rezzonico, Fabio; Braun-Kiewnick, Andrea; Mann, Rachel A; Rodoni, Brendan; Goesmann, Alexander; Duffy, Brion; Smits, Theo H M

    2012-10-01

    Comparative genomic analysis revealed differences in the lipopolysaccharide (LPS) biosynthesis gene cluster between the Rubus-infecting strain ATCC BAA-2158 and the Spiraeoideae-infecting strain CFBP 1430 of Erwinia amylovora. These differences corroborate rpoB-based phylogenetic clustering of E. amylovora into four different groups and enable the discrimination of Spiraeoideae- and Rubus-infecting strains. The structure of the differences between the two groups supports the hypothesis that adaptation to Rubus spp. took place after species separation of E. amylovora and E. pyrifoliae that contrasts with a recently proposed scenario, based on CRISPR data, in which the shift to domesticated apple would have caused an evolutionary bottleneck in the Spiraeoideae-infecting strains of E. amylovora which would be a much earlier event. In the core region of the LPS biosynthetic gene cluster, Spiraeoideae-infecting strains encode three glycosyltransferases and an LPS ligase (Spiraeoideae-type waaL), whereas Rubus-infecting strains encode two glycosyltransferases and a different LPS ligase (Rubus-type waaL). These coding domains share little to no homology at the amino acid level between Rubus- and Spiraeoideae-infecting strains, and this genotypic difference was confirmed by polymerase chain reaction analysis of the associated DNA region in 31 Rubus- and Spiraeoideae-infecting strains. The LPS biosynthesis gene cluster may thus be used as a molecular marker to distinguish between Rubus- and Spiraeoideae-infecting strains of E. amylovora using primers designed in this study.

  19. Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis

    PubMed Central

    Garin, Intza; Edghill, Emma L.; Akerman, Ildem; Rubio-Cabezas, Oscar; Rica, Itxaso; Locke, Jonathan M.; Maestro, Miguel Angel; Alshaikh, Adnan; Bundak, Ruveyde; del Castillo, Gabriel; Deeb, Asma; Deiss, Dorothee; Fernandez, Juan M.; Godbole, Koumudi; Hussain, Khalid; O’Connell, Michele; Klupa, Thomasz; Kolouskova, Stanislava; Mohsin, Fauzia; Perlman, Kusiel; Sumnik, Zdenek; Rial, Jose M.; Ugarte, Estibaliz; Vasanthi, Thiruvengadam; Johnstone, Karen; Flanagan, Sarah E.; Martínez, Rosa; Castaño, Carlos; Patch, Ann-Marie; Fernández-Rebollo, Eduardo; Raile, Klemens; Morgan, Noel; Harries, Lorna W.; Castaño, Luis; Ellard, Sian; Ferrer, Jorge; de Nanclares, Guiomar Perez; Hattersley, Andrew T.

    2010-01-01

    Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (−3.2 SD score vs. −2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man. PMID:20133622

  20. RirA is the iron response regulator of the rhizobactin 1021 biosynthesis and transport genes in Sinorhizobium meliloti 2011.

    PubMed

    Viguier, Caroline; O Cuív, Páraic; Clarke, Paul; O'Connell, Michael

    2005-05-15

    The genes encoding the biosynthesis and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti, are negatively regulated by iron. Mutagenesis of rirA, the rhizobial iron regulator, resulted in abolition of the iron responsive regulation of the biosynthesis and transport genes. Bioassay analysis revealed that the siderophore is produced in the presence of iron in a rirA mutant. RNA analysis and GFP fusions supported the conclusion that RirA is the mediator of iron-responsive transcriptional repression of the two transcripts encoding the biosynthesis and transport genes. RirA in S. meliloti appears to fulfil the role often observed for Fur in other bacterial species. The regulator was found to mediate the iron-responsive expression of two additional genes, smc02726 and dppA1, repressing the former while activating the latter. The rirA mutant nodulated the host plant Medicago sativa (alfalfa) and fixed nitrogen as effectively as the wild type.

  1. The albonoursin gene Cluster of S noursei biosynthesis of diketopiperazine metabolites independent of nonribosomal peptide synthetases.

    PubMed

    Lautru, Sylvie; Gondry, Muriel; Genet, Roger; Pernodet, Jean Luc

    2002-12-01

    Albonoursin [cyclo(deltaPhe-DeltaLeu)], an antibacterial peptide produced by Streptomyces noursei, is one of the simplest representatives of the large diketopiperazine (DKP) family. Formation of alpha,beta unsaturations was previously shown to occur on cyclo(L-Phe-L-Leu), catalyzed by the cyclic dipeptide oxidase (CDO). We used CDO peptide sequence information to isolate a 3.8 kb S. noursei DNA fragment that directs albonoursin biosynthesis in Streptomyces lividans. This fragment encompasses four complete genes: albA and albB, necessary for CDO activity; albC, sufficient for cyclic dipeptide precursor formation, although displaying no similarity to non ribosomal peptide synthetase (NRPS) genes; and albD, encoding a putative membrane protein. This first isolated DKP biosynthetic gene cluster should help to elucidate the mechanism of DKP formation, totally independent of NRPS, and to characterize novel DKP biosynthetic pathways that could be engineered to increase the molecular diversity of DKP derivatives.

  2. Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis.

    PubMed Central

    Guasch, J F; Piqué, N; Climent, N; Ferrer, S; Merino, S; Rubires, X; Tomas, J M; Regué, M

    1996-01-01

    Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomás, and M. Requé, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b. PMID:8824620

  3. Diversification of genes for carotenoid biosynthesis in aphids following an ancient transfer from a fungus.

    PubMed

    Nováková, Eva; Moran, Nancy A

    2012-01-01

    The pea aphid genome was recently found to harbor genes for carotenoid biosynthesis, reflecting an ancestral transfer from a fungus. To explore the evolution of the carotene desaturase gene family within aphids, sequences were retrieved from a set of 34 aphid species representing numerous deeply diverging lineages of aphids and analyzed together with fungal sequences retrieved from databases. All aphids have at least one copy of this gene and some aphid species have up to seven, whereas fungal genomes consistently have a single copy. The closest relatives of aphids, adelgids, also have carotene desaturase; these sequences are most closely related to those from aphids, supporting a shared origin from a fungal to insect transfer predating the divergence of adelgids and aphids. Likewise, all aphids, and adelgids, have carotenoid profiles that are consistent with their biosynthesis using the acquired genes of fungal origin rather than derivation from food plants. The carotene desaturase was acquired from a fungal species outside of Ascomycota or Basidiomycota and closest to Mucoromycotina among sequences available in databases. In aphids, an ongoing pattern of gene duplication is indicated by the presence of both anciently and recently diverged paralogs within genomes and by the presence of a high frequency of pseudogenes that appear to be recently inactivated. Recombination among paralogs is evident, making analyses of patterns of selection difficult, but tests of selection for a nonrecombining region indicates that duplications tend to be followed by bouts of positive selection. Species of Macrosiphini, which often show color polymorphisms, typically have a larger number of desaturase copies relative to other species sampled in the study. These results indicate that aphid evolution has been accompanied by ongoing evolution of carotenogenic genes, which have undergone duplication, recombination, and occasional positive selection to yield a wide variety of carotenoid

  4. Functional Characterization of 4′OMT and 7OMT Genes in BIA Biosynthesis

    PubMed Central

    Gurkok, Tugba; Ozhuner, Esma; Parmaksiz, Iskender; Özcan, Sebahattin; Turktas, Mine; İpek, Arif; Demirtas, Ibrahim; Okay, Sezer; Unver, Turgay

    2016-01-01

    Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L.), the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA) including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes remain to be revealed. In this study, expressions of 3-hydroxy-N-methylcoclaurine 4′–methyltransferase (4′OMT) and reticuline 7-O-methyltransferase (7OMT) genes were subjected to manipulation to functionally characterize their roles in BIA biosynthesis. Measurements of alkaloid accumulation were performed in leaf, stem, and capsule tissues accordingly. Suppression of 4′OMT expression caused reduction in the total alkaloid content in stem tissue whereas total alkaloid content was significantly induced in the capsule. Silencing of the 7OMT gene also caused repression in total alkaloid content in the stem. On the other hand, over-expression of 4′OMT and 7OMT resulted in higher morphine accumulation in the stem but suppressed amount in the capsule. Moreover, differential expression in several BIA synthesis genes (CNMT, TYDC, 6OMT, SAT, COR, 4′OMT, and 7OMT) were observed upon manipulation of 4′OMT and 7OMT expression. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4′OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues. PMID:26909086

  5. Cloning and Characterization of an Aspergillus nidulans Gene Involved in the Regulation of Penicillin Biosynthesis

    PubMed Central

    Van den Brulle, Jan; Steidl, Stefan; Brakhage, Axel A.

    1999-01-01

    To identify regulators of penicillin biosynthesis, a previously isolated mutant of Aspergillus nidulans (Prg-1) which carried the trans-acting prgA1 mutation was used. This mutant also contained fusions of the penicillin biosynthesis genes acvA and ipnA with reporter genes (acvA-uidA and ipnA-lacZ) integrated in a double-copy arrangement at the chromosomal argB gene. The prgA1 mutant strain exhibited only 20 to 50% of the ipnA-lacZ and acvA-uidA expression exhibited by the wild-type strain and had only 20 to 30% of the penicillin produced by the wild-type strain. Here, using complementation with a genomic cosmid library, we isolated a gene (suAprgA1) which complemented the prgA1 phenotype to the wild-type phenotype; i.e., the levels of expression of both gene fusions and penicillin production were nearly wild-type levels. Analysis of the suAprgA1 gene in the prgA1 mutant did not reveal any mutation in the suAprgA1 gene or unusual transcription of the gene. This suggested that the suAprgA1 gene is a suppressor of the prgA1 mutation. The suAprgA1 gene is 1,245 bp long. Its five exons encode a deduced protein that is 303 amino acids long. The putative SUAPRGA1 protein was similar to both the human p32 protein and Mam33p of Saccharomyces cerevisiae. Analysis of the ordered gene library of A. nidulans indicated that suAprgA1 is located on chromosome VI. Deletion of the suAprgA1 gene resulted in an approximately 50% reduction in ipnA-lacZ expression and in a slight reduction in acvA-uidA expression. The ΔsuAprgA1 strain produced about 60% of the amount of penicillin produced by the wild-type strain. PMID:10583968

  6. A functional gene cluster for toxoflavin biosynthesis in the genome of the soil bacterium Pseudomonas protegens Pf-5

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Toxoflavin is a broad-spectrum toxin best known for its role in virulence of Burkholderia glumae, which causes panicle blight of rice. A gene cluster containing homologs of toxoflavin biosynthesis genes (toxA-E) of B. glumae is present in the genome of Pseudomonas protegens Pf-5, a biological contr...

  7. Modular synthase-encoding gene involved in α-olefin biosynthesis in Synechococcus sp. strain PCC 7002.

    PubMed

    Mendez-Perez, Daniel; Begemann, Matthew B; Pfleger, Brian F

    2011-06-01

    A gene involved in the production of medium-chain α-olefins was identified in the cyanobacterium Synechococcus sp. strain PCC 7002. The gene encodes a large multidomain protein with homology to type I polyketide synthases, suggesting a route for hydrocarbon biosynthesis from fatty acids via an elongation decarboxylation mechanism.

  8. Identification of a trichothecene gene cluster and description of the harzianum A biosynthesis pathway in the fungus Trichoderma arundinaceum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Trichothecenes are sesquiterpenes that act like mycotoxins. Their biosynthesis has been mainly studied in the fungal genera Fusarium, where most of the biosynthetic genes (tri) are grouped in a cluster regulated by ambient conditions and regulatory genes. Unexpectedly, few studies are available abou...

  9. Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway and their regulation during reproduction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

  10. Genetic Profiling of the Isoprenoid and Sterol Biosynthesis Pathway Genes of Trypanosoma cruzi

    PubMed Central

    Cosentino, Raúl O.; Agüero, Fernán

    2014-01-01

    In Trypanosoma cruzi the isoprenoid and sterol biosynthesis pathways are validated targets for chemotherapeutic intervention. In this work we present a study of the genetic diversity observed in genes from these pathways. Using a number of bioinformatic strategies, we first identified genes that were missing and/or were truncated in the T. cruzi genome. Based on this analysis we obtained the complete sequence of the ortholog of the yeast ERG26 gene and identified a non-orthologous homolog of the yeast ERG25 gene (sterol methyl oxidase, SMO), and we propose that the orthologs of ERG25 have been lost in trypanosomes (but not in Leishmanias). Next, starting from a set of 16 T. cruzi strains representative of all extant evolutionary lineages, we amplified and sequenced ∼24 Kbp from 22 genes, identifying a total of 975 SNPs or fixed differences, of which 28% represent non-synonymous changes. We observed genes with a density of substitutions ranging from those close to the average (∼2.5/100 bp) to some showing a high number of changes (11.4/100 bp, for the putative lathosterol oxidase gene). All the genes of the pathway are under apparent purifying selection, but genes coding for the sterol C14-demethylase, the HMG-CoA synthase, and the HMG-CoA reductase have the lowest density of missense SNPs in the panel. Other genes (TcPMK, TcSMO-like) have a relatively high density of non-synonymous SNPs (2.5 and 1.9 every 100 bp, respectively). However, none of the non-synonymous changes identified affect a catalytic or ligand binding site residue. A comparative analysis of the corresponding genes from African trypanosomes and Leishmania shows similar levels of apparent selection for each gene. This information will be essential for future drug development studies focused on this pathway. PMID:24828104

  11. Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates.

    PubMed

    Murray, Shauna A; Diwan, Rutuja; Orr, Russell J S; Kohli, Gurjeet S; John, Uwe

    2015-11-01

    A group of marine dinoflagellates (Alveolata, Eukaryota), consisting of ∼10 species of the genus Alexandrium, Gymnodinium catenatum and Pyrodinium bahamense, produce the toxin saxitoxin and its analogues (STX), which can accumulate in shellfish, leading to ecosystem and human health impacts. The genes, sxt, putatively involved in STX biosynthesis, have recently been identified, however, the evolution of these genes within dinoflagellates is not clear. There are two reasons for this: uncertainty over the phylogeny of dinoflagellates; and that the sxt genes of many species of Alexandrium and other dinoflagellate genera are not known. Here, we determined the phylogeny of STX-producing and other dinoflagellates based on a concatenated eight-gene alignment. We determined the presence, diversity and phylogeny of sxtA, domains A1 and A4 and sxtG in 52 strains of Alexandrium, and a further 43 species of dinoflagellates and thirteen other alveolates. We confirmed the presence and high sequence conservation of sxtA, domain A4, in 40 strains (35 Alexandrium, 1 Pyrodinium, 4 Gymnodinium) of 8 species of STX-producing dinoflagellates, and absence from non-producing species. We found three paralogs of sxtA, domain A1, and a widespread distribution of sxtA1 in non-STX producing dinoflagellates, indicating duplication events in the evolution of this gene. One paralog, clade 2, of sxtA1 may be particularly related to STX biosynthesis. Similarly, sxtG appears to be generally restricted to STX-producing species, while three amidinotransferase gene paralogs were found in dinoflagellates. We investigated the role of positive (diversifying) selection following duplication in sxtA1 and sxtG, and found negative selection in clades of sxtG and sxtA1, clade 2, suggesting they were functionally constrained. Significant episodic diversifying selection was found in some strains in clade 3 of sxtA1, a clade that may not be involved in STX biosynthesis, indicating pressure for diversification

  12. MpigE, a gene involved in pigment biosynthesis in Monascus ruber M7.

    PubMed

    Liu, Qingpei; Xie, Nana; He, Yi; Wang, Li; Shao, Yanchun; Zhao, Hongzhou; Chen, Fusheng

    2014-01-01

    Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries. However, MP biosynthesis pathway is still a controversy, and only few related genes have been reported. In this study, the function of MpigE, a gene involved in MP biosynthesis in Monascus ruber M7, was analyzed. The results revealed that the disruption, complementation, and overexpression of MpigE in M. ruber M7 had very little effects on the growth and phenotypes except MPs. The MpigE deletion strain (∆MpigE) just yielded four kinds of yellow MPs and very little red pigments, while the wild-type strain M. ruber M7 produced a MP complex mixture including three (orange, red, and yellow) categories of MP compounds. Two of the four yellow MPs produced by ∆MpigE were the same as those yielded by M. ruber M7. The MpigE complementation strain (∆MpigE::MpigE) recovered the ability to generate orange and red MPs as M. ruber M7. The MP types produced by the MpigE overexpression strain (M7::PtrpC-MpigE) were consistent with those of M. ruber M7, while the color value was about 1.3-fold as that of M. ruber M7 (3,129 U/g red kojic). For the production of citrinin, the disruption of MpigE almost had no influence on the strain, whereas the overexpression of MpigE made citrinin decrease drastically in YES fermentation. This work will make a contribution to the study on the biosynthesis pathway of MPs in M. ruber.

  13. Differential expression of flavonoid biosynthesis genes and accumulation of phenolic compounds in common buckwheat (Fagopyrum esculentum).

    PubMed

    Li, Xiaohua; Park, Nam Il; Xu, Hui; Woo, Sun-Hee; Park, Cheol Ho; Park, Sang Un

    2010-12-08

    Common buckwheat (Fagopyrum esculentum) is a short-season grain crop that is a source of rutin and other phenolic compounds. In this study, we isolated the cDNAs of 11 F. esculentum enzymes in the flavonoid biosynthesis pathway, namely, phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL) 1 and 2, chalcone synthase (CHS), chalcone isomerase (CHI), flavone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonol synthase (FLS) 1 and 2, and anthocyanidin synthase (ANS). Quantitative real-time polymerase chain reaction analysis showed that these genes were most highly expressed in the stems and roots. However, high performance liquid chromatography analysis indicated that their flavonoid products, such as rutin and catechin, accumulated in the flowers and leaves. These results suggested that flavonoids may be transported within F. esculentum. In addition, light and dark growth conditions affected the expression levels of the biosynthesis genes and accumulation of phenolic compounds in F. esculentum sprouts.

  14. Expression profiling of genes involved in ascorbate biosynthesis and recycling during fleshy root development in radish.

    PubMed

    Xu, Yao; Zhu, Xianwen; Chen, Yinglong; Gong, Yiqin; Liu, Liwang

    2013-09-01

    Ascorbate is a primary antioxidant and an essential enzyme cofactor in plants, which has an important effect on the development of plant root system. To investigate the molecular mechanisms of ascorbate accumulation during root development and reveal the key genes of the ascorbate biosynthesis and recycling pathways, the expression of 16 related genes together with ascorbate abundance were analyzed in the flesh and skin of radish (Raphanus sativus L.) fleshy root. The content of ascorbate decreased with root growth in both the flesh and skin. Expression of GDP-d-mannose pyrophosphorylase, GDP-d-mannose-3',5'-epimerase and d-galacturonate reductase were also decreased and correlated with ascorbate levels in the flesh. In the skin, the expression of GDP-d-mannose pyrophosphorylase and l-galactose dehydrogenase was correlated with ascorbate levels. These results suggested that ascorbate accumulation is affected mainly by biosynthesis rather than recycling in radish root, and the l-galactose pathway may be the major biosynthetic route of ascorbate, and moreover, the salvage pathway may also contribute to ascorbate accumulation. The data suggested that GDP-d-mannose pyrophosphorylase could play an important role in the regulation of ascorbate accumulation during radish fleshy taproot development.

  15. Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis.

    PubMed

    Xu, Zhong; Wang, Yemin; Chater, Keith F; Ou, Hong-Yu; Xu, H Howard; Deng, Zixin; Tao, Meifeng

    2017-03-15

    Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation

  16. Cadmium-mediated disruption of cortisol biosynthesis involves suppression of corticosteroidogenic genes in rainbow trout.

    PubMed

    Sandhu, Navdeep; Vijayan, Mathilakath M

    2011-05-01

    Cadmium is widely distributed in the aquatic environment and is toxic to fish even at sublethal concentrations. This metal is an endocrine disruptor, and one well established role in teleosts is the suppression of adrenocorticotrophic hormone (ACTH)-stimulated cortisol biosynthesis by the interrenal tissue. However the mechanism(s) leading to this steroid suppression is poorly understood. We tested the hypothesis that cadmium targets genes encoding proteins critical for corticosteroid biosynthesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc), in rainbow trout (Oncorhynchus mykiss). To test this, head kidney slices (containing the interrenal tissues) were incubated in vitro with cadmium chloride (0, 10, 100 and 1000nM) for 4h either in the presence or absence of ACTH (0.5IU/mL). In the unstimulated head kidney slices, cadmium exposure did not affect basal cortisol secretion and the mRNA levels of MC2R and P450scc, while StAR gene expression was significantly reduced. Cadmium exposure significantly suppressed ACTH-stimulated cortisol production in a dose-related fashion. This cadmium-mediated suppression in corticosteroidogenesis corresponded with a significant reduction in MC2R, StAR and P450scc mRNA levels in trout head kidney slices. The inhibition of ACTH-stimulated cortisol production and suppression of genes involved in corticosteroidogenesis by cadmium were completely abolished in the presence of 8-Bromo-cAMP (a cAMP analog). Overall, cadmium disrupts the expression of genes critical for corticosteroid biosynthesis in rainbow trout head kidney slices. However, the rescue of cortisol production as well as StAR and P450scc gene expressions by cAMP analog suggests that cadmium impact occurs upstream of cAMP production. We propose that MC2R signaling, the primary step in ACTH-induced cortocosteroidogenesis, is a key target for cadmium-mediated disruption of

  17. Manipulation of regulatory genes reveals complexity and fidelity in hormaomycin biosynthesis.

    PubMed

    Cai, Xiaofeng; Teta, Roberta; Kohlhaas, Christoph; Crüsemann, Max; Ueoka, Reiko; Mangoni, Alfonso; Freeman, Michael F; Piel, Jörn

    2013-06-20

    Hormaomycin (HRM) is a structurally remarkable peptide produced by Streptomyces griseoflavus W-384 that acts as a Streptomyces signaling metabolite and exhibits potent antibiotic activity against coryneform actinomycetes. HRM biosynthetic studies have been hampered by inconsistent and low production. To enhance fermentation titers, the role of its cluster-encoded regulatory genes was investigated. Extra copies of the putative regulators hrmA and hrmB were introduced into the wild-type strain, resulting in an increase of HRM production and its analogs up to 135-fold. For the HrmB overproducer, six bioactive analogs were isolated and characterized. This study demonstrates that HrmA and HrmB are positive regulators in HRM biosynthesis. A third gene, hrmH, was identified as encoding a protein capable of shifting the metabolic profile of HRM and its derivatives. Its manipulation resulted in the generation of an additional HRM analog.

  18. Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development.

    PubMed

    Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra; Peck, Matthew L; Vega-Sánchez, Miguel E; Williams, Brian; Chiniquy, Dawn M; Saha, Prasenjit; Pattathil, Sivakumar; Conlin, Brian; Zhu, Lan; Hahn, Michael G; Willats, William G T; Scheller, Henrik V; Ronald, Pamela C; Bartley, Laura E

    2016-10-01

    Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture.

  19. The ctnG gene encodes carbonic anhydrase involved in mycotoxin citrinin biosynthesis from Monascus aurantiacus.

    PubMed

    Li, Yan-Ping; Tang, Xiao; Wu, Wei; Xu, Yang; Huang, Zhi-Bing; He, Qing-Hua

    2015-01-01

    Citrinin, a fungal secondary metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. Citrinin is synthesised by condensation of acetyl-CoA and malonyl-CoA. Six genes involved in the citrinin biosynthesis, including pksCT, ctnA and ctnB, have been cloned in Monascus purpureus. The pksCT gene encodes a polyketide synthase; ctnA is a regulatory factor; and ctnB encodes an oxidoreductase. When the three genes were respectively disrupted, the disruption strains drastically decreased citrinin production or barely produced citrinin. Ten new genes have been discovered in Monascus aurantiacus besides the above six genes. One of these gene displayed the highest similarity to the β-carbonic anhydrase gene from Aspergillus oryzae (74% similarity) and was designated ctnG. To learn more about the citrinin biosynthetic pathway, a ctnG-replacement vector was constructed to disrupt ctnG with the hygromycin resistance gene as the selection marker, then transformed into M. aurantiacus Li AS3.4384 by a protoplast-PEG method. The citrinin content of three disruptants was reduced to about 50%, meanwhile pigment production decreased by 23%, respectively, over those of the wild-type strains. ctnG was deduced to be involved in the formation of malonyl-CoA as a common precursor of red pigments and citrinin. Therefore, the disruption of the ctnG gene decreased citrinin and pigment production. M. aurantiacus Li AS3.4384 can produce higher concentrations of citrinin than other strains such as M. purpureus and M. ruber. Establishing the function of citrinin biosynthetic genes in M. aurantiacus is helpful in understanding the citrinin synthetic pathway and adopting some strategies to control contamination.

  20. The Ethylene Biosynthesis Gene CitACS4 Regulates Monoecy/Andromonoecy in Watermelon (Citrullus lanatus)

    PubMed Central

    Manzano, Susana; Aguado, Encarnación; Martínez, Cecilia; Megías, Zoraida; García, Alicia; Jamilena, Manuel

    2016-01-01

    Monoecious and andromonoecious cultivars of watermelon are characterised by the production of male and female flower or male and hermaphrodite flowers, respectively. The segregation analysis in the offspring of crosses between monoecious and andromonoecious lines has demonstrated that this trait is controlled by a single gene pair, being the monoecious allele M semi-dominant to the andromonoecious allele A. The two studied F1 hybrids (MA) had a predominantly monoecious phenotype since both produced not only female flowers, but also bisexual flowers with incomplete stamens, and hermaphrodite flowers with pollen. Given that in other cucurbit species andromonoecy is conferred by mutations in the ethylene biosynthesis genes CmACS7, CsACS2 and CpACS27A we have cloned and characterised CitACS4, the watermelon gene showing the highest similarity with the formers. CitACS4 encoded for a type ACS type III enzyme that is predominantly expressed in pistillate flowers of watermelon. In the andromonoecious line we have detected a missense mutation in a very conserved residue of CitACS4 (C364W) that cosegregates with the andromonoecious phenotype in two independent F2 populations, concomitantly with a reduction in ethylene production in the floral buds that will develop as hermaphrodite flowers. The gene does not however co-segregates with other sex expression traits regulated by ethylene in this species, including pistillate flowering transition and the number of pistillate flowers per plant. These data indicate that CitAC4 is likely to be involved in the biosynthesis of the ethylene required for stamen arrest during the development of female flowers. The C364W mutation would reduce the production of ethylene in pistillate floral buds, promoting the conversion of female into hermaphrodite flowers, and therefore of monoecy into andromonoecy. PMID:27149159

  1. Aflatoxin variability in pistachios.

    PubMed Central

    Mahoney, N E; Rodriguez, S B

    1996-01-01

    Pistachio fruit components, including hulls (mesocarps and epicarps), seed coats (testas), and kernels (seeds), all contribute to variable aflatoxin content in pistachios. Fresh pistachio kernels were individually inoculated with Aspergillus flavus and incubated 7 or 10 days. Hulled, shelled kernels were either left intact or wounded prior to inoculation. Wounded kernels, with or without the seed coat, were readily colonized by A. flavus and after 10 days of incubation contained 37 times more aflatoxin than similarly treated unwounded kernels. The aflatoxin levels in the individual wounded pistachios were highly variable. Neither fungal colonization nor aflatoxin was detected in intact kernels without seed coats. Intact kernels with seed coats had limited fungal colonization and low aflatoxin concentrations compared with their wounded counterparts. Despite substantial fungal colonization of wounded hulls, aflatoxin was not detected in hulls. Aflatoxin levels were significantly lower in wounded kernels with hulls than in kernels of hulled pistachios. Both the seed coat and a water-soluble extract of hulls suppressed aflatoxin production by A. flavus. PMID:8919781

  2. Exhaustive analysis of BH4 and dopamine biosynthesis genes in patients with Dopa-responsive dystonia.

    PubMed

    Clot, Fabienne; Grabli, David; Cazeneuve, Cécile; Roze, Emmanuel; Castelnau, Pierre; Chabrol, Brigitte; Landrieu, Pierre; Nguyen, Karine; Ponsot, Gérard; Abada, Myriem; Doummar, Diane; Damier, Philippe; Gil, Roger; Thobois, Stéphane; Ward, Alana J; Hutchinson, Michael; Toutain, Annick; Picard, Fabienne; Camuzat, Agnès; Fedirko, Estelle; Sân, Chankannira; Bouteiller, Delphine; LeGuern, Eric; Durr, Alexandra; Vidailhet, Marie; Brice, Alexis

    2009-07-01

    Dopa-responsive dystonia is a childhood-onset dystonic disorder, characterized by a dramatic response to low dose of L-Dopa. Dopa-responsive dystonia is mostly caused by autosomal dominant mutations in the GCH1 gene (GTP cyclohydrolase1) and more rarely by autosomal recessive mutations in the TH (tyrosine hydroxylase) or SPR (sepiapterin reductase) genes. In addition, mutations in the PARK2 gene (parkin) which causes autosomal recessive juvenile parkinsonism may present as Dopa-responsive dystonia. In order to evaluate the relative frequency of the mutations in these genes, but also in the genes involved in the biosynthesis and recycling of BH4, and to evaluate the associated clinical spectrum, we have studied a large series of index patients (n = 64) with Dopa-responsive dystonia, in whom dystonia improved by at least 50% after L-Dopa treatment. Fifty seven of these patients were classified as pure Dopa-responsive dystonia and seven as Dopa-responsive dystonia-plus syndromes. All patients were screened for point mutations and large rearrangements in the GCH1 gene, followed by sequencing of the TH and SPR genes, then PTS (pyruvoyl tetrahydropterin synthase), PCBD (pterin-4a-carbinolamine dehydratase), QDPR (dihydropteridin reductase) and PARK2 (parkin) genes. We identified 34 different heterozygous point mutations in 40 patients, and six different large deletions in seven patients in the GCH1 gene. Except for one patient with mental retardation and a large deletion of 2.3 Mb encompassing 10 genes, all patients had stereotyped clinical features, characterized by pure Dopa-responsive dystonia with onset in the lower limbs and an excellent response to low doses of L-Dopa. Dystonia started in the first decade of life in 40 patients (85%) and before the age of 1 year in one patient (2.2%). Three of the 17 negative GCH1 patients had mutations in the TH gene, two in the SPR gene and one in the PARK2 gene. No mutations in the three genes involved in the biosynthesis and

  3. Cloning and heterologous expression of ectoine biosynthesis genes from Bacillus halodurans in Escherichia coli.

    PubMed

    Anbu Rajan, Lawrance; Joseph, Toms C; Thampuran, Nirmala; James, Roswin; Ashok Kumar, Kesavan; Viswanathan, Chinnusamy; Bansal, Kailash C

    2008-08-01

    The genes involved in the biosynthetic pathway of ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) from Bacillus halodurans were cloned as an operon and expressed in E. coli. Analysis of the deduced ectoine biosynthesis cluster amino acid sequence revealed that the ectoine operon contain 2,389 bp, encoded by three genes; ectA, ectB and ectC that encode proteins of 189, 427 and 129 amino acids with deduced molecular masses of 21,048, 47,120 and 14,797 Da respectively. Extracts of induced cells showed two bands at 41 kDa and 17 kDa, possibly corresponding to the products of the later two genes. However the expression of ectA gene could not be ascertained by SDS-PAGE. The activity of the ectA protein was confirmed by an acylation assay. The transgenic E. coli accumulated upto 4.6 mg ectoine/l culture. This is the first report of an engineered E. coli strain carrying the ectoine genes of the alkaliphilic bacterium, B. halodurans.

  4. Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis

    PubMed Central

    Julsing, Mattijs K.; Rijpkema, Michael; Woerdenbag, Herman J.; Quax, Wim J.

    2007-01-01

    In comparison to other bacteria Bacillus subtilis emits the volatile compound isoprene in high concentrations. Isoprene is the smallest representative of the natural product group of terpenoids. A search in the genome of B. subtilis resulted in a set of genes with yet unknown function, but putatively involved in the methylerythritol phosphate (MEP) pathway to isoprene. Further identification of these genes would give the possibility to engineer B. subtilis as a host cell for the production of terpenoids like the valuable plant-produced drugs artemisinin and paclitaxel. Conditional knock-out strains of putative genes were analyzed for the amount of isoprene emitted. Differences in isoprene emission were used to identify the function of the enzymes and of the corresponding selected genes in the MEP pathway. We give proof on a biochemical level that several of these selected genes from this species are involved in isoprene biosynthesis. This opens the possibilities to investigate the physiological function of isoprene emission and to increase the endogenous flux to the terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for the heterologous production of more complex terpenoids in B. subtilis. PMID:17458547

  5. Microcyclamide Biosynthesis in Two Strains of Microcystis aeruginosa: from Structure to Genes and Vice Versa▿ †

    PubMed Central

    Ziemert, Nadine; Ishida, Keishi; Quillardet, Philippe; Bouchier, Christiane; Hertweck, Christian; de Marsac, Nicole Tandeau; Dittmann, Elke

    2008-01-01

    Comparative analysis of related biosynthetic gene clusters can provide new insights into the versatility of these pathways and allow the discovery of new natural products. The freshwater cyanobacterium Microcystis aeruginosa NIES298 produces the cytotoxic peptide microcyclamide. Here, we provide evidence that the cyclic hexapeptide is formed by a ribosomal pathway through the activity of a set of processing enzymes closely resembling those recently shown to be involved in patellamide biosynthesis in cyanobacterial symbionts of ascidians. Besides two subtilisin-type proteases and a heterocyclization enzyme, the gene cluster discovered in strain NIES298 encodes six further open reading frames, two of them without similarity to enzymes encoded by the patellamide gene cluster. Analyses of genomic data of a second cyanobacterial strain, M. aeruginosa PCC 7806, guided the discovery and structural elucidation of two novel peptides of the microcyclamide family. The identification of the microcyclamide biosynthetic genes provided an avenue by which to study the regulation of peptide synthesis at the transcriptional level. The precursor genes were strongly and constitutively expressed throughout the growth phase, excluding the autoinduction of these peptides, as has been observed for several peptide pheromone families in bacteria. PMID:18245249

  6. Microcyclamide biosynthesis in two strains of Microcystis aeruginosa: from structure to genes and vice versa.

    PubMed

    Ziemert, Nadine; Ishida, Keishi; Quillardet, Philippe; Bouchier, Christiane; Hertweck, Christian; de Marsac, Nicole Tandeau; Dittmann, Elke

    2008-03-01

    Comparative analysis of related biosynthetic gene clusters can provide new insights into the versatility of these pathways and allow the discovery of new natural products. The freshwater cyanobacterium Microcystis aeruginosa NIES298 produces the cytotoxic peptide microcyclamide. Here, we provide evidence that the cyclic hexapeptide is formed by a ribosomal pathway through the activity of a set of processing enzymes closely resembling those recently shown to be involved in patellamide biosynthesis in cyanobacterial symbionts of ascidians. Besides two subtilisin-type proteases and a heterocyclization enzyme, the gene cluster discovered in strain NIES298 encodes six further open reading frames, two of them without similarity to enzymes encoded by the patellamide gene cluster. Analyses of genomic data of a second cyanobacterial strain, M. aeruginosa PCC 7806, guided the discovery and structural elucidation of two novel peptides of the microcyclamide family. The identification of the microcyclamide biosynthetic genes provided an avenue by which to study the regulation of peptide synthesis at the transcriptional level. The precursor genes were strongly and constitutively expressed throughout the growth phase, excluding the autoinduction of these peptides, as has been observed for several peptide pheromone families in bacteria.

  7. Pristinamycin I biosynthesis in Streptomyces pristinaespiralis: molecular characterization of the first two structural peptide synthetase genes.

    PubMed Central

    de Crécy-Lagard, V; Blanc, V; Gil, P; Naudin, L; Lorenzon, S; Famechon, A; Bamas-Jacques, N; Crouzet, J; Thibaut, D

    1997-01-01

    Two genes involved in the biosynthesis of the depsipeptide antibiotics pristinamycins I (PI) produced by Streptomyces pristinaespiralis were cloned and sequenced. The 1.7-kb snbA gene encodes a 3-hydroxypicolinic acid:AMP ligase, and the 7.7-kb snbC gene encodes PI synthetase 2, responsible for incorporating L-threonine and L-aminobutyric acid in the PI macrocycle. snbA and snbC, which encode the two first structural enzymes of PI synthesis, are not contiguous. Both genes are located in PI-specific transcriptional units, as disruption of one gene or the other led to PI-deficient strains producing normal levels of the polyunsaturated macrolactone antibiotic pristinamycin II, also produced by S. pristinaespiralis. Analysis of the deduced amino acid sequences showed that the SnbA protein is a member of the adenylate-forming enzyme superfamily and that the SnbC protein contains two amino acid-incorporating modules and a C-terminal epimerization domain. A model for the initiation of PI synthesis analogous to the established model of initiation of fatty acid synthesis is proposed. PMID:9006024

  8. Identification of a Novel Gene for Biosynthesis of a Bacteroid-Specific Electron Carrier Menaquinone

    PubMed Central

    Xie, Fuli; Cheng, Guojun; Xu, Hui; Wang, Zhi; Lei, Lei; Li, Youguo

    2011-01-01

    Ubiquinone (UQ) has been considered as an electron mediator in electron transfer that generates ATP in Rhizobium under both free-living and symbiosis conditions. When mutated, the dmtH gene has a symbiotic phenotype of forming ineffective nodules on Astragalus sinicus. The gene was isolated from a Mesorhizobium huakuii 7653R transposon-inserted mutant library. The DNA sequence and conserved protein domain analyses revealed that dmtH encodes demethylmenaquinone (DMK) methyltransferase, which catalyzes the terminal step of menaquinone (MK) biosynthesis. Comparative analysis indicated that dmtH homologs were present in only a few Rhizobia. Real-time quantitative PCR showed dmtH is a bacteroid-specific gene. The highest expression was seen at 25 days after inoculation of strain 7653R. Gene disruption and complementation tests demonstrated that the dmtH gene was essential for bacteroid development and symbiotic nitrogen fixation ability. MK and UQ were extracted from the wild type strain 7653R and mutant strain HK116. MK-7 was accumulated under microaerobic condition and UQ-10 was accumulated under aerobic condition in M. huakuii 7653R. The predicted function of DmtH protein was confirmed by the measurement of methyltransferase activity in vitro. These results revealed that MK-7 was used as an electron carrier instead of UQ in M. huakuii 7653R bacteroids. PMID:22194970

  9. Identification of a novel gene for biosynthesis of a bacteroid-specific electron carrier menaquinone.

    PubMed

    Xie, Fuli; Cheng, Guojun; Xu, Hui; Wang, Zhi; Lei, Lei; Li, Youguo

    2011-01-01

    Ubiquinone (UQ) has been considered as an electron mediator in electron transfer that generates ATP in Rhizobium under both free-living and symbiosis conditions. When mutated, the dmtH gene has a symbiotic phenotype of forming ineffective nodules on Astragalus sinicus. The gene was isolated from a Mesorhizobium huakuii 7653R transposon-inserted mutant library. The DNA sequence and conserved protein domain analyses revealed that dmtH encodes demethylmenaquinone (DMK) methyltransferase, which catalyzes the terminal step of menaquinone (MK) biosynthesis. Comparative analysis indicated that dmtH homologs were present in only a few Rhizobia. Real-time quantitative PCR showed dmtH is a bacteroid-specific gene. The highest expression was seen at 25 days after inoculation of strain 7653R. Gene disruption and complementation tests demonstrated that the dmtH gene was essential for bacteroid development and symbiotic nitrogen fixation ability. MK and UQ were extracted from the wild type strain 7653R and mutant strain HK116. MK-7 was accumulated under microaerobic condition and UQ-10 was accumulated under aerobic condition in M. huakuii 7653R. The predicted function of DmtH protein was confirmed by the measurement of methyltransferase activity in vitro. These results revealed that MK-7 was used as an electron carrier instead of UQ in M. huakuii 7653R bacteroids.

  10. The Genome Sequence of the Cyanobacterium Oscillatoria sp. PCC 6506 Reveals Several Gene Clusters Responsible for the Biosynthesis of Toxins and Secondary Metabolites▿

    PubMed Central

    Méjean, Annick; Mazmouz, Rabia; Mann, Stéphane; Calteau, Alexandra; Médigue, Claudine; Ploux, Olivier

    2010-01-01

    We report a draft sequence of the genome of Oscillatoria sp. PCC 6506, a cyanobacterium that produces anatoxin-a and homoanatoxin-a, two neurotoxins, and cylindrospermopsin, a cytotoxin. Beside the clusters of genes responsible for the biosynthesis of these toxins, we have found other clusters of genes likely involved in the biosynthesis of not-yet-identified secondary metabolites. PMID:20675499

  11. Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora.

    PubMed

    Yang, Fan; Korban, Schuyler S; Pusey, P Lawrence; Elofsson, Michael; Sundin, George W; Zhao, Youfu

    2014-01-01

    The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E. amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E. amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein-encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small-molecule inhibitors that disable T3SS function could be explored to control fire blight disease.

  12. MAIZEWALL. Database and Developmental Gene Expression Profiling of Cell Wall Biosynthesis and Assembly in Maize 1[W

    PubMed Central

    Guillaumie, Sabine; San-Clemente, Hélène; Deswarte, Caroline; Martinez, Yves; Lapierre, Catherine; Murigneux, Alain; Barrière, Yves; Pichon, Magalie; Goffner, Deborah

    2007-01-01

    An extensive search for maize (Zea mays) genes involved in cell wall biosynthesis and assembly has been performed and 735 sequences have been centralized in a database, MAIZEWALL (http://www.polebio.scsv.ups-tlse.fr/MAIZEWALL). MAIZEWALL contains a bioinformatic analysis for each entry and gene expression data that are accessible via a user-friendly interface. A maize cell wall macroarray composed of a gene-specific tag for each entry was also constructed to monitor global cell wall-related gene expression in different organs and during internode development. By using this macroarray, we identified sets of genes that exhibit organ and internode-stage preferential expression profiles. These data provide a comprehensive fingerprint of cell wall-related gene expression throughout the maize plant. Moreover, an in-depth examination of genes involved in lignin biosynthesis coupled to biochemical and cytological data from different organs and stages of internode development has also been undertaken. These results allow us to trace spatially and developmentally regulated, putative preferential routes of monolignol biosynthesis involving specific gene family members and suggest that, although all of the gene families of the currently accepted monolignol biosynthetic pathway are conserved in maize, there are subtle differences in family size and a high degree of complexity in spatial expression patterns. These differences are in keeping with the diversity of lignified cell types throughout the maize plant. PMID:17098859

  13. High expression of cholesterol biosynthesis genes is associated with resistance to statin treatment and inferior survival in breast cancer

    PubMed Central

    Kimbung, Siker; Lettiero, Barbara; Feldt, Maria; Bosch, Ana; Borgquist, Signe

    2016-01-01

    There is sufficient evidence that statins have a protective role against breast cancer proliferation and recurrence, but treatment predictive biomarkers are lacking. Breast cancer cell lines displaying diverse sensitivity to atorvastatin were subjected to global transcriptional profiling and genes significantly altered by statin treatment were identified. Atorvastatin treatment strongly inhibited proliferation in estrogen receptor (ER) negative cell lines and a commensurate response was also evident on the genome-wide transcriptional scale, with ER negative cells displaying a robust deregulation of genes involved in the regulation of cell cycle progression and apoptosis. Interestingly, atorvastatin upregulated genes involved in the cholesterol biosynthesis pathway in all cell lines, irrespective of sensitivity to statin treatment. However, the level of pathway induction; measured as the fold change in transcript levels, was inversely correlated to the effect of statin treatment on cell growth. High expression of cholesterol biosynthesis genes before treatment was associated with resistance to statin therapy in cell lines and clinical biopsies. Furthermore, high expression of cholesterol biosynthesis genes was independently prognostic for a shorter recurrence-free and overall survival, especially among ER positive tumors. Dysregulation of cholesterol biosynthesis is therefore predictive for both sensitivity to anti-cancer statin therapy and prognosis following primary breast cancer diagnosis. PMID:27458152

  14. Identification of two aflatrem biosynthesis gene loci in Aspergillus flavus and metabolic engineering of Penicillium paxilli to elucidate their function.

    PubMed

    Nicholson, Matthew J; Koulman, Albert; Monahan, Brendon J; Pritchard, Beth L; Payne, Gary A; Scott, Barry

    2009-12-01

    Aflatrem is a potent tremorgenic toxin produced by the soil fungus Aspergillus flavus, and a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Gene clusters for indole-diterpene biosynthesis have recently been described in several species of filamentous fungi. A search of Aspergillus complete genome sequence data identified putative aflatrem gene clusters in the genomes of A. flavus and Aspergillus oryzae. In both species the genes for aflatrem biosynthesis cluster at two discrete loci; the first, ATM1, is telomere proximal on chromosome 5 and contains a cluster of three genes, atmG, atmC, and atmM, and the second, ATM2, is telomere distal on chromosome 7 and contains five genes, atmD, atmQ, atmB, atmA, and atmP. Reverse transcriptase PCR in A. flavus demonstrated that aflatrem biosynthesis transcript levels increased with the onset of aflatrem production. Transfer of atmP and atmQ into Penicillium paxilli paxP and paxQ deletion mutants, known to accumulate paxilline intermediates paspaline and 13-desoxypaxilline, respectively, showed that AtmP is a functional homolog of PaxP and that AtmQ utilizes 13-desoxypaxilline as a substrate to synthesize aflatrem pathway-specific intermediates, paspalicine and paspalinine. We propose a scheme for aflatrem biosynthesis in A. flavus based on these reconstitution experiments in P. paxilli and identification of putative intermediates in wild-type cultures of A. flavus.

  15. Characterization of cellulose structure of Populus plants modified in candidate cellulose biosynthesis genes

    DOE PAGES

    Bali, Garima; Khunsupat, Ratayakorn; Akinosho, Hannah; ...

    2016-09-10

    Here, the recalcitrant nature of lignocellulosic biomass is a combined effect of several factors such as high crystallinity and high degree of polymerization of cellulose, lignin content and structure, and the available surface area for enzymatic degradation (i.e., accessibility). Genetic improvement of feedstock cell wall properties is a path to reducing recalcitrance of lignocellulosic biomass and improving conversion to various biofuels. An advanced understanding of the cellulose biosynthesis pathway is essential to precisely modify cellulose properties of plant cell walls. Here we report on the impact of modified expression of candidate cellulose biosynthesis pathway genes on the ultra-structure of cellulose,more » a key carbohydrate polymer of Populus cell wall using advanced nuclear magnetic resonance approaches. Noteworthy changes were observed in the cell wall characteristics of downregulated KORRIGAN 1 (KOR) and KOR 2 transgenic plants in comparison to the wild-type control. It was observed that all of the transgenic lines showed variation in cellulose ultrastructure, increase in cellulose crystallinity and decrease in the cellulose degree of polymerization. Additionally, the properties of cellulose allomorph abundance and accessibility were found to be variable. Application of such cellulose characterization techniques beyond the traditional measurement of cellulose abundance to comprehensive studies of cellulose properties in larger transgenic and naturally variable populations is expected to provide deeper insights into the complex nature of lignocellulosic material, which can significantly contribute to the development of precisely tailored plants for enhanced biofuels production.« less

  16. Characterization of cellulose structure of Populus plants modified in candidate cellulose biosynthesis genes

    SciTech Connect

    Bali, Garima; Khunsupat, Ratayakorn; Akinosho, Hannah; Payyavula, Raja S.; Samuel, Reichel; Tuskan, Gerald A.; Kalluri, Udaya C.; Ragauskas, Arthur J.

    2016-09-10

    Here, the recalcitrant nature of lignocellulosic biomass is a combined effect of several factors such as high crystallinity and high degree of polymerization of cellulose, lignin content and structure, and the available surface area for enzymatic degradation (i.e., accessibility). Genetic improvement of feedstock cell wall properties is a path to reducing recalcitrance of lignocellulosic biomass and improving conversion to various biofuels. An advanced understanding of the cellulose biosynthesis pathway is essential to precisely modify cellulose properties of plant cell walls. Here we report on the impact of modified expression of candidate cellulose biosynthesis pathway genes on the ultra-structure of cellulose, a key carbohydrate polymer of Populus cell wall using advanced nuclear magnetic resonance approaches. Noteworthy changes were observed in the cell wall characteristics of downregulated KORRIGAN 1 (KOR) and KOR 2 transgenic plants in comparison to the wild-type control. It was observed that all of the transgenic lines showed variation in cellulose ultrastructure, increase in cellulose crystallinity and decrease in the cellulose degree of polymerization. Additionally, the properties of cellulose allomorph abundance and accessibility were found to be variable. Application of such cellulose characterization techniques beyond the traditional measurement of cellulose abundance to comprehensive studies of cellulose properties in larger transgenic and naturally variable populations is expected to provide deeper insights into the complex nature of lignocellulosic material, which can significantly contribute to the development of precisely tailored plants for enhanced biofuels production.

  17. The NlaIV restriction and modification genes of Neisseria lactamica are flanked by leucine biosynthesis genes.

    PubMed

    Lau, P C; Forghani, F; Labbé, D; Bergeron, H; Brousseau, R; Höltke, H J

    1994-04-01

    The genes encoding the Neisseria lactamica restriction endonuclease IV (R.NlaIV) and its cognate DNA methyltransferase (M.NlaIV), both of which recognize the sequence GGNNCC, have been cloned in Escherichia coli and overexpressed using the T7 polymerase/promoter system. Analysis of a sequenced 3.58 kb fragment established the gene order, leuD-M.NlaIV-R.NlaIV-leuB. The predicted primary sequence of M.NlaIV (423 amino acids) shows the highest degree of identity to a pair of cytosine-specific methyltransferases, M.BanI (44.9%) and M.HgiCI (44.3%), which recognize the sequence GGYRCC (Y, pyrimidines; R, purines). In contrast, the R.NlaIV protein sequence (243 amino acids) is unique in the existing data-base, a situation that holds for most endonucleases. Flanking the NlaIV modification and restriction genes are homologues of the leuD and leuB genes of enteric bacteria, which code for enzymes in the leucine biosynthesis pathway. This gene context implies a possible new mode of gene regulation for the RM.NlaIV system, which would involve a mechanism similar to the recently discovered leucine/Lrp regulon in E. coli.

  18. Current Bacterial Gene Encoding Capsule Biosynthesis Protein CapI Contains Nucleotides Derived from Exonization

    PubMed Central

    Wang, Yong; Tao, Xia-Fang; Su, Zhi-Xi; Liu, A-Ke; Liu, Tian-Lei; Sun, Ling; Yao, Qin; Chen, Ke-Ping; Gu, Xun

    2016-01-01

    Since the proposition of introns-early hypothesis, although many studies have shown that most eukaryotic ancestors possessed intron-rich genomes, evidence of intron existence in genomes of ancestral bacteria has still been absent. While not a single intron has been found in all protein-coding genes of current bacteria, analyses on bacterial genes horizontally transferred into eukaryotes at ancient time may provide evidence of intron existence in bacterial ancestors. In this study, a bacterial gene encoding capsule biosynthesis protein CapI was found in the genome of sea anemone, Nematostella vectensis. This horizontally transferred gene contains a phase 1 intron of 40 base pairs. The nucleotides of this intron have high sequence identity with those encoding amino acids in current bacterial CapI gene, indicating that the intron and the amino acid-coding nucleotides are originated from the same ancestor sequence. Moreover, 5′-splice site of this intron is located in a GT-poor region associated with a closely following AG-rich region, suggesting that deletion mutation at 5′-splice site has been employed to remove this intron and the intron-like amino acid-coding nucleotides in current bacterial CapI gene are derived from exonization. These data suggest that bacterial CapI gene contained intron(s) at ancient time. This is the first report providing the result of sequence analysis to suggest possible existence of spliceosomal introns in ancestral bacterial genes. The methodology employed in this study may be used to identify more such evidence that would aid in settlement of the dispute between introns-early and introns-late theories. PMID:27980385

  19. A gene cluster required for coordinated biosynthesis of lipopolysaccharide and extracellular polysaccharide also affects virulence of Pseudomonas solanacearum.

    PubMed Central

    Kao, C C; Sequeira, L

    1991-01-01

    Bacterial cell surface components can be important determinants of virulence. At least three gene clusters important for extracellular polysaccharide (EPS) biosynthesis have been previously identified in the plant pathogen Pseudomonas solanacearum. We have found that one of these gene clusters, named ops, is also required for lipopolysaccharide (LPS) biosynthesis. Mutations in any complementation unit of this cluster decreased EPS production, prevented the binding of an LPS-specific phage, and altered the mobility of purified LPS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, restoration of LPS biosynthesis alone was not sufficient to restore virulence to the wild-type level, suggesting that EPS is important for pathogenesis. Images FIG. 2 FIG. 3 PMID:1744040

  20. Comparative Hepatotoxicity of Aflatoxin B1 among Workers Exposed to Different Organic Dust with Emphasis on Polymorphism Role of Glutathione S-Transferase Gene

    PubMed Central

    Saad-Hussein, Amal; Shahy, Eman M.; Shaheen, Weam; Taha, Mona M.; Mahdy-Abdallah, Heba; Ibrahim, Khadiga S.; Hafez, Salwa F.; Fadl, Nevein N.; El-Shamy, Karima A.

    2016-01-01

    AIM: The study aimed to investigate effects of organic dust exposure from different sources on aflatoxin B1-albumin adducts (AFB1/Alb), and role of glutathione S-transferase (GST) gene polymorphism in hepatotoxicity of (AFB1) among exposed workers. MATERIAL AND METHODS: Liver enzymes, AFB1/Alb, and GST polymorphism were estimated in 132 wheat flour dust and 87 woods sawmill workers, and 156 controls. RESULTS: Results revealed that AFB1/Alb and liver enzymes were significantly elevated in exposed workers compared to controls, and were significantly higher in sawmill workers compared to flour workers. AFB1/Alb in flour and sawmill workers with GSTT1 and GSTM1&GSTT1 null genotypes were significantly higher than controls, and in sawmill workers with GSTM1&GSTT1 null than flour workers. Liver enzymes (ALT and AST) in sawmill workers were significantly higher than flour workers and controls in all GST polymorphism; except in GSTT1 polymorphism, where these enzymes were significantly higher in the two exposed groups than controls. CONCLUSIONS: In conclusion, organic dust exposure may cause elevation in AFB1/Alb and liver enzymes of exposed workers, and GST gene polymorphism plays an important role in susceptibility to hepatic parenchymal cell injury; except in workers with GSTT1&GSTM1 null genotype, gene susceptibility seemed to have little role and the main role was for environmental exposures. PMID:27335608

  1. Identification of a putative FR901469 biosynthesis gene cluster in fungal sp. No. 11243 and enhancement of the productivity by overexpressing the transcription factor gene frbF.

    PubMed

    Matsui, Makoto; Yokoyama, Tatsuya; Nemoto, Kaoru; Kumagai, Toshitaka; Terai, Goro; Tamano, Koichi; Machida, Masayuki; Shibata, Takashi

    2017-02-01

    FR901469 is an antifungal antibiotic produced by fungal sp. No. 11243. Here, we searched for FR901469 biosynthesis genes in the genome of No. 11243. Based on the molecular structure of FR901469 and endogenous functional motifs predicted in each genomic NRPS gene, a putative FR901469 biosynthesis gene cluster harboring the most plausible NRPS gene was identified. A transcription factor gene, designated frbF, was found in the cluster. To improve FR901469 productivity, we constructed a strain in which frbF was overexpressed and named it TFH2-2. FR901469 productivity of TFH2-2 was 3.4 times higher than that of the wild-type strain. Transcriptome analysis revealed that most of the genes in the putative FR901469 biosynthesis gene cluster were upregulated in TFH2-2. It also showed that the expression of genes related to ergosterol biosynthesis, β-1,3-glucan catabolism, and chitin synthesis was inclined to exhibit significant differences in TFH2-2.

  2. Effects of Aflatoxin on Seeding Growth and Ultrastructure in Plants

    PubMed Central

    Crisan, Eli V.

    1973-01-01

    Nineteen plants belonging to 11 species of the cruciferae were studied to determine the effects of aflatoxin B1 on seed germination and seedling development. Germination was not inhibited in any test organism at a concentration of 100 μg of aflatoxin per ml of agar substrate. Inhibition of elongation of the hypocotyls and roots in the species studied varied from 29 to 93% and from 22 to 91% in the respective tissues. Lepidium sativum was the most susceptible plant studied and exhibited the maximal inhibitory response noted above at concentrations of 8 μg of aflatoxin per ml. The ultrastructure of Lepidium root cells treated with crystalline aflatoxin B1 exhibited morphological changes characteristic of those found in aflatoxin-treated animal cells. In addition to changes in the cytoplasmic organelles, numerous ring-shaped nucleoli with prominent nucleolar caps were produced. The effect of aflatoxin on plant cells is compared with similar effects induced by actinomycin D. Seed germination and seedling development is discussed in relation to the effects of both compounds on deoxyribonucleic acid-dependent ribonucleic acid biosynthesis. Images PMID:4767301

  3. Down-regulation of lignin biosynthesis in transgenic Leucaena leucocephala harboring O-methyltransferase gene.

    PubMed

    Rastogi, Smita; Dwivedi, Upendra Nath

    2006-01-01

    In the present study, a 0.47 kb OMT gene construct from aspen, encoding for an enzyme O-methyltransferase (OMT, EC 2.1.1.6), in antisense orientation was used to down-regulate lignin biosynthesis in Leucaena leucocephala. The plants were transformed with Agrobacterium tumefaciens strain harboring the antisense gene, and the transformation was confirmed by PCR amplification of the npt II gene. The integration of a heterologous antisense OMT gene construct in transformed plants led to a maximum of 60% reduction in OMT activity relative to control. The evaluation of total lignin content by the Klason method revealed a maximum of 28% reduction. Histochemical analyses of stem sections depicted a reduction in lignin content and normal xylem development. The results also suggested a probable increase in aldehyde levels and a decrease in syringyl units. Lignin down-regulation was accompanied by an increase in methanol soluble phenolics to an extent that had no impact on wood discoloration, and the plants displayed a normal phenotype. Concomitantly, an increase of up to 9% in cellulose content was also observed. Upon alkali extraction, modified lignin was more extractable as evident from reduced Klason lignin in saponified residue and increased alkali soluble phenolics. The results together suggested that the extent of down-regulation of OMT activity achieved may lead to quality amelioration of Leucaena with respect to its applicability in pulp and paper manufacture as well as nutritive and easily digestible forage production.

  4. The effect of nutrition pattern alteration on Chlorella pyrenoidosa growth, lipid biosynthesis-related gene transcription.

    PubMed

    Fan, Jianhua; Cui, Yanbin; Zhou, Yang; Wan, Minxi; Wang, Weiliang; Xie, Jingli; Li, Yuanguang

    2014-07-01

    Heterotrophy to photoautotrophy transition leads to the accumulation of lipids in Chlorella, which has potential to produce both healthy food and biofuels. Therefore, it is of key interest to study the metabolism shift and gene expression changes that influenced by the transition. Both total and neutral lipids contents were increased rapidly within 48 h after the switch to light environment, from 24.5% and 18.0% to 35.3% and 27.4%, respectively, along with the sharp decline of starch from 42.3% to 10.4% during 24h photoinduction phase. By analyzing the correlation between lipid content and gene expression, results revealed several genes viz. me g3137, me g6562, pepc g6833, dgat g3280 and dgat g7566, which encode corresponding enzymes in the de novo lipid biosynthesis pathway, are highly related to lipid accumulation and might be exploited as target genes for genetic modification. These results represented the feasibility of lipid production through trophic converting cultivation.

  5. Duplicate Maize Wrinkled1 Transcription Factors Activate Target Genes Involved in Seed Oil Biosynthesis1[C][W

    PubMed Central

    Pouvreau, Benjamin; Baud, Sébastien; Vernoud, Vanessa; Morin, Valérie; Py, Cyrille; Gendrot, Ghislaine; Pichon, Jean-Philippe; Rouster, Jacques; Paul, Wyatt; Rogowsky, Peter M.

    2011-01-01

    WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs. PMID:21474435

  6. Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes.

    PubMed

    Itkin, M; Heinig, U; Tzfadia, O; Bhide, A J; Shinde, B; Cardenas, P D; Bocobza, S E; Unger, T; Malitsky, S; Finkers, R; Tikunov, Y; Bovy, A; Chikate, Y; Singh, P; Rogachev, I; Beekwilder, J; Giri, A P; Aharoni, A

    2013-07-12

    Steroidal glycoalkaloids (SGAs) such as α-solanine found in solanaceous food plants--as, for example, potato--are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.

  7. Developing tools for investigating the multiple roles of ethylene: Identification and mapping genes for ethylene biosynthesis and reception in barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The plant hormone ethylene is important to many plant processes from germination through senescence, including responses to in vitro growth and plant regeneration. Knowledge of the number of genes, and of their function, that are involved in ethylene biosynthesis and reception is necessary to determ...

  8. Gibberellin 3-oxidase gene expression patterns influence gibberellin biosynthesis, growth, and development in pea.

    PubMed

    Reinecke, Dennis M; Wickramarathna, Aruna D; Ozga, Jocelyn A; Kurepin, Leonid V; Jin, Alena L; Good, Allen G; Pharis, Richard P

    2013-10-01

    Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth.

  9. A Malus crabapple chalcone synthase gene, McCHS, regulates red petal color and flavonoid biosynthesis.

    PubMed

    Tai, Deqiang; Tian, Ji; Zhang, Jie; Song, Tingting; Yao, Yuncong

    2014-01-01

    Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, 'Royalty' and 'Flame', have dark red and white petals respectively, while the intermediate cultivar 'Radiant' has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in 'Radiant'. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple.

  10. A Malus Crabapple Chalcone Synthase Gene, McCHS, Regulates Red Petal Color and Flavonoid Biosynthesis

    PubMed Central

    Song, Tingting; Yao, Yuncong

    2014-01-01

    Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, ‘Royalty’ and ‘Flame’, have dark red and white petals respectively, while the intermediate cultivar ‘Radiant’ has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in ‘Radiant’. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple. PMID:25357207

  11. Transcriptional Profiles of Hybrid Eucalyptus Genotypes with Contrasting Lignin Content Reveal That Monolignol Biosynthesis-related Genes Regulate Wood Composition

    PubMed Central

    Shinya, Tomotaka; Iwata, Eiji; Nakahama, Katsuhiko; Fukuda, Yujiroh; Hayashi, Kazunori; Nanto, Kazuya; Rosa, Antonio C.; Kawaoka, Akiyoshi

    2016-01-01

    Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, α-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants. PMID

  12. Comparative glandular trichome transcriptome based gene characterization reveals reasons for differential (-)-menthol biosynthesis in Mentha species.

    PubMed

    Akhtar, Md Qussen; Qamar, Nida; Yadav, Pallavi; Kulkarni, Pallavi; Kumar, Ajay; Shasany, Ajit Kumar

    2017-02-11

    The genes involved in menthol biosynthesis are reported earlier in Mentha × piperita. But the information on these genes is not available in Mentha arvensis. To bridge the gap in knowledge on differential biosynthesis of monoterpenes leading to compositional variation in the essential oil of these species, a comparative transcriptome analysis of the glandular trichome was carried out. In addition to the MVA and MEP pathway genes, about 210 and 196 different terpene synthases (TPS) transcripts were identified from annotation in M. arvensis and M. × piperita, respectively, and correlated to several monoterpenes present in the essential oil. Six isoforms of (-)-menthol dehydrogenases (MD), the last enzyme of the menthol biosynthetic pathway, were identified, cloned and characterized from the transcriptome data (3 from each species). Varied expression levels and differential enzyme kinetics of these isoforms indicated the nature and composition of the product, as these isoforms generate both (-)-menthol and (+)-neomenthol from (-)-menthone and converts (-)-menthol to (-)-menthone in the reverse reaction, and hence together determine the quantity of (-)-menthol in the essential oil in these two species. Several genes for high value minor monoterpenes could also be identified from the transcriptome data. Abbreviations - AACT, acetyl-CoA C-acetyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; DGE, digital gene expression; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; FPPS, farnesyl pyrophosphate synthase; GC, gas chromatography; GPPS, geranyl pyrophosphate synthase; GT, glandular trichome; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; HMGS, hydroxymethylglutaryl-CoA synthase; IDI, isopentenyl-diphosphate delta-isomerase; IPD, isopiperitenol dehydrogenase; IPI

  13. Lipopolysaccharide biosynthesis-related genes are required for colony pigmentation of Porphyromonas gingivalis.

    PubMed

    Sato, Keiko; Kido, Nobuo; Murakami, Yukitaka; Hoover, Charles I; Nakayama, Koji; Yoshimura, Fuminobu

    2009-04-01

    The periodontopathic bacterium Porphyromonas gingivalis forms pigmented colonies when incubated on blood agar plates as a result of accumulation of mu-oxo haem dimer on the cell surface. Gingipain-adhesin complexes are responsible for production of mu-oxo haem dimer from haemoglobin. Non-pigmented mutants (Tn6-5, Tn7-1, Tn7-3 and Tn10-4) were isolated from P. gingivalis by Tn4351 transposon mutagenesis [Hoover & Yoshimura (1994), FEMS Microbiol Lett 124, 43-48]. In this study, we found that the Tn6-5, Tn7-1 and Tn7-3 mutants carried Tn4351 DNA in a gene homologous to the ugdA gene encoding UDP-glucose 6-dehydrogenase, a gene encoding a putative group 1 family glycosyltransferase and a gene homologous to the rfa gene encoding ADP heptose-LPS heptosyltransferase, respectively. The Tn10-4 mutant carried Tn4351 DNA at the same position as that for Tn7-1. Gingipain activities associated with cells of the Tn7-3 mutant (rfa) were very weak, whereas gingipain activities were detected in the culture supernatants. Immunoblot and mass spectrometry analyses also revealed that gingipains, including their precursor forms, were present in the culture supernatants. A lipopolysaccharide (LPS) fraction of the rfa deletion mutant did not show the ladder pattern that was usually seen for the LPS of the wild-type P. gingivalis. A recombinant chimera gingipain was able to bind to an LPS fraction of the wild-type P. gingivalis in a dose-dependent manner. These results suggest that the rfa gene product is associated with biosynthesis of LPS and/or cell-surface polysaccharides that can function as an anchorage for gingipain-adhesin complexes.

  14. Wheat drought-responsive WXPL transcription factors regulate cuticle biosynthesis genes.

    PubMed

    Bi, Huihui; Luang, Sukanya; Li, Yuan; Bazanova, Natalia; Borisjuk, Nikolai; Hrmova, Maria; Lopato, Sergiy

    2017-02-04

    The cuticle forms a hydrophobic waxy layer that covers plant organs and provides protection from biotic and abiotic stresses. Transcription of genes responsible for cuticle formation is regulated by several types of transcription factors (TFs). Five orthologous to WAX PRODUCTION (WXP1 and WXP2) genes from Medicago truncatula were isolated from a cDNA library prepared from flag leaves and spikes of drought tolerant wheat (Triticum aestivum, breeding line RAC875) and designated TaWXP-like (TaWXPL) genes. Tissue-specific and drought-responsive expression of TaWXPL1D and TaWXPL2B was investigated by quantitative RT-PCR in two Australian wheat genotypes, RAC875 and Kukri, with contrasting glaucousness and drought tolerance. Rapid dehydration and/or slowly developing cyclic drought induced specific expression patterns of WXPL genes in flag leaves of the two cultivars RAC875 and Kukri. TaWXPL1D and TaWXPL2B proteins acted as transcriptional activators in yeast and in wheat cell cultures, and conserved sequences in their activation domains were localised at their C-termini. The involvement of wheat WXPL TFs in regulation of cuticle biosynthesis was confirmed by transient expression in wheat cells, using the promoters of wheat genes encoding two cuticle biosynthetic enzymes, the 3-ketoacyl-CoA-synthetase and the cytochrome P450 monooxygenase. Using the yeast 1-hybrid (Y1H) assay we also demonstrated the differential binding preferences of TaWXPL1D and TaWXPL2B towards three stress-related DNA cis-elements. Protein structural determinants underlying binding selectivity were revealed using comparative 3D molecular modelling of AP2 domains in complex with cis-elements. A scheme is proposed, which links the roles of WXPL and cuticle-related MYB TFs in regulation of genes responsible for the synthesis of cuticle components.

  15. Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

    PubMed Central

    Wang, Jianrong; Li, Yangyuan; Liu, Danni

    2014-01-01

    Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos. PMID:25474088

  16. Anthocyanin accumulation and molecular analysis of anthocyanin biosynthesis-associated genes in eggplant (Solanum melongena L.).

    PubMed

    Zhang, Yanjie; Hu, Zongli; Chu, Guihua; Huang, Cheng; Tian, Shibing; Zhao, Zhiping; Chen, Guoping

    2014-04-02

    Eggplant (Solanum melongena L.) is an edible fruit vegetable cultivated and consumed worldwide. The purple eggplant is more eye-catching and popular for the health-promoting anthocyanins contained in the fruit skin. Two kinds of anthocyanin were separated and identified from purple cultivar (Zi Chang) by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. To investigate the molecular mechanisms of anthocyanin accumulation in eggplant, the transcripts of anthocyanin biosynthetic and regulatory genes were analyzed in the fruit skin and the flesh of the purple cultivar and the white cultivar (Bai Xue). Compared with the other tissues, SmMYB1 and all anthocyanin biosynthetic genes except PAL were dramatically upregulated in the fruit skin of the purple cultivar. Overexpression of SmMYB1 activated abundant anthocyanin accumulation in the regenerating shoots of eggplant. These results prove that transcriptional activation of SmMYB1 accounts for constitutive upregulation of most anthocyanin biosynthetic genes and the onset of anthocyanin biosynthesis in the purple cultivar.

  17. Aflatoxins: A Global Concern for Food Safety, Human Health and Their Management

    PubMed Central

    Kumar, Pradeep; Mahato, Dipendra K.; Kamle, Madhu; Mohanta, Tapan K.; Kang, Sang G.

    2017-01-01

    The aflatoxin producing fungi, Aspergillus spp., are widely spread in nature and have severely contaminated food supplies of humans and animals, resulting in health hazards and even death. Therefore, there is great demand for aflatoxins research to develop suitable methods for their quantification, precise detection and control to ensure the safety of consumers’ health. Here, the chemistry and biosynthesis process of the mycotoxins is discussed in brief along with their occurrence, and the health hazards to humans and livestock. This review focuses on resources, production, detection and control measures of aflatoxins to ensure food and feed safety. The review is informative for health-conscious consumers and research experts in the fields. Furthermore, providing knowledge on aflatoxins toxicity will help in ensure food safety and meet the future demands of the increasing population by decreasing the incidence of outbreaks due to aflatoxins. PMID:28144235

  18. Regulation of carotenoid and bacteriochlorophyll biosynthesis genes and identification of an evolutionarily conserved gene required for bacteriochlorophyll accumulation.

    PubMed

    Armstrong, G A; Cook, D N; Ma, D; Alberti, M; Burke, D H; Hearst, J E

    1993-05-01

    The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.

  19. Gene regulation of anthocyanin biosynthesis in two blood-flesh peach (Prunus persica (L.) Batsch) cultivars during fruit development.

    PubMed

    Jiao, Yun; Ma, Rui-juan; Shen, Zhi-jun; Yan, Juan; Yu, Ming-liang

    2014-09-01

    The blood-flesh peach has become popular in China due to its attractive anthocyanin-induced pigmentation and antioxidant properties. In this study, we investigated the molecular mechanisms underlying anthocyanin accumulation by examining the expression of nine genes of the anthocyanin biosynthesis pathway found in the peach mesocarp. Expression was measured at six developmental stages in fruit of two blood-flesh and one white-flesh peach cultivars, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results show that the expression of the chalcone synthase (CHS) gene was closely related to anthocyanin accumulation in both of the blood-flesh peaches. In the white-flesh peach, we found that the transcription level of phenylalanine ammonia-lyase (PAL) during fruit development was much lower than that in the blood-flesh peach, even though all other genes of the anthocyanin biosynthesis pathway were highly expressed, suggesting that the PAL gene may be limiting in anthocyanin production in the white-flesh peach. Moreover, the transcription levels of the CHS and UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT) genes were markedly up-regulated at three days after bag removal (DABR) in the blood-flesh peach, suggesting that CHS and UFGT are the key genes in the process of anthocyanin biosynthesis for both of the blood-flesh peaches. The present study will be of great help in improving understanding of the molecular mechanisms involved in anthocyanin accumulation in blood-flesh peaches.

  20. Transcriptome Analysis of Syringa oblata Lindl. Inflorescence Identifies Genes Associated with Pigment Biosynthesis and Scent Metabolism

    PubMed Central

    Zheng, Jian; Hu, Zenghui; Guan, Xuelian; Dou, Dequan; Bai, Guo; Wang, Yu; Guo, Yingtian; Li, Wei; Leng, Pingsheng

    2015-01-01

    Syringa oblata Lindl. is a woody ornamental plant with high economic value and characteristics that include early flowering, multiple flower colors, and strong fragrance. Despite a long history of cultivation, the genetics and molecular biology of S. oblata are poorly understood. Transcriptome and expression profiling data are needed to identify genes and to better understand the biological mechanisms of floral pigments and scents in this species. Nine cDNA libraries were obtained from three replicates of three developmental stages: inflorescence with enlarged flower buds not protruded, inflorescence with corolla lobes not displayed, and inflorescence with flowers fully opened and emitting strong fragrance. Using the Illumina RNA-Seq technique, 319,425,972 clean reads were obtained and were assembled into 104,691 final unigenes (average length of 853 bp), 41.75% of which were annotated in the NCBI non-redundant protein database. Among the annotated unigenes, 36,967 were assigned to gene ontology categories and 19,956 were assigned to eukaryoticorthologous groups. Using the Kyoto Encyclopedia of Genes and Genomes pathway database, 12,388 unigenes were sorted into 286 pathways. Based on these transcriptomic data, we obtained a large number of candidate genes that were differentially expressed at different flower stages and that were related to floral pigment biosynthesis and fragrance metabolism. This comprehensive transcriptomic analysis provides fundamental information on the genes and pathways involved in flower secondary metabolism and development in S. oblata, providing a useful database for further research on S. oblata and other plants of genus Syringa. PMID:26587670

  1. Influences of Climate on Aflatoxin Producing Fungi and Aflatoxin Contamination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aflatoxins are potent mycotoxins that cause developmental and immune system suppression, cancer, and death. As a result of regulations intended to reduce human exposure, crop contamination with aflatoxins causes significant economic loss for producers, marketers, and processors of diverse susceptibl...

  2. Key gene regulating cell wall biosynthesis and recalcitrance in Populus, gene Y

    SciTech Connect

    Chen, Jay; Engle, Nancy; Gunter, Lee E.; Jawdy, Sara; Tschaplinski, Timothy J.; Tuskan, Gerald A.

    2015-12-08

    This disclosure provides methods and transgenic plants for improved production of renewable biofuels and other plant-derived biomaterials by altering the expression and/or activity of Gene Y, an O-acetyltransferase. This disclosure also provides expression vectors containing a nucleic acid (Gene Y) which encodes the polypeptide of SEQ ID NO: 1 and is operably linked to a heterologous promoter.

  3. High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

    PubMed Central

    Massot, Capucine; Bancel, Doriane; Lopez Lauri, Félicie; Truffault, Vincent; Baldet, Pierre; Stevens, Rebecca; Gautier, Hélène

    2013-01-01

    Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m-2 s-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling. PMID:24367665

  4. Profiling and Quantifying Differential Gene Transcription Provide Insights into Ganoderic Acid Biosynthesis in Ganoderma lucidum in Response to Methyl Jasmonate

    PubMed Central

    Shi, Liang; Mu, Da-Shuai; Jiang, Ai-Liang; Han, Qin; Zhao, Ming-Wen

    2013-01-01

    Ganoderma lucidum is a mushroom with traditional medicinal properties that has been widely used in China and other countries in Eastern Asia. Ganoderic acids (GA) produced by G. lucidum exhibit important pharmacological activities. Previous studies have demonstrated that methyl jasmonate (MeJA) is a potent inducer of GA biosynthesis and the expression of genes involved in the GA biosynthesis pathway in G. lucidum. To further explore the mechanism of GA biosynthesis, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) was used to identify genes that are differentially expressed in response to MeJA. Using 64 primer combinations, over 3910 transcriptionally derived fragments (TDFs) were obtained. Reliable sequence data were obtained for 390 of 458 selected TDFs. Ninety of these TDFs were annotated with known functions through BLASTX searching the GenBank database, and 12 annotated TDFs were assigned into secondary metabolic pathways by searching the KEGGPATHWAY database. Twenty-five TDFs were selected for qRT-PCR analysis to confirm the expression patterns observed with cDNA-AFLP. The qRT-PCR results were consistent with the altered patterns of gene expression revealed by the cDNA-AFLP technique. Additionally, the transcript levels of 10 genes were measured at the mycelium, primordia, and fruiting body developmental stages of G. lucidum. The greatest expression levels were reached during primordia for all of the genes except cytochrome b2 reached its highest expression level in the mycelium stage. This study not only identifies new candidate genes involved in the regulation of GA biosynthesis but also provides further insight into MeJA-induced gene expression and secondary metabolic response in G. lucidum. PMID:23762280

  5. Candidate genes involved in tanshinone biosynthesis in hairy roots of Salvia miltiorrhiza revealed by cDNA microarray.

    PubMed

    Cui, Guanghong; Huang, Luqi; Tang, Xiaojing; Zhao, Jingxue

    2011-04-01

    Salvia miltiorrhiza is a valuable Chinese herb (Danshen) that is widely used in traditional Chinese medicine. Diterpene quinones, known as tanshinones, are the main bioactive components of S. miltiorrhiza; however, there is only limited information regarding the molecular mechanisms underlying secondary metabolism in this plant. We used cDNA microarray analysis to identify changes in the gene expression profile at different stages of hairy root development in S. miltiorrhiza. A total of 203 genes were singled out from 4,354 cDNA clones on the microarray, and 114 unique differentially expressed cDNA clones were identified: six genes differentially expressed in 45-day hairy root compared with 30-day hairy root; 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root; and 12 genes unstably expressed at different stages. Among the 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root, a total of 57 genes were up-regulated, and 26 genes represent 29 metabolism-related enzymes. Copalyl diphosphate synthase, which catalyzes the conversion of the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate to copalyl diphosphate, was up-regulated 6.63 fold, and another six genes involved in tanshinone biosynthesis and eight candidate P450 genes were also differentially expressed. These data provide new insights for further identification of the enzymes involved in tanshinone biosynthesis.

  6. Cloning and characterization of the gene cluster for biosynthesis of ectoine from Nesterenkonia halobia DSM 20541.

    PubMed

    Zhang, Bo; Bao, Xin; Wang, Lei; Yang, Su Sheng

    2008-06-01

    The ectABC genes encoding the biosynthesis of ectoine were identified from Nesterenkonia halobia DSM 20541. The intergenic regions of the ectABC genes from N. halobia DSM 20541 were more loosely spaced than those that had been reported before. The amino acid sequence deduced from ectABC of the strain was highly homologous to the EctABC of Brevibacterium linens BL2 (EctA 50%, EctB 70%, and EctC 68% identities). The osmoprotection of ectABC was studied in the Escherichia coli KNabc and E. coli XL1-Blue. The results revealed that ectABC could shorten the lag phase and enhance the final OD600 of E. coli XL1-Blue in MM63 medium containing 0.68 M NaCl, and could initiate KNabc growth in 0.2 M NaCl. Ectoine was proven to be accumulated in E. coli KNabc/pGEM-Nect using HPLC-UV, and validated by LC-MSD-Trap-VL.

  7. Enhancement of doxorubicin production by expression of structural sugar biosynthesis and glycosyltransferase genes in Streptomyces peucetius.

    PubMed

    Malla, Sailesh; Niraula, Narayan Prasad; Liou, Kwangkyoung; Sohng, Jae Kyung

    2009-08-01

    To enhance doxorubicin (DXR) production, the structural sugar biosynthesis genes desIII and desIV from Streptomyces venezuelae ATCC 15439 and the glycosyltransferase pair dnrS/dnrQ from Streptomyces peucetius ATCC 27952 were cloned into the expression vector pIBR25, which contains a strong ermE promoter. The recombinant plasmids pDnrS25 and pDnrQS25 were constructed for overexpression of dnrS and the dnrS/dnrQ pair, whereas pDesSD25 and pDesQS25 were constructed to express desIII/desIV and dnrS/dnrQ-desIII/desIV, respectively. All of these recombinant plasmids were introduced into S. peucetius ATCC 27952. The recombinant strains produced more DXR than the S. peucetius parental strain: a 1.2-fold increase with pDnrS25, a 2.8-fold increase with pDnrQS25, a 2.6-fold increase with pDesSD25, and a 5.6-fold increase with pDesQS25. This study showed that DXR production was significantly enhanced by overexpression of potential biosynthetic sugar genes and glycosyltransferase.

  8. Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions.

    PubMed

    Luo, Chuping; Liu, Xuehui; Zhou, Huafei; Wang, Xiaoyu; Chen, Zhiyi

    2015-01-01

    Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way.

  9. Gene coexpression network analysis of oil biosynthesis in an interspecific backcross of oil palm.

    PubMed

    Guerin, Chloé; Joët, Thierry; Serret, Julien; Lashermes, Philippe; Vaissayre, Virginie; Agbessi, Mawussé D T; Beulé, Thierry; Severac, Dany; Amblard, Philippe; Tregear, James; Durand-Gasselin, Tristan; Morcillo, Fabienne; Dussert, Stéphane

    2016-09-01

    Global demand for vegetable oils is increasing at a dramatic rate, while our understanding of the regulation of oil biosynthesis in plants remains limited. To gain insights into the mechanisms that govern oil synthesis and fatty acid (FA) composition in the oil palm fruit, we used a multilevel approach combining gene coexpression analysis, quantification of allele-specific expression and joint multivariate analysis of transcriptomic and lipid data, in an interspecific backcross population between the African oil palm, Elaeis guineensis, and the American oil palm, Elaeis oleifera, which display contrasting oil contents and FA compositions. The gene coexpression network produced revealed tight transcriptional coordination of fatty acid synthesis (FAS) in the plastid with sugar sensing, plastidial glycolysis, transient starch storage and carbon recapture pathways. It also revealed a concerted regulation, along with FAS, of both the transfer of nascent FA to the endoplasmic reticulum, where triacylglycerol assembly occurs, and of the production of glycerol-3-phosphate, which provides the backbone of triacylglycerols. Plastid biogenesis and auxin transport were the two other biological processes most tightly connected to FAS in the network. In addition to WRINKLED1, a transcription factor (TF) known to activate FAS genes, two novel TFs, termed NF-YB-1 and ZFP-1, were found at the core of the FAS module. The saturated FA content of palm oil appeared to vary above all in relation to the level of transcripts of the gene coding for β-ketoacyl-acyl carrier protein synthase II. Our findings should facilitate the development of breeding and engineering strategies in this and other oil crops.

  10. Transcriptomic analysis of Camellia ptilophylla and identification of genes associated with flavonoid and caffeine biosynthesis.

    PubMed

    Li, M M; Xue, J Y; Wen, Y L; Guo, H S; Sun, X Q; Zhang, Y M; Hang, Y Y

    2015-12-29

    Camellia ptilophylla, or cocoa tea, is naturally decaffeinated and its predominant catechins and purine alkaloids are trans-catechins and theobromine Regular tea [Camellia sinensis (L.) O. Ktze.] is evolutionarily close to cocoa tea and produces cis-catechins and caffeine. Here, the transcriptome of C. ptilophylla was sequenced using the 101-bp paired-end technique. The quality of the raw data was assessed to yield 70,227,953 cleaned reads totaling 7.09 Gbp, which were assembled de novo into 56,695 unique transcripts and then clustered into 44,749 unigenes. In catechin biosynthesis, leucoanthocyanidin reductase (LAR) catalyzes the transition of leucoanthocyanidin to trans-catechins, while anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR) catalyze cis-catechin production. Our data demonstrate that two LAR genes (CpLAR1 and CpLAR2) by C. ptilophylla may be advantageous due to the combined effects of this quantitative trait, permitting increased leucoanthocyanidin consumption for the synthesis of trans-catechins. In contrast, the only ANS gene observed in C. sinensis (CsANS) shared high identity (99.2%) to one homolog from C. ptilophylla (CpANS1), but lower identity (~80%) to another (CpANS2). We hypothesized that the diverged CpANS2 might have lost its ability to synthesize cis-catechins. C. ptilophylla and C. sinensis each contain two copies of ANR, which share high identity and may share the same function. Transcriptomic sequencing captured two N-methyl nucleosidase genes named NMT1 and NMT2. NMT2 was highly identical to three orthologous genes TCS2, PCS2, and ICS2, which did not undergo methylation in vitro; in contrast, NMT1 was less identical to TCS, PCS and ICS, indicating that NMT1 may undergo neofunctionalization.

  11. Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds.

    PubMed

    Tagashira, Yusuke; Shimizu, Tomoe; Miyamoto, Masanobu; Nishida, Sho; Yoshida, Kaoru T

    2015-04-24

    The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP₆) biosynthesis-related genes, as InsP₆ is a major storage form of P in seeds. The rice (Oryza sativa L.) low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP₆ biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP₆ and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP₆ biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP₆ biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

  12. Polysaccharide biosynthesis-related genes explain phenotype-genotype correlation of Microcystis colonies in Meiliang Bay of Lake Taihu, China

    PubMed Central

    Xu, Shutu; Sun, Qianqian; Zhou, Xiaohua; Tan, Xiao; Xiao, Man; Zhu, Wei; Li, Ming

    2016-01-01

    The 16S rDNA, 16S-23S rDNA-ITS, cpcBA-IGS, mcy gene and several polysaccharide biosynthesis-related genes (epsL and TagH) were analyzed along with the identification of the morphology of Microcystis colonies collected in Lake Taihu in 2014. M. wesenbergii colonies could be distinguished directly from other colonies using espL. TagH divided all of the samples into two clusters but failed to distinguish different phenotypes. Our results indicated that neither morphology nor molecular tools including 16S rDNA, 16S-23S ITS and cpcBA-IGS could distinguish toxic and non-toxic species among the identified Microcystis species. No obvious relationship was detected between the phenotypes of Microcystis and their genotypes using 16S, 16S-23S and cpcBA-IGS, but polysaccharide biosynthesis-related genes may distinguish the Microcystis phenotypes. Furthermore, the sequences of the polysaccharide biosynthesis-related genes (espL and TagH) extracted from Microcystis scums collected throughout 2015 was analyzed. Samples dominated by M. ichthyoblabe (60–100%) and M. wesenbergii (60–100%) were divided into different clade by both espL and TagH, respectively. Therefore, it was confirmed that M. wesenbergii and M. ichthyoblabe could be distinguished by the polysaccharide biosynthesis-related genes (espL and TagH). This study is of great significance in filling the gap between classification of molecular biology and the morphological taxonomy of Microcystis. PMID:27752091

  13. Polysaccharide biosynthesis-related genes explain phenotype-genotype correlation of Microcystis colonies in Meiliang Bay of Lake Taihu, China

    NASA Astrophysics Data System (ADS)

    Xu, Shutu; Sun, Qianqian; Zhou, Xiaohua; Tan, Xiao; Xiao, Man; Zhu, Wei; Li, Ming

    2016-10-01

    The 16S rDNA, 16S-23S rDNA-ITS, cpcBA-IGS, mcy gene and several polysaccharide biosynthesis-related genes (epsL and TagH) were analyzed along with the identification of the morphology of Microcystis colonies collected in Lake Taihu in 2014. M. wesenbergii colonies could be distinguished directly from other colonies using espL. TagH divided all of the samples into two clusters but failed to distinguish different phenotypes. Our results indicated that neither morphology nor molecular tools including 16S rDNA, 16S-23S ITS and cpcBA-IGS could distinguish toxic and non-toxic species among the identified Microcystis species. No obvious relationship was detected between the phenotypes of Microcystis and their genotypes using 16S, 16S-23S and cpcBA-IGS, but polysaccharide biosynthesis-related genes may distinguish the Microcystis phenotypes. Furthermore, the sequences of the polysaccharide biosynthesis-related genes (espL and TagH) extracted from Microcystis scums collected throughout 2015 was analyzed. Samples dominated by M. ichthyoblabe (60–100%) and M. wesenbergii (60–100%) were divided into different clade by both espL and TagH, respectively. Therefore, it was confirmed that M. wesenbergii and M. ichthyoblabe could be distinguished by the polysaccharide biosynthesis-related genes (espL and TagH). This study is of great significance in filling the gap between classification of molecular biology and the morphological taxonomy of Microcystis.

  14. Dynamic changes in catechin levels and catechin biosynthesis-related gene expression in albino tea plants (Camellia sinensis L.).

    PubMed

    Xiong, Ligui; Li, Juan; Li, Yinhua; Yuan, Ling; Liu, Shuoqian; Huang, Jian'an; Liu, Zhonghua

    2013-10-01

    Tea (Camellia sinensis (L.) O. Kuntze) leaves are a major source of flavonoids that mainly belong to the flavan-3-ols or catechins and are implicated in a wide range of health benefits. Although the catechins in tea leaves were identified long ago, the regulatory mechanisms governing catechin biosynthesis remain unclear. In the present work, the dynamic changes of catechin levels and the expression profiles of catechin-related genes in albino tea plants were intensively examined. The amounts of most catechins decreased to their lowest levels in the albino phase, when epigallocatechingallate was the highest of the catechins compared to all catechins, and catechin the lowest. Enzyme assays indicated that phenylalanine ammonia-lyase (PAL) activity was positively correlated with the concentration of catechins (r = 0.673). Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that the transcript abundance of flavonoid biosynthetic genes followed a tightly regulated biphasic pattern, and was affected by albinism. These genes (PAL, C4H, 4CL, CHS, CHI, F3H, FLS, F3'H, F3'5'H, DFR, LAR, ANS and ANR) encode enzymes in flavonoid biosynthesis. The expression levels of PAL, F3H and FLS were correlated with the concentration of catechins and the correlation coefficients were -0.683, 0.687 and -0.602, respectively. Therefore, these results indicate that PAL might be a core regulator in the control of catechin biosynthesis in albino tea plants.

  15. Comparative Transcriptome Analysis of Penicillium citrinum Cultured with Different Carbon Sources Identifies Genes Involved in Citrinin Biosynthesis

    PubMed Central

    Li, Taotao; Jiang, Guoxiang; Qu, Hongxia; Wang, Yong; Xiong, Yehui; Jian, Qijie; Wu, Yu; Duan, Xuewu; Zhu, Xiangrong; Hu, Wenzhong; Wang, Jiasheng; Gong, Liang; Jiang, Yueming

    2017-01-01

    Citrinin is a toxic secondary metabolite of Penicillium citrinum and its contamination in many food items has been widely reported. However, research on the citrinin biosynthesis pathway and its regulation mechanism in P. citrinum is rarely reported. In this study, we investigated the effect of different carbon sources on citrinin production by P. citrinum and used transcriptome analysis to study the underlying molecular mechanism. Our results indicated that glucose, used as the sole carbon source, could significantly promote citrinin production by P. citrinum in Czapek’s broth medium compared with sucrose. A total of 19,967 unigenes were annotated by BLAST in Nr, Nt, Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Transcriptome comparison between P. citrinum cultured with sucrose and glucose revealed 1085 differentially expressed unigenes. Among them, 610 were upregulated while 475 were downregulated under glucose as compared to sucrose. KEGG pathway and Gene ontology (GO) analysis indicated that many metabolic processes (e.g., carbohydrate, secondary metabolism, fatty acid and amino acid metabolism) were affected, and potentially interesting genes that encoded putative components of signal transduction, stress response and transcription factor were identified. These genes obviously had important impacts on their regulation in citrinin biosynthesis, which provides a better understanding of the molecular mechanism of citrinin biosynthesis by P. citrinum. PMID:28230802

  16. De novo transcriptome of safflower and the identification of putative genes for oleosin and the biosynthesis of flavonoids.

    PubMed

    Li, Haiyan; Dong, Yuanyuan; Yang, Jing; Liu, Xiuming; Wang, Yanfang; Yao, Na; Guan, Lili; Wang, Nan; Wu, Jinyu; Li, Xiaokun

    2012-01-01

    Safflower (Carthamus tinctorius L.) is one of the most extensively used oil crops in the world. However, little is known about how its compounds are synthesized at the genetic level. In this study, Solexa-based deep sequencing on seed, leaf and petal of safflower produced a de novo transcriptome consisting of 153,769 unigenes. We annotated 82,916 of the unigenes with gene annotation and assigned functional terms and specific pathways to a subset of them. Metabolic pathway analysis revealed that 23 unigenes were predicted to be responsible for the biosynthesis of flavonoids and 8 were characterized as seed-specific oleosins. In addition, a large number of differentially expressed unigenes, for example, those annotated as participating in anthocyanin and chalcone synthesis, were predicted to be involved in flavonoid biosynthesis pathways. In conclusion, the de novo transcriptome investigation of the unique transcripts provided candidate gene resources for studying oleosin-coding genes and for investigating genes related to flavonoid biosynthesis and metabolism in safflower.

  17. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    PubMed

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-02

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

  18. A maize QTL for silk maysin levels contains duplicated Myb-homologous genes which jointly regulate flavone biosynthesis.

    PubMed

    Zhang, Peifen; Wang, Yibin; Zhang, Jianbo; Maddock, Sheila; Snook, Maurice; Peterson, Thomas

    2003-05-01

    The maize p1 locus coincides with a major QTL (quantitative trait locus) determining levels of maysin, a C-glycosyl flavone that deters feeding by corn ear-worm. The p1 gene is tightly linked with a second gene, p2, and both genes encode similar Myb-domain proteins. We show here that maize cell cultures transformed with either the p1 or p2 genes expressed under a constitutive promoter accumulate transcripts for flavonoid biosynthetic genes, and synthesize phenylpropanoids and C-glycosyl flavones related to maysin. Additionally, maize plants that are deleted for the p1 gene have reduced maysin levels and moderate silk-browning reaction, whereas plants with a deletion of both p1 and p2 have non-detectable silk maysin and non-browning silks. We conclude that both p1 and p2 induce maysin biosynthesis in silk, although the two genes differ in their expression and pigmentation effects in other tissues. These results show that a QTL for flavone biosynthesis actually comprises two tightly linked genes with related functions.

  19. The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana.

    PubMed

    Zhang, Gaoyang; Zhang, Yujia; Xu, Jiantang; Niu, Xiaoping; Qi, Jianmin; Tao, Aifen; Zhang, Liwu; Fang, Pingping; Lin, LiHui; Su, Jianguang

    2014-08-10

    The Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a key enzyme in lignin biosynthesis in plants. In this study we cloned the full-length cDNA of the Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) gene from jute using homology clone (primers were designed according to the sequence of CCoAOMT gene of other plants), and a modified RACE technique, subsequently named "CcCCoAOMT1". Bioinformatic analyses showed that the gene is a member of the CCoAOMT gene family. Real-time PCR analysis revealed that the CcCCoAOMT1 gene is constitutively expressed in all tissues, and the expression level was greatest in stem, followed by stem bark, roots and leaves. In order to understand this gene's function, we transformed it into Arabidopsis thaliana; integration (one insertion site) was confirmed following PCR and southern hybridization. The over-expression of CcCCoAOMT1 in these transgenic A.thaliana plants resulted in increased plant height and silique length relative to non-transgenic plants. Perhaps the most important finding was that the transgenic Arabidopsis plants contained more lignin (20.44-21.26%) than did control plants (17.56%), clearly suggesting an important role of CcCCoAOMT1 gene in lignin biosynthesis. These data are important for the success of efforts to reduce jute lignin content (thereby increasing fiber quality) via CcCCoAOMT1 gene inhibition.

  20. The gene cluster for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway homologues and a monomodular non-ribosomal peptide synthetase gene.

    PubMed

    He, J; Magarvey, N; Piraee, M; Vining, L C

    2001-10-01

    Regions of the Streptomyces venezuelae ISP5230 chromosome flanking pabAB, an amino-deoxychorismate synthase gene needed for chloramphenicol (Cm) production, were examined for involvement in biosynthesis of the antibiotic. Three of four ORFs in the sequence downstream of pabAB resembled genes involved in the shikimate pathway. BLASTX searches of GenBank showed that the deduced amino acid sequences of ORF3 and ORF4 were similar to proteins encoded by monofunctional genes for chorismate mutase and prephenate dehydrogenase, respectively, while the sequence of the ORF5 product resembled deoxy-arabino-heptulosonate-7-phosphate (DAHP) synthase, the enzyme that initiates the shikimate pathway. A relationship to Cm biosynthesis was indicated by sequence similarities between the ORF6 product and membrane proteins associated with Cm export. BLASTX searches of GenBank for matches with the translated sequence of ORF1 in chromosomal DNA immediately upstream of pabAB did not detect products relevant to Cm biosynthesis. However, the presence of Cm biosynthesis genes in a 7.5 kb segment of the chromosome beyond ORF1 was inferred when conjugal transfer of the DNA into a blocked S. venezuelae mutant restored Cm production. Deletions in the 7.5 kb segment of the wild-type chromosome eliminated Cm production, confirming the presence of Cm biosynthesis genes in this region. Sequencing and analysis located five ORFs, one of which (ORF8) was deduced from BLAST searches of GenBank, and from characteristic motifs detected in alignments of its deduced amino acid sequence, to be a monomodular nonribosomal peptide synthetase. GenBank searches did not identify ORF7, but matched the translated sequences of ORFs 9, 10 and 11 with short-chain ketoreductases, the ATP-binding cassettes of ABC transporters, and coenzyme A ligases, respectively. As has been shown for ORF2, disrupting ORF3, ORF7, ORF8 or ORF9 blocked Cm production.

  1. Ageratum conyzoides essential oil as aflatoxin suppressor of Aspergillus flavus.

    PubMed

    Nogueira, Juliana H C; Gonçalez, Edlayne; Galleti, Silvia R; Facanali, Roseane; Marques, Márcia O M; Felício, Joana D

    2010-01-31

    Aflatoxin B(1) (AFB(1)) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. Inhibitory effects of essential oil of Ageratum conyzoides, on the mycelial growth and aflatoxin B(1) production by Aspergillus flavus were studied. Cultures were incubated in yeast extract-sucrose (YES) broth for days at 25 degrees C at the following different concentrations of the essential oil (from 0.0 to 30mug/mL). The essential oil inhibited fungal growth to different extents depending on the concentration, and completely inhibited aflatoxin production at concentrations above 0.10microg/mL. The analysis of the oil by GC/MS showed that its main components are precocene II (46.35%), precocene I (42.78%), cumarine (5.01%) and Trans-caryophyllene (3.02%). Comparison by transmission electron microscopy of the fungal cells, control and those incubated with different concentrations of essential oil, showed ultra-structural changes which were concentration dependent of the essential oil of A. conyzoides. Such ultra-structural changes were more evident in the endomembrane system, affecting mainly the mitochondria. Degradation was also observed in both surrounding fibrils. The ability to inhibit aflatoxin production as a new biological activity of A.conyzoides L. indicates that it may be considered as a useful tool for a better understanding of the complex pathway of aflatoxin biosynthesis.

  2. Phosphate control of pabS gene transcription during candicidin biosynthesis.

    PubMed

    Asturias, J A; Liras, P; Martín, J F

    1990-09-01

    The pabS gene of Streptomyces griseus IMRU3570 encodes the enzyme p-aminobenzoic acid synthase, which synthesizes p-aminobenzoic acid (PABA), a precursor of the antibiotic candicidin (Cd). The pabS transcript reached a peak at 12 h of incubation in batch cultures, preceding the formation of PABA synthase and the antibiotic itself. A decay of the pabS transcript was observed with an apparent half-life of 35 min. Inorganic phosphate (Pi; 7.5 mM) reduced the synthesis of the pabS transcript by 90-95%, and consequently the formation of PABA synthase and Cd. Thirty min after addition of 7.5 mM Pi, the cells synthesized only about 15% as much pabS transcript compared to control cultures. However, Pi stimulated two- to threefold total RNA synthesis. The 1.7-kb pabS transcript shown by Northern hybridization was greatly reduced in amount in cells grown in 7.5 mM phosphate. Pi-deregulated mutants, described previously, were impaired in the transcriptional control exerted by Pi. It is concluded that Pi control of PABA synthase and Cd biosynthesis is exerted by repression of formation of the pabS mRNA.

  3. [Enhancement of artemisinin biosynthesis in transgenic Artemisia annua L. by overexpressed HDR and ADS genes].

    PubMed

    Wang, Ya-Xiong; Long, Shi-Ping; Zeng, Li-Xia; Xiang, Li-En; Lin, Zhi; Chen, Min; Liao, Zhi-Hua

    2014-09-01

    Artemisnin is a novel sesquiterpene lactone with an internal peroxide bridge structure, which is extracted from traditional Chinese herb Artemisia annua L. (Qinghao). Recommended by World Health Organization, artemisinin is the first-line drug in the treatment of encephalic and chloroquine-resistant malaria. In the present study, transgenic A. annua plants were developed by overexpressing the key enzymes involved in the biosynthetic pathway of artemisinin. Based on Agrobacterium-mediated transformation methods, transgenic plants of A. annua with overexpression of both HDR and ADS were obtained through hygromycin screening. The genomic PCR analysis confirmed six transgenic lines in which both HDR and ADS were integrated into genome. The gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had higher expression levels of HDR and ADS than the non-transgenic control (except ah3 in which the expression level of ADS showed no significant difference compared with control); and the HPLC analysis of artemisinin demonstrated that transgenic A. annua plants produced artemisinin at significantly higher level than non-transgenic plants. Especially, the highest content of artemisinin was found in transgenic line ah70, in which the artemisinin content was 3.48 times compared with that in non-transgenic lines. In summary, overexpression of HDR and ADS facilitated artemisinin biosynthesis and this method could be applied to develop transgenic plants of A. annua with higher yield of artemisinin.

  4. Aflatoxins and safe storage

    PubMed Central

    Villers, Philippe

    2014-01-01

    The paper examines both field experience and research on the prevention of the exponential growth of aflatoxins during multi-month post-harvest storage in hot, humid countries. The approach described is the application of modern safe storage methods using flexible, Ultra Hermetic™ structures that create an unbreatheable atmosphere through insect and microorganism respiration alone, without use of chemicals, fumigants, or pumps. Laboratory and field data are cited and specific examples are given describing the uses of Ultra Hermetic storage to prevent the growth of aflatoxins with their significant public health consequences. Also discussed is the presently limited quantitative information on the relative occurrence of excessive levels of aflatoxin (>20 ppb) before vs. after multi-month storage of such crops as maize, rice, and peanuts when under high humidity, high temperature conditions and, consequently, the need for further research to determine the frequency at which excessive aflatoxin levels are reached in the field vs. after months of post-harvest storage. The significant work being done to reduce aflatoxin levels in the field is mentioned, as well as its probable implications on post-harvest storage. Also described is why, with some crops such as peanuts, using Ultra Hermetic storage may require injection of carbon dioxide, or use of an oxygen absorber as an accelerant. The case of peanuts is discussed and experimental data is described. PMID:24782846

  5. Aflatoxins and safe storage.

    PubMed

    Villers, Philippe

    2014-01-01

    The paper examines both field experience and research on the prevention of the exponential growth of aflatoxins during multi-month post-harvest storage in hot, humid countries. The approach described is the application of modern safe storage methods using flexible, Ultra Hermetic™ structures that create an unbreatheable atmosphere through insect and microorganism respiration alone, without use of chemicals, fumigants, or pumps. Laboratory and field data are cited and specific examples are given describing the uses of Ultra Hermetic storage to prevent the growth of aflatoxins with their significant public health consequences. Also discussed is the presently limited quantitative information on the relative occurrence of excessive levels of aflatoxin (>20 ppb) before vs. after multi-month storage of such crops as maize, rice, and peanuts when under high humidity, high temperature conditions and, consequently, the need for further research to determine the frequency at which excessive aflatoxin levels are reached in the field vs. after months of post-harvest storage. The significant work being done to reduce aflatoxin levels in the field is mentioned, as well as its probable implications on post-harvest storage. Also described is why, with some crops such as peanuts, using Ultra Hermetic storage may require injection of carbon dioxide, or use of an oxygen absorber as an accelerant. The case of peanuts is discussed and experimental data is described.

  6. Comprehensive assessment of the genes involved in withanolide biosynthesis from Withania somnifera: chemotype-specific and elicitor-responsive expression.

    PubMed

    Agarwal, Aditya Vikram; Gupta, Parul; Singh, Deeksha; Dhar, Yogeshwar Vikram; Chandra, Deepak; Trivedi, Prabodh Kumar

    2017-03-11

    Withania somnifera (L.) Dunal (Family, Solanaceae), is among the most valuable medicinal plants used in Ayurveda owing to its rich reservoir of pharmaceutically active secondary metabolites known as withanolides. Withanolides are C28-steroidal lactones having a triterpenoidal metabolic origin synthesised via mevalonate (MVA) pathway and methyl-D-erythritol-4-phosphate (MEP) pathway involving metabolic intermediacy of 24-methylene (C30-terpenoid) cholesterol. Phytochemical studies suggest differences in the content and/or nature of withanolides in different tissues of different chemotypes. Though development of genomic resources has provided information about putative genes encoding enzymes for biosynthesis of intermediate steps of terpenoid backbone, not much is known about their regulation and response to elicitation. In this study, we generated detailed molecular information about genes catalysing key regulatory steps of withanolide biosynthetic pathway. The full-length sequences of genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis and their paralogs have been characterized for their functional and structural properties as well as phylogeny using bioinformatics approach. The expression analysis suggests that these genes are differentially expressed in different tissues (with maximal expression in young leaf), chemotypes and in response to salicylic acid (SA) and methyl jasmonate (MJ) treatments. Sub-cellular localization studies suggest that both paralogs of sterol ∆-7 reductase (WsDWF5-1 and WsDWF5-2) are localized in the endoplasmic reticulum (ER) thus supporting their indispensible role in withanolide biosynthesis. Comprehensive information developed, in this study, will lead to elucidation of chemotype- as well as tissue-specific withanolide biosynthesis and development of new tools for functional genomics in this important medicinal plant.

  7. Genome-Scale Discovery of Cell Wall Biosynthesis Genes in Populus (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

    ScienceCinema

    Muchero, Wellington [Oak Ridge National Laboratory

    2016-07-12

    Wellington Muchero from Oak Ridge National Laboratory gives a talk titled "Discovery of Cell Wall Biosynthesis Genes in Populus" at the JGI 7th Annual Users Meeting: Genomics of Energy & Environment Meeting on March 22, 2012 in Walnut Creek, California.

  8. Evolutionary origin of the NCSI gene subfamily encoding norcoclaurine synthase is associated with the biosynthesis of benzylisoquinoline alkaloids in plants

    PubMed Central

    Vimolmangkang, Sornkanok; Deng, Xianbao; Owiti, Albert; Meelaph, Thitirat; Ogutu, Collins; Han, Yuepeng

    2016-01-01

    Sacred lotus is rich in biologically active compounds, particularly benzylisoquinoline alkaloids (BIAs). Here, we report on isolation of genes encoding (S)-norcoclaurine synthase (NCS) in sacred lotus, which is a key entry-enzyme in BIA biosynthesis. Seven NCS genes, designated NnNCS1 through NnNCS7, were identified in the sacred lotus genome, and five are located next to each other within a 83 kb region on scaffold 8. The NCS genes are divided into two subfamilies, designated NCSI and NCSII. The NCSII genes are universal in plants, while the NCSI genes are only identified in a limited number of dicotyledonous taxa that produce BIAs. In sacred lotus, only NnNCS4 belongs to the NCSII subfamily, whilst the rest NCS genes within the NCSI subfamily. Overall, the NnNCS7 gene was predominantly expressed in all tested tissues, and its expression is significantly correlated with alkaloid content in leaf. In contrast, the NnNCS4 expression shows no significant correlation with alkaloid accumulation in leaf, and its lack of expression cannot inhibit alkaloid accumulation. Taken together, these results suggest that the NCSI subfamily is crucial for BIA biosynthesis, and its origin may represent an important evolutionary event that allows certain plant taxa to produce BIAs. PMID:27189519

  9. Identification of Arabidopsis GPAT9 (At5g60620) as an Essential Gene Involved in Triacylglycerol Biosynthesis1[OPEN

    PubMed Central

    Browse, John

    2016-01-01

    The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER)-localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here, we present the results of a series of in vivo, in vitro, and in silico experiments in Arabidopsis (Arabidopsis thaliana) designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and the transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together, these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis. PMID:26586834

  10. De Novo Transcriptome Assembly in Chili Pepper (Capsicum frutescens) to Identify Genes Involved in the Biosynthesis of Capsaicinoids

    PubMed Central

    Liu, Shaoqun; Li, Wanshun; Wu, Yimin; Chen, Changming; Lei, Jianjun

    2013-01-01

    The capsaicinoids are a group of compounds produced by chili pepper fruits and are used widely in many fields, especially in medical purposes. The capsaicinoid biosynthetic pathway has not yet been established clearly. To understand more knowledge in biosynthesis of capsaicinoids, we applied RNA-seq for the mixture of placenta and pericarp of pungent pepper (Capsicum frutescens L.). We have assessed the effect of various assembly parameters using different assembly software, and obtained one of the best strategies for de novo assembly of transcriptome data. We obtained a total 54,045 high-quality unigenes (transcripts) using Trinity software. About 92.65% of unigenes showed similarity to the public protein sequences, genome of potato and tomato and pepper (C. annuum) ESTs databases. Our results predicted 3 new structural genes (DHAD, TD, PAT), which filled gaps of the capsaicinoid biosynthetic pathway predicted by Mazourek, and revealed new candidate genes involved in capsaicinoid biosynthesis based on KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. A significant number of SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) markers were predicted in C. frutescens and C. annuum sequences, which will be helpful in the identification of polymorphisms within chili pepper populations. These data will provide new insights to the pathway of capsaicinoid biosynthesis and subsequent research of chili peppers. In addition, our strategy of de novo transcriptome assembly is applicable to a wide range of similar studies. PMID:23349661

  11. Deep sequencing and transcriptome analyses to identify genes involved in secoiridoid biosynthesis in the Tibetan medicinal plant Swertia mussotii.

    PubMed

    Liu, Yue; Wang, Yi; Guo, Fengxian; Zhan, Lin; Mohr, Toni; Cheng, Prisca; Huo, Naxin; Gu, Ronghui; Pei, Danning; Sun, Jiaqing; Tang, Li; Long, Chunlin; Huang, Luqi; Gu, Yong Q

    2017-02-22

    Swertia mussotii Franch. is an important traditional Tibetan medicinal plant with pharmacological properties effective in the treatment of various ailments including hepatitis. Secoiridoids are the major bioactive compounds in S. mussotii. To better understand the secoiridoid biosynthesis pathway, we generated transcriptome sequences from the root, leaf, stem, and flower tissues, and performed de novo sequence assembly, yielding 98,613 unique transcripts with an N50 of 1,085 bp. Putative functions could be assigned to 35,029 transcripts (35.52%) based on BLAST searches against annotation databases including GO and KEGG. The expression profiles of 39 candidate transcripts encoding the key enzymes for secoiridoid biosynthesis were examined in different S. mussotii tissues, validated by qRT-PCR, and compared with the homologous genes from S. japonica, a species in the same family, unveiling the gene expression, regulation, and conservation of the pathway. The examination of the accumulated levels of three bioactive compounds, sweroside, swertiamarin, and gentiopicroside, revealed their considerable variations in different tissues, with no significant correlation with the expression profiles of key genes in the pathway, suggesting complex biological behaviours in the coordination of metabolite biosynthesis and accumulation. The genomic dataset and analyses presented here lay the foundation for further research on this important medicinal plant.

  12. Deep sequencing and transcriptome analyses to identify genes involved in secoiridoid biosynthesis in the Tibetan medicinal plant Swertia mussotii

    PubMed Central

    Liu, Yue; Wang, Yi; Guo, Fengxian; Zhan, Lin; Mohr, Toni; Cheng, Prisca; Huo, Naxin; Gu, Ronghui; Pei, Danning; Sun, Jiaqing; Tang, Li; Long, Chunlin; Huang, Luqi; Gu, Yong Q.

    2017-01-01

    Swertia mussotii Franch. is an important traditional Tibetan medicinal plant with pharmacological properties effective in the treatment of various ailments including hepatitis. Secoiridoids are the major bioactive compounds in S. mussotii. To better understand the secoiridoid biosynthesis pathway, we generated transcriptome sequences from the root, leaf, stem, and flower tissues, and performed de novo sequence assembly, yielding 98,613 unique transcripts with an N50 of 1,085 bp. Putative functions could be assigned to 35,029 transcripts (35.52%) based on BLAST searches against annotation databases including GO and KEGG. The expression profiles of 39 candidate transcripts encoding the key enzymes for secoiridoid biosynthesis were examined in different S. mussotii tissues, validated by qRT-PCR, and compared with the homologous genes from S. japonica, a species in the same family, unveiling the gene expression, regulation, and conservation of the pathway. The examination of the accumulated levels of three bioactive compounds, sweroside, swertiamarin, and gentiopicroside, revealed their considerable variations in different tissues, with no significant correlation with the expression profiles of key genes in the pathway, suggesting complex biological behaviours in the coordination of metabolite biosynthesis and accumulation. The genomic dataset and analyses presented here lay the foundation for further research on this important medicinal plant. PMID:28225035

  13. Organ- and Growing Stage-Specific Expression of Solanesol Biosynthesis Genes in Nicotiana tabacum Reveals Their Association with Solanesol Content.

    PubMed

    Yan, Ning; Zhang, Hongbo; Zhang, Zhongfeng; Shi, John; Timko, Michael P; Du, Yongmei; Liu, Xinmin; Liu, Yanhua

    2016-11-15

    Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase (DXS), two 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD), four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE), two 2-C-methyl-d-erythritol 2,4-cyclo-diphosphate synthase (IspF), four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH), six isopentenyl diphosphate isomerase (IPI), and two solanesyl diphosphate synthase (SPS) candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2), which possessed two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs and of leaves from four growing stages of tobacco plants corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthesis genes and the accumulation of solanesol, thus providing valuable insight into the regulation of solanesol biosynthesis in tobacco.

  14. De novo transcriptome assembly in chili pepper (Capsicum frutescens) to identify genes involved in the biosynthesis of capsaicinoids.

    PubMed

    Liu, Shaoqun; Li, Wanshun; Wu, Yimin; Chen, Changming; Lei, Jianjun

    2013-01-01

    The capsaicinoids are a group of compounds produced by chili pepper fruits and are used widely in many fields, especially in medical purposes. The capsaicinoid biosynthetic pathway has not yet been established clearly. To understand more knowledge in biosynthesis of capsaicinoids, we applied RNA-seq for the mixture of placenta and pericarp of pungent pepper (Capsicum frutescens L.). We have assessed the effect of various assembly parameters using different assembly software, and obtained one of the best strategies for de novo assembly of transcriptome data. We obtained a total 54,045 high-quality unigenes (transcripts) using Trinity software. About 92.65% of unigenes showed similarity to the public protein sequences, genome of potato and tomato and pepper (C. annuum) ESTs databases. Our results predicted 3 new structural genes (DHAD, TD, PAT), which filled gaps of the capsaicinoid biosynthetic pathway predicted by Mazourek, and revealed new candidate genes involved in capsaicinoid biosynthesis based on KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. A significant number of SSR (Simple Sequence Repeat) and SNP (Single Nucleotide Polymorphism) markers were predicted in C. frutescens and C. annuum sequences, which will be helpful in the identification of polymorphisms within chili pepper populations. These data will provide new insights to the pathway of capsaicinoid biosynthesis and subsequent research of chili peppers. In addition, our strategy of de novo transcriptome assembly is applicable to a wide range of similar studies.

  15. Molecular cloning and characterization of genes involved in rosmarinic acid biosynthesis from Prunella vulgaris.

    PubMed

    Kim, Yeon Bok; Shin, YouJin; Tuan, Pham Anh; Li, Xiaohua; Park, Yunji; Park, Nam-il; Park, Sang Un

    2014-01-01

    Prunella vulgaris L., commonly known as "self-heal" or "heal-all," is a perennial herb with a long history of medicinal use. Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme-A (CoA) ligase (4CL) are important enzymes in the phenylpropanoid pathway and in the accumulation of rosmarinic acid (RA), which is a major secondary metabolite in P. vulgaris. In this study, we isolated cDNAs encoding PvPAL, PvC4H, and Pv4CL from P. vulgaris using rapid amplification of cDNA ends polymerase chain reaction (PCR). The amino acid sequence alignments of PvPAL, PvC4H, and Pv4CL showed high sequence identity to those of other plants. Quantitative real-time PCR analysis was used to determine the transcript levels of genes involved in RA biosynthesis in the flowers, leaves, stems, and roots of P. vulgaris. The transcript levels of PvPAL, PvC4H, and Pv4CL1 were the highest in flowers, whereas Pv4CL2 was the highest in roots. High-performance liquid chromatography analysis also showed the highest RA content in the flowers (3.71 mg/g dry weight). We suggest that the expression of the PvPAL, PvC4H, and Pv4CL1 genes is correlated with the accumulation of RA. Our results revealed that P. vulgaris flowers are appropriate for medicinal usage, and our findings provide support for increasing RA production in this plant.

  16. The mtmVUC genes of the mithramycin gene cluster in Streptomyces argillaceus are involved in the biosynthesis of the sugar moieties.

    PubMed

    González, A; Remsing, L L; Lombó, F; Fernández, M J; Prado, L; Braña, A F; Künzel, E; Rohr, J; Méndez, C; Salas, J A

    2001-02-01

    Mithramycin is a glycosylated aromatic polyketide produced by Streptomyces argillaceus, and is used as an antitumor drug. Three genes (mtmV, mtmU and mtmC) from the mithramycin gene cluster have been cloned, and characterized by DNA sequencing and by analysis of the products that accumulate in nonproducing mutants, which were generated by insertional inactivation of these genes. The mtm V gene codes for a 2,3-dehydratase that catalyzes early and common steps in the biosynthesis of the three sugars found in mithramycin (D-olivose, D-oliose and D-mycarose); its inactivation caused the accumulation of the nonglycosylated intermediate premithramycinone. The mtmU gene codes for a 4-ketoreductase involved in D-oliose biosynthesis, and its inactivation resulted in the accumulation of premithramycinone and premithramycin A , the first glycosylated intermediate which contains a D-olivose unit. The third gene, mtmC, is involved in D-mycarose biosynthesis and codes for a C-methyltransferase. Two mutants with lesions in the mtmC gene accumulated mithramycin intermediates lacking the D-mycarose moiety but containing D-olivose units attached to C-12a in which the 4-keto group is unreduced. This suggests that mtmC could code for a second enzyme activity, probably a D-olivose 4-ketoreductase, and that the glycosyltransferase responsible for the incorporation of D-olivose (MtmGIV) shows some degree of flexibility with respect to its sugar co-substrate, since the 4-ketoanalog is also transferred. A pathway is proposed for the biosynthesis of the three sugar moieties in mithramycin.

  17. Production of Aflatoxin on Rice

    PubMed Central

    Shotwell, Odette L.; Hesseltine, C. W.; Stubblefield, R. D.; Sorenson, W. G.

    1966-01-01

    A method has been developed for the production of aflatoxin by growing Aspergillus flavus strain NRRL 2999 on the solid substrate rice. Optimal yields, more than 1 mg of aflatoxin B1 per g of starting material, were obtained in 5 days at 28 C. A crude product containing aflatoxins was isolated by chloroform extraction and precipitation with hexane from concentrated solutions. The crude product consisted of 50% aflatoxin in the following ratio: B1-B2-G1-G2, 100:0.15:0.22:0.02. Aflatoxin B1 was separated from almost all the impurities and from the other aflatoxins by chromatography on silica gel with 1% ethyl alcohol in chloroform. Analytically pure aflatoxin B1 was recrystallized from chloroform-hexane mixtures. Images Fig. 1 PMID:5970829

  18. Identification of candidate genes in Arabidopsis and Populus cell wall biosynthesis using text-mining, co-expression network analysis and comparative genomics.

    PubMed

    Yang, Xiaohan; Ye, Chu-Yu; Bisaria, Anjali; Tuskan, Gerald A; Kalluri, Udaya C

    2011-12-01

    Populus is an important bioenergy crop for bioethanol production. A greater understanding of cell wall biosynthesis processes is critical in reducing biomass recalcitrance, a major hindrance in efficient generation of biofuels from lignocellulosic biomass. Here, we report the identification of candidate cell wall biosynthesis genes through the development and application of a novel bioinformatics pipeline. As a first step, via text-mining of PubMed publications, we obtained 121 Arabidopsis genes that had the experimental evidence supporting their involvement in cell wall biosynthesis or remodeling. The 121 genes were then used as bait genes to query an Arabidopsis co-expression database, and additional genes were identified as neighbors of the bait genes in the network, increasing the number of genes to 548. The 548 Arabidopsis genes were then used to re-query the Arabidopsis co-expression database and re-construct a network that captured additional network neighbors, expanding to a total of 694 genes. The 694 Arabidopsis genes were computationally divided into 22 clusters. Queries of the Populus genome using the Arabidopsis genes revealed 817 Populus orthologs. Functional analysis of gene ontology and tissue-specific gene expression indicated that these Arabidopsis and Populus genes are high likelihood candidates for functional characterization in relation to cell wall biosynthesis.

  19. Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

    PubMed Central

    2011-01-01

    Background Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. Results We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. Conclusions The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome

  20. Aflatoxin decomposition in various soils

    SciTech Connect

    Angle, J.S.

    1986-08-01

    The persistence of aflatoxin in the soil environment could potentially result in a number of adverse environmental consequences. To determine the persistence of aflatoxin in soil, /sup 14/C-labeled aflatoxin B1, was added to silt loam, sandy loam, and silty clay loam soils and the subsequent release of /sup 14/CO/sub 2/ was determined. After 120 days of incubation, 8.1% of the original aflatoxin added to the silt loam soil was released as CO/sub 2/. Aflatoxin decomposition in the sandy loam soil proceeded more quickly than the other two soils for the first 20 days of incubation. After this time, the decomposition rate declined and by the end of the study, 4.9% of the aflatoxin was released as CO/sub 2/. Aflatoxin decomposition proceeded most slowly in the silty clay loam soil. Only 1.4% of aflatoxin added to the soil was released as CO/sub 2/ after 120 days incubation. To determine whether aflatoxin was bound to the silty clay loam soil, aflatoxin B1 was added to this soil and incubated for 20 days. The soil was periodically extracted and the aflatoxin species present were determined using thin layer chromatographic (TLC) procedures. After one day of incubation, the degradation products, aflatoxins B2 and G2, were observed. It was also found that much of the aflatoxin extracted from the soil was not mobile with the TLC solvent system used. This indicated that a conjugate may have formed and thus may be responsible for the lack of aflatoxin decomposition.

  1. Wounding of potato tubers induces increases in ABA biosynthesis and catabolism and alters expression of ABA metabolic genes.

    PubMed

    Suttle, Jeffrey C; Lulai, Edward C; Huckle, Linda L; Neubauer, Jonathan D

    2013-04-15

    The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato tubers. An increase in ABA and ABA metabolite content was observed 48h after wounding and remained elevated through 96h. Wounding induced dramatic increases in the expression of the ABA metabolic genes encoding zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and ABA-8'-hydroxylase. Although the patterns of wound-induced expression of individual genes varied, increased gene expression was observed within 3h of wounding and remained elevated through 96h. An apparent correlation between expression of the gene encoding ZEP and the increase in ABA content suggested that the wound-induced increase in ABA biosynthesis was regulated by both substrate availability and increased NCED activity. Suppression of wound-induced jasmonic acid accumulation by rinsing the wounded tissue with water did not inhibit the subsequent increase in ABA content. Exogenous ethylene completely suppressed the wound-induced increase in ABA content and dramatically reduced wound-induced up-regulation of ABA metabolic genes. This study is the first to identify the molecular bases for increased ABA accumulation following physical trauma in potato tubers and highlights the complex physiological interactions between various wound-induced hormones.

  2. Fluorometric assay for aflatoxins

    SciTech Connect

    Chakrabarti, A.G.

    1984-11-01

    The method that is now widely adopted by the government laboratories for the assay of individual aflatoxin components (B/sub 1/, B/sub 2/, G/sub 1/, and G/sub 2/) utilizes a TLC technique. The extraction and clean-up steps of this technique were further researched but the method is still time consuming. It is, therefore, very important to develop a rapid and accurate assay technique for aflatoxins. The current research proposes a technique which utilizes a Turner Fluorometer.

  3. Three Novel Rice Genes Closely Related to the Arabidopsis IRX9, IRX9L, and IRX14 Genes and Their Roles in Xylan Biosynthesis

    PubMed Central

    Chiniquy, Dawn; Varanasi, Patanjali; Oh, Taeyun; Harholt, Jesper; Katnelson, Jacob; Singh, Seema; Auer, Manfred; Simmons, Blake; Adams, Paul D.; Scheller, Henrik V.; Ronald, Pamela C.

    2013-01-01

    Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx) mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9), Os01g48440 (OsIRX9L), and Os06g47340 (OsIRX14), from glycosyltransferase family 43 as putative orthologs to the putative β-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the over-expression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx) mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase (XylT) activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in XylT activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength. PMID:23596448

  4. Regulatory genes and environmental regulation of amylovoran biosynthesis in Erwinia amylovora

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The requirement of the exopolysaccharide amylovoran for Erwinia amylovora pathogenesis is well documented. However, regulation of amylovoran biosynthesis has not been comprehensively studied. We have previously reported that amylovoran production is strain-dependent in E. amylovora isolates. We have...

  5. Spatial transcriptome analysis provides insights of key gene(s) involved in steroidal saponin biosynthesis in medicinally important herb Trillium govanianum

    PubMed Central

    Singh, Pradeep; Singh, Gagandeep; Bhandawat, Abhishek; Singh, Gopal; Parmar, Rajni; Seth, Romit; Sharma, Ram Kumar

    2017-01-01

    Trillium govanianum, an endangered medicinal herb native to the Himalaya, is less studied at the molecular level due to the non-availability of genomic resources. To facilitate the basic understanding of the key genes and regulatory mechanism of pharmaceutically important biosynthesis pathways, first spatial transcriptome sequencing of T. govanianum was performed. 151,622,376 (~11.5 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 69,174 transcripts. Functional annotation with multiple public databases identified array of genes involved in steroidal saponin biosynthesis and other secondary metabolite pathways including brassinosteroid, carotenoid, diterpenoid, flavonoid, phenylpropanoid, steroid and terpenoid backbone biosynthesis, and important TF families (bHLH, MYB related, NAC, FAR1, bZIP, B3 and WRKY). Differentially expressed large number of transcripts, together with CYPs and UGTs suggests involvement of these candidates in tissue specific expression. Combined transcriptome and expression analysis revealed that leaf and fruit tissues are the main site of steroidal saponin biosynthesis. In conclusion, comprehensive genomic dataset created in the current study will serve as a resource for identification of potential candidates for genetic manipulation of targeted bioactive metabolites and also contribute for development of functionally relevant molecular marker resource to expedite molecular breeding and conservation efforts in T. govanianum. PMID:28349986

  6. Data on the presence or absence of genes encoding essential proteins for ochratoxin and fumonisin biosynthesis in Aspergillus niger and Aspergillus welwitschiae

    PubMed Central

    Massi, Fernanda Pelisson; Sartori, Daniele; Ferranti, Larissa de Souza; Iamanaka, Beatriz Thie; Taniwaki, Marta Hiromi; Vieira, Maria Lucia Carneiro; Fungaro, Maria Helena Pelegrinelli

    2016-01-01

    We present the multiplex PCR data for the presence/absence of genes involved in OTA and FB2 biosynthesis in Aspergillus niger/Aspergillus welwitschiae strains isolated from different food substrates in Brazil. Among the 175 strains analyzed, four mPCR profiles were found: Profile 1 (17%) highlights strains harboring in their genome the pks, radH and the fum8 genes. Profile 2 (3.5%) highlights strains harboring genes involved in OTA biosynthesis i.e. radH and pks. Profile 3 (51.5%) highlights strains harboring the fum8 gene. Profile 4 (28%) highlights strains not carrying the genes studied herein. This research content is supplemental to our original research article, “Prospecting for the incidence of genes involved in ochratoxin and fumonisin biosynthesis in Brazilian strains of A. niger and A. welwitschiae” [1]. PMID:27054181

  7. Expression profiling of the triterpene saponin biosynthesis genes FPS, SS, SE, and DS in the medicinal plant Panax notoginseng.

    PubMed

    Niu, Yunyun; Luo, Hongmei; Sun, Chao; Yang, Tae-Jin; Dong, Linlin; Huang, Linfang; Chen, Shilin

    2014-01-01

    Panax notoginseng (Burk) F. H. Chen, an economically significant medicinal plant with hemostatic and health tonic activities, has been used in Traditional Chinese Medicine (TCM) for more than 3,000 years. Triterpene saponins are responsible for most of the pharmacological activities of P. notoginseng. Here, we cloned five cDNA sequences encoding the key enzymes involved in triterpene saponin biosynthesis, namely, PnFPS, PnSS, PnSE1, PnSE2, and PnDS, and analyzed the conserved domains and phylogenetics of their corresponding proteins. Their organ-specific expression patterns in four-year-old P. notoginseng were detected by real-time PCR, showing that they were all most highly expressed in flowers. In addition, four of the genes, excluding PnSE2, were upregulated in leaves following stimulation with methyl jasmonate. This study is the first comprehensive analysis of the expression patterns of pivotal genes for triterpene saponin biosynthesis in P. notoginseng and provides a basis to further elucidate the molecular mechanism for the biosynthesis of these medically important compounds.

  8. Transcriptional regulator LsrB of Sinorhizobium meliloti positively regulates the expression of genes involved in lipopolysaccharide biosynthesis.

    PubMed

    Tang, Guirong; Wang, Ying; Luo, Li

    2014-09-01

    Rhizobia induce nitrogen-fixing nodules on host legumes, which is important in agriculture and ecology. Lipopolysaccharide (LPS) produced by rhizobia is required for infection or bacteroid survival in host cells. Genes required for LPS biosynthesis have been identified in several Rhizobium species. However, the regulation of their expression is not well understood. Here, Sinorhizobium meliloti LsrB, a member of the LysR family of transcriptional regulators, was found to be involved in LPS biosynthesis by positively regulating the expression of the lrp3-lpsCDE operon. An lsrB in-frame deletion mutant displayed growth deficiency, sensitivity to the detergent sodium dodecyl sulfate, and acidic pH compared to the parent strain. This mutant produced slightly less LPS due to lower expression of the lrp3 operon. Analysis of the transcriptional start sites of the lrp3 and lpsCDE gene suggested that they constitute one operon. The expression of lsrB was positively autoregulated. The promoter region of lrp3 was specifically precipitated by anti-LsrB antibodies in vivo. The promoter DNA fragment containing TN11A motifs was bound by the purified LsrB protein in vitro. These new findings suggest that S. meliloti LsrB is associated with LPS biosynthesis, which is required for symbiotic nitrogen fixation on some ecotypes of alfalfa plants.

  9. Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase.

    PubMed

    Vidal-Meireles, André; Neupert, Juliane; Zsigmond, Laura; Rosado-Souza, Laise; Kovács, László; Nagy, Valéria; Galambos, Anikó; Fernie, Alisdair R; Bock, Ralph; Tóth, Szilvia Z

    2017-04-01

    Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H2 O2 and (1) O2 resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.

  10. De Novo Transcriptome and Expression Profile Analysis to Reveal Genes and Pathways Potentially Involved in Cantharidin Biosynthesis in the Blister Beetle Mylabris cichorii

    PubMed Central

    Huang, Yi; Wang, Zhongkang; Zha, Shenfang; Wang, Yu; Jiang, Wei; Liao, Yufeng; Song, Zhangyong; Qi, Zhaoran; Yin, Youping

    2016-01-01

    The dried body of Mylabris cichorii is well-known Chinese traditional medicine. The sesquiterpenoid cantharidin, which is secreted mostly by adult male beetles, has recently been used as an anti-cancer drug. However, little is known about the mechanisms of cantharidin biosynthesis. Furthermore, there is currently no genomic or transcriptomic information for M. cichorii. In this study, we performed de novo assembly transcriptome of M. cichorii using the Illumina Hiseq2000. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences (about 81%). We also constructed two expression profile libraries (20–25 day-old adult males and 20–25 day-old adult females) and discovered 2,465 significantly differentially-expressed genes. Putative genes and pathways involved in the biosynthesis of cantharidin were then characterized. We also found that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate (MVA) pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP/DOXP) pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone (JH) biosynthesis or degradation. The results of transcriptome and expression profiling analysis provide a comprehensive sequence resource for M. cichorii that could facilitate the in-depth study of candidate genes and pathways involved in cantharidin biosynthesis, and may thus help to improve our understanding of the mechanisms of cantharidin biosynthesis in blister beetles. PMID:26752526

  11. De Novo Transcriptome Analysis of Plant Pathogenic Fungus Myrothecium roridum and Identification of Genes Associated with Trichothecene Mycotoxin Biosynthesis

    PubMed Central

    Ye, Wei; Liu, Taomei; Zhu, Muzi; Zhang, Weimin; Li, Haohua; Huang, Zilei; Li, Saini

    2017-01-01

    Myrothecium roridum is a plant pathogenic fungus that infects different crops and decreases the yield of economical crops, including soybean, cotton, corn, pepper, and tomato. Until now, the pathogenic mechanism of M. roridum has remained unclear. Different types of trichothecene mycotoxins were isolated from M. roridum, and trichothecene was considered as a plant pathogenic factor of M. roridum. In this study, the transcriptome of M. roridum in different incubation durations was sequenced using an Illumina Hiseq 2000. A total of 35,485 transcripts and 25,996 unigenes for M. roridum were obtained from 8.0 Gb clean reads. The protein–protein network of the M. roridum transcriptome indicated that the mitogen-activated protein kinases signal pathway also played an important role in the pathogenicity of M. roridum. The genes related to trichothecene biosynthesis were annotated. The expression levels of these genes were also predicted and validated through quantitative real-time polymerase chain reaction. Tri5 gene encoding trichodiene synthase was cloned and expressed, and the purified trichodiene synthase was able to catalyze farnesyl pyrophosphate into different kinds of sesquiterpenoids.Tri4 and Tri11 genes were expressed in Escherichia coli, and their corresponding enzymatic properties were characterized. The phylogenetic tree of trichodiene synthase showed a great discrepancy between the trichodiene synthase from M. roridum and other species. Our study on the genes related to trichothecene biosynthesis establishes a foundation for the M. roridum hazard prevention, thus improving the yields of economical crops. PMID:28245611

  12. [Aflatoxins--health risk factors].

    PubMed

    Miliţă, Nicoleta Manuela; Mihăescu, Gr; Chifiriuc, Carmen

    2010-01-01

    Aflatoxins are secondary metabolites produced by a group of strains, mainly Aspergillus and Penicillium species. These mycotoxins are bifurano-coumarin derivatives group with four major products B1, B2, G1 and G2 according to blue or green fluorescence emitted in ultraviolet light and according to chromatographic separation. After metabolism of aflatoxin B1 and B2 in the mammalian body, result two metabolites M1 and M2 as hydroxylated derivatives of the parent compound. Aflatoxins have high carcinogenic potential, the most powerful carcinogens in different species of animals and humans. International Agency for Research on Cancer has classified aflatoxin B1 in Group I carcinogens. The target organ for aflatoxins is the liver. In chronic poisoning, aflatoxin is a risk to health, for a long term causing cancer (hepatocellular carcinoma), and in acute intoxications aflatoxin is lethal. This work purpose to discuss aflatoxins issue: the synthesis, absorption and elimination of aflatoxins, the toxicity mechanisms, and measures to limit the content of aflatoxins in food

  13. Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene.

    PubMed

    Buch, Aditi D; Archana, G; Kumar, G Naresh

    2009-08-01

    Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant approximately 2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2- and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3- and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525

  14. Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

    PubMed Central

    Ushimaru, Kazunori; Mizuno, Shoji

    2015-01-01

    Recombinant Ralstonia eutropha strain PHB−4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB−4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB−4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone. PMID:25645560

  15. A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans

    DOE PAGES

    Stonebloom, Solomon; Ebert, Berit; Xiong, Guangyan; ...

    2016-04-18

    We report pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth. T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wildmore » type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content. Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.« less

  16. A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans

    SciTech Connect

    Stonebloom, Solomon; Ebert, Berit; Xiong, Guangyan; Pattathil, Sivakumar; Birdseye, Devon; Lao, Jeemeng; Pauly, Markus; Hahn, Michael G.; Heazlewood, Joshua L.; Scheller, Henrik Vibe

    2016-04-18

    We report pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth. T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content. Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

  17. Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis in Streptomyces griseus DSM40695

    PubMed Central

    Smith, Wyatt C.; Xiang, Longkuan; Shen, Ben

    2000-01-01

    The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR and nonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of the nonS mutant by the expression of nonS in trans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of the nonS mutant in the presence of exogenous (±)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonS gene and fermentation of the nonS mutant in the presence of exogenously added (±)-NA suggests that NonS catalyzes the formation of (−)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis in S. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis. PMID:10858335

  18. Comparative mRNA Expression Profiles of Riboflavin Biosynthesis Genes in Lactobacilli Isolated from Human Feces and Fermented Bamboo Shoots

    PubMed Central

    Thakur, Kiran; Tomar, Sudhir K.; Wei, Zhao-Jun

    2017-01-01

    With the aim to bioprospect potent riboflavin producing lactobacilli, the present study was carried out to evaluate the relative mRNA expression of riboflavin biosynthesis genes namely Rib 1, Rib 2, Rib 3, and Rib 4 from potent riboflavin producers obtained from our previous studies. All the four genes were successfully cloned and sequenced for further analysis by in silico procedures. As studied by non-denaturing Polyacrylamide gel electrophoresis, no difference in size of all the four genes among those of various lactobacilli was observed. The relative fold increase in mRNA expression in Rib 1, Rib 2, Rib 3, and Rib 4 genes has been observed to be 10-, 1-, 0.7-, and 8.5-fold, respectively. Due to increase in relative mRNA expression for all the Rib genes as well as phenotypic production attribute, KTLF1 strain was used further for expression studies in milk and whey. The fold increase in mRNA expression for all the four Rib genes was higher at 12 and 18 h in milk and whey respectively. After exposure to roseoflavin, resistant variant of KTLF1 showed considerable increase in expression of all the targets genes. This is the first ever study to compare the mRNA expression of riboflavin biosynthesis pathway genes in lactobacilli and it also under lines the effect of media and harvesting time which significantly affect the expression of rib genes. The use of roseoflavin-resistant strains capable of synthesizing riboflavin in milk and whey paves a way for an exciting and economically viable biotechnological approach to develop novel riboflavin bio-enriched functional foods. PMID:28367143

  19. Identification of Loci and Functional Characterization of Trichothecene Biosynthesis Genes in Filamentous Fungi of the Genus Trichoderma▿†

    PubMed Central

    Cardoza, R. E.; Malmierca, M. G.; Hermosa, M. R.; Alexander, N. J.; McCormick, S. P.; Proctor, R. H.; Tijerino, A. M.; Rumbero, A.; Monte, E.; Gutiérrez, S.

    2011-01-01

    Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function. PMID:21642405

  20. The major volatile compound 2-phenylethanol from the biocontrol yeast Pichia anomala inhibits growth and expression of aflatoxin biosynthetic genes of Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus is a globally distributed fungus and an important food contaminant because it produces the most potent natural carcinogenic compound known as aflatoxin (AF) B1. The major volatile from a yeast strain, Pichia anomala WRL-076 was identified by SPEM-GC/MS analysis to be 2-phenylethan...

  1. De Novo Transcriptome Assembly in Shiraia bambusicola to Investigate Putative Genes Involved in the Biosynthesis of Hypocrellin A

    PubMed Central

    Zhao, Ning; Lin, Xi; Qi, Shan-Shan; Luo, Zhi-Mei; Chen, Shuang-Lin; Yan, Shu-Zhen

    2016-01-01

    Shiraia bambusicola is a species of the monotypic genus Shiraia in the phylum Ascomycota. In China, it is known for its pharmacological properties that are used to treat rheumatic arthritis, sciatica, pertussis, tracheitis and so forth. Its major medicinal active metabolite is hypocrellin A, which exhibits excellent antiviral and antitumor properties. However, the genes involved in the hypocrellin A anabolic pathways were still unknown due to the lack of genomic information for this species. To investigate putative genes that are involved in the biosynthesis of hypocrellin A and determine the pathway, we performed transcriptome sequencing for Shiraia bambusicola S4201-W and the mutant S4201-D1 for the first time. S4201-W has excellent hypocrellin A production, while the mutant S4201-D1 does not. Then, we obtained 38,056,034 and 39,086,896 clean reads from S4201-W and S4201-D1, respectively. In all, 17,923 unigenes were de novo assembled, and the N50 length was 1970 bp. Based on the negative binomial distribution test, 716 unigenes were found to be upregulated, and 188 genes were downregulated in S4201-D1, compared with S4201-W. We have found seven unigenes involved in the biosynthesis of hypocrellin A and proposed a putative hypocrellin A biosynthetic pathway. These data will provide a valuable resource and theoretical basis for future molecular studies of hypocrellin A, help identify the genes involved in the biosynthesis of hypocrellin A and help facilitate functional studies for enhancing hypocrellin A production. PMID:26927096

  2. Activation of a Silent Fungal Polyketide Biosynthesis Pathway through Regulatory Cross Talk with a Cryptic Nonribosomal Peptide Synthetase Gene Cluster ▿ †

    PubMed Central

    Bergmann, Sebastian; Funk, Alexander N.; Scherlach, Kirstin; Schroeckh, Volker; Shelest, Ekaterina; Horn, Uwe; Hertweck, Christian; Brakhage, Axel A.

    2010-01-01

    Filamentous fungi produce numerous natural products that constitute a consistent source of potential drug leads, yet it seems that the majority of natural products are overlooked since most biosynthesis gene clusters are silent under standard cultivation conditions. Screening secondary metabolite genes of the model fungus Aspergillus nidulans, we noted a silent gene cluster on chromosome II comprising two nonribosomal peptide synthetase (NRPS) genes, inpA and inpB, flanked by a regulatory gene that we named scpR for secondary metabolism cross-pathway regulator. The induced expression of the scpR gene using the promoter of the alcohol dehydrogenase AlcA led to the transcriptional activation of both the endogenous scpR gene and the NRPS genes. Surprisingly, metabolic profiling of the supernatant of mycelia overexpressing scpR revealed the production of the polyketide asperfuranone. Through transcriptome analysis we found that another silent secondary metabolite gene cluster located on chromosome VIII coding for asperfuranone biosynthesis was specifically induced. Quantitative reverse transcription-PCR proved the transcription not only of the corresponding polyketide synthase (PKS) biosynthesis genes, afoE and afoG, but also of their activator, afoA, under alcAp-scpR-inducing conditions. To exclude the possibility that the product of the inp cluster induced the asperfuranone gene cluster, a strain carrying a deletion of the NRPS gene inpB and, in addition, the alcAp-scpR overexpression cassette was generated. In this strain, under inducing conditions, transcripts of the biosynthesis genes of both the NRPS-containing gene cluster inp and the asperfuranone gene cluster except gene inpB were detected. Moreover, the existence of the polyketide product asperfuranone indicates that the transcription factor ScpR controls the expression of the asperfuranone biosynthesis gene cluster. This expression as well as the biosynthesis of asperfuranone was abolished after the deletion

  3. In Silico Identification and Comparative Genomics of Candidate Genes Involved in Biosynthesis and Accumulation of Seed Oil in Plants

    PubMed Central

    Sharma, Arti; Chauhan, Rajinder Singh

    2012-01-01

    Genes involved in fatty acids biosynthesis, modification and oil body formation are expected to be conserved in structure and function in different plant species. However, significant differences in the composition of fatty acids and total oil contents in seeds have been observed in different plant species. Comparative genomics was performed on 261 genes involved in fatty acids biosynthesis, TAG synthesis, and oil bodies formation in Arabidopsis, Brassica rapa, castor bean and soybean. In silico expression analysis revealed that stearoyl desaturase, FatB, FAD2, oleosin and DGAT are highly abundant in seeds, thereby considered as ideal candidates for mining of favorable alleles in natural population. Gene structure analysis for major genes, ACCase, FatA, FatB, FAD2, FAD3 and DGAT, which are known to play crucial role in oil synthesis revealed that there are uncommon variations (SNPs and INDELs) which lead to varying content and composition of fatty acids in seed oil. The predicted variations can provide good targets for seed oil QTL identification, understanding the molecular mechanism of seed oil accumulation, and genetic modification to enhance seed oil yield in plants. PMID:22312320

  4. Multiplexed Integrating Plasmids for Engineering of the Erythromycin Gene Cluster for Expression in Streptomyces spp. and Combinatorial Biosynthesis

    PubMed Central

    Fayed, Bahgat; Ashford, David A.; Hashem, Amal M.; Amin, Magdy A.; El Gazayerly, Omaima N.; Gregory, Matthew A.

    2015-01-01

    Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ϕC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated. PMID:26431970

  5. De Novo Transcriptome Analysis to Identify Anthocyanin Biosynthesis Genes Responsible for Tissue-Specific Pigmentation in Zoysiagrass (Zoysia japonica Steud.)

    PubMed Central

    Ahn, Jong Hwa; Kim, June-Sik; Kim, Seungill; Soh, Hye Yeon; Shin, Hosub; Jang, Hosung; Ryu, Ju Hyun; Kim, Ahyeong; Yun, Kil-Young; Kim, Shinje; Kim, Ki Sun; Choi, Doil; Huh, Jin Hoe

    2015-01-01

    Zoysiagrass (Zoysia japonica Steud.) is commonly found in temperate climate regions and widely used for lawns, in part, owing to its uniform green color. However, some zoysiagrass cultivars accumulate red to purple pigments in their spike and stolon tissues, thereby decreasing the aesthetic value. Here we analyzed the anthocyanin contents of two zoysiagrass cultivars ‘Anyang-jungji’ (AJ) and ‘Greenzoa’ (GZ) that produce spikes and stolons with purple and green colors, respectively, and revealed that cyanidin and petunidin were primarily accumulated in the pigmented tissues. In parallel, we performed a de novo transcriptome assembly and identified differentially expressed genes between the two cultivars. We found that two anthocyanin biosynthesis genes encoding anthocyanidin synthase (ANS) and dihydroflavonol 4-reductase (DFR) were preferentially upregulated in the purple AJ spike upon pigmentation. Both ANS and DFR genes were also highly expressed in other zoysiagrass cultivars with purple spikes and stolons, but their expression levels were significantly low in the cultivars with green tissues. We observed that recombinant ZjDFR1 and ZjANS1 proteins successfully catalyze the conversions of dihydroflavonols into leucoanthocyanidins and leucoanthocyanidins into anthocyanidins, respectively. These findings strongly suggest that upregulation of ANS and DFR is responsible for tissue-specific anthocyanin biosynthesis and differential pigmentation in zoysiagrass. The present study also demonstrates the feasibility of a de novo transcriptome analysis to identify the key genes associated with specific traits, even in the absence of reference genome information. PMID:25905914

  6. Morphological characteristics, anatomical structure, and gene expression: novel insights into gibberellin biosynthesis and perception during carrot growth and development.

    PubMed

    Wang, Guang-Long; Xiong, Fei; Que, Feng; Xu, Zhi-Sheng; Wang, Feng; Xiong, Ai-Sheng

    2015-01-01

    Gibberellins (GAs) are considered potentially important regulators of cell elongation and expansion in plants. Carrot undergoes significant alteration in organ size during its growth and development. However, the molecular mechanisms underlying gibberellin accumulation and perception during carrot growth and development remain unclear. In this study, five stages of carrot growth and development were investigated using morphological and anatomical structural techniques. Gibberellin levels in leaf, petiole, and taproot tissues were also investigated for all five stages. Gibberellin levels in the roots initially increased and then decreased, but these levels were lower than those in the petioles and leaves. Genes involved in gibberellin biosynthesis and signaling were identified from the carrotDB, and their expression was analyzed. All of the genes were evidently responsive to carrot growth and development, and some of them showed tissue-specific expression. The results suggested that gibberellin level may play a vital role in carrot elongation and expansion. The relative transcription levels of gibberellin pathway-related genes may be the main cause of the different bioactive GAs levels, thus exerting influences on gibberellin perception and signals. Carrot growth and development may be regulated by modification of the genes involved in gibberellin biosynthesis, catabolism, and perception.

  7. A Sorangium cellulosum (myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes.

    PubMed Central

    Schupp, T; Toupet, C; Cluzel, B; Neff, S; Hill, S; Beck, J J; Ligon, J M

    1995-01-01

    A 40-kb region of DNA from Sorangium cellulosum So ce26, which contains polyketide synthase (PKS) genes for synthesis of the antifungal macrolide antibiotic soraphen A, was cloned. These genes were detected by homology to Streptomyces violaceoruber genes encoding components of granaticin PKS, thus extending this powerful technique for the identification of bacterial PKS genes, which has so far been applied only to actinomycetes, to the gram-negative myxobacteria. Functional analysis by gene disruption has indicated that about 32 kb of contiguous DNA of the cloned region contains genes involved in soraphen A biosynthesis. The nucleotide sequence of a 6.4-kb DNA fragment, derived from the region with homology to granaticin PKS genes, was determined. Analysis of this sequence has revealed the presence of a single large open reading frame beginning and ending outside the 6.4-kb fragment. The deduced amino acid sequence indicates the presence of a domain with a high level of similarity to beta-ketoacyl synthases that are involved in polyketide synthesis. Other domains with high levels of similarity to regions of known polyketide biosynthetic functions were identified, including those for acyl transferase, acyl carrier protein, ketoreductase, and dehydratase. We present data which indicate that soraphen A biosynthesis is catalyzed by large, multifunctional enzymes analogous to other bacterial PKSs of type I. PMID:7601830

  8. Growth, serum biochemistry, complement activity, and liver gene expression responses of Pekin ducklings to graded levels of cultured aflatoxin B1.

    PubMed

    Chen, X; Horn, N; Cotter, P F; Applegate, T J

    2014-08-01

    A 14-d study was conducted to evaluate the effects of cultured aflatoxin B1 (AFB1) on performance, serum biochemistry, serum natural antibody and complement activity, and hepatic gene expression parameters in Pekin ducklings. A total of 144 male Pekin ducklings were weighed, tagged, and randomly allotted to 4 dietary treatments containing 4 concentrations of AFB1 (0, 0.11, 0.14, and 0.21 mg/kg) from 0 to 14 d of age (6 cages per diet; 6 ducklings per cage). Compared with the control group, there was a 10.9, 31.7, and 47.4% (P < 0.05) decrease in cumulative BW gain with 0.11, 0.14, and 0.21 mg of AFB1/kg of diet, respectively, but feed efficiency was not affected. Increasing concentrations of AFB1 reduced cumulative BW gain and feed intake both linearly and quadratically, and regression equations were developed with r(2) ≥0.73. Feeding 0.11 to 0.21 mg of AFB1/kg reduced serum glucose, creatinine, albumin, total protein, globulin, Ca, P, and creatine phosphokinase linearly, whereas serum urea N, Cl, alkaline phosphatase, and aspartate amino transferase concentrations increased linearly with increasing AFB1 (P < 0.05). Additionally, 0.11 to 0.21 mg of AFB1/kg diets impaired classical and alternative complement pathways in the duckling serum when tested by lysis of rabbit, human type O, and horse erythrocytes, and decreased rabbit and horse agglutinins (P < 0.05). Liver peroxisome proliferator activated receptor α (PPARα) expression was linearly downregulated by AFB1 (P < 0.01). Results from this study indicate that for every 0.10 mg/kg increase in dietary AFB1, cumulative feed intake and BW gain decrease approximately 230 and 169 g per duckling from hatch to 14 d; and that AFB1 at very low concentrations can significantly impair liver function and gene expression, and innate immune dynamics in Pekin ducklings.

  9. Differential expression of ethylene biosynthesis genes in drupelets and receptacle of raspberry (Rubus idaeus).

    PubMed

    Fuentes, Lida; Monsalve, Liliam; Morales-Quintana, Luis; Valdenegro, Mónika; Martínez, Juan-Pablo; Defilippi, Bruno G; González-Agüero, Mauricio

    2015-05-01

    Red Raspberry (Rubus idaeus) is traditionally classified as non-climacteric, and the role of ethylene in fruit ripening is not clear. The available information indicates that the receptacle, a modified stem that supports the drupelets, is involved in ethylene production of ripe fruits. In this study, we report receptacle-related ethylene biosynthesis during the ripening of fruits of cv. Heritage. In addition, the expression pattern of ethylene biosynthesis transcripts was evaluated during the ripening process. The major transcript levels of 1-aminocyclopropane-1-carboxylic acid synthase (RiACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (RiACO1) were concomitant with ethylene production, increased total soluble solids (TSS) and decreased titratable acidity (TA) and fruit firmness. Moreover, ethylene biosynthesis and transcript levels of RiACS1 and RiACO1 were higher in the receptacle, sustaining the receptacle's role as a source of ethylene in regulating the ripening of raspberry.

  10. Identification of Genes Involved in Indole-3-Acetic Acid Biosynthesis by Gluconacetobacter diazotrophicus PAL5 Strain Using Transposon Mutagenesis

    PubMed Central

    Rodrigues, Elisete P.; Soares, Cleiton de Paula; Galvão, Patrícia G.; Imada, Eddie L.; Simões-Araújo, Jean L.; Rouws, Luc F. M.; de Oliveira, André L. M.; Vidal, Márcia S.; Baldani, José I.

    2016-01-01

    Gluconacetobacter diazotrophicus is a beneficial nitrogen-fixing endophyte found in association with sugarcane plants and other important crops. Beneficial effects of G. diazotrophicus on sugarcane growth and productivity have been attributed to biological nitrogen fixation process and production of phytohormones especially indole-3-acetic acid (IAA); however, information about the biosynthesis and function of IAA in G. diazotrophicus is still scarce. Therefore, the aim of this work was to identify genes and pathways involved in IAA biosynthesis in this bacterium. In our study, the screening of two independent Tn5 mutant libraries of PAL5T strain using the Salkowski colorimetric assay revealed two mutants (Gdiaa34 and Gdiaa01), which exhibited 95% less indolic compounds than the parental strain when grown in LGIP medium supplemented with L-tryptophan. HPLC chromatograms of the wild-type strain revealed the presence of IAA and of the biosynthetic intermediates indole-3-pyruvic acid (IPyA) and indole-3-lactate (ILA). In contrast, the HPLC profiles of both mutants showed no IAA but only a large peak of non-metabolized tryptophan and low levels of IPyA and ILA were detected. Molecular characterization revealed that Gdiaa01 and Gdiaa34 mutants had unique Tn5 insertions at different sites within the GDI2456 open read frame, which is predicted to encode a L-amino acid oxidase (LAAO). GDI2456 (lao gene) forms a cluster with GDI2455 and GDI2454 ORFs, which are predicted to encode a cytochrome C and an RidA protein, respectively. RT-qPCR showed that transcript levels of lao. cccA, and ridA genes were reduced in the Gdiaa01 as compared to PAL5T. In addition, rice plants inoculated with Gdiaa01 showed significantly smaller root development (length, surface area, number of forks and tips) than those plants inoculated with PAL5T. In conclusion, our study demonstrated that G. diazotrophicus PAL5T produces IAA via the IPyA pathway in cultures supplemented with tryptophan and

  11. Silencing of BnTT1 family genes affects seed flavonoid biosynthesis and alters seed fatty acid composition in Brassica napus.

    PubMed

    Lian, Jianping; Lu, Xiaochun; Yin, Nengwen; Ma, Lijuan; Lu, Jing; Liu, Xue; Li, Jiana; Lu, Jun; Lei, Bo; Wang, Rui; Chai, Yourong

    2017-01-01

    TRANSPARENT TESTA1 (TT1) is a zinc finger protein that contains a WIP domain. It plays important roles in controlling differentiation and pigmentation of the seed coat endothelium, and can affect the expression of early biosynthetic genes and late biosynthetic genes of flavonoid biosynthesis in Arabidopsis thaliana. In Brassica napus (AACC, 2n=38), the functions of BnTT1 genes remain unknown and few studies have focused on their roles in fatty acid (FA) biosynthesis. In this study, BnTT1 family genes were silenced by RNA interference, which resulted in yellow rapeseed, abnormal testa development (a much thinner testa), decreased seed weight, and altered seed FA composition in B. napus. High-throughput sequencing of genes differentially expressed between developing transgenic B. napus and wild-type seeds revealed altered expression of numerous genes involved in flavonoid and FA biosynthesis. As a consequence of this altered expression, we detected a marked decrease of oleic acid (C18:1) and notable increases of linoleic acid (C18:2) and α-linolenic acid (C18:3) in mature transgenic B. napus seeds by gas chromatography and near-infrared reflectance spectroscopy. Meanwhile, liquid chromatography-mass spectrometry showed reduced accumulation of flavonoids in transgenic seeds. Therefore, we propose that BnTT1s are involved in the regulation of flavonoid biosynthesis, and may also play a role in FA biosynthesis in B. napus.

  12. Inhibition of the mevalonate pathway enhances carvacrol biosynthesis and DXR gene expression in shoot cultures of Satureja khuzistanica Jamzad.

    PubMed

    Ramak, Parvin; Kazempour Osaloo, Shahrokh; Ebrahimzadeh, Hassan; Sharifi, Mozafar; Behmanesh, Mehrdad

    2013-09-01

    Carvacrol is a major component of Satureja khuzistanica Jamzad (≤90%) that has significant antimicrobial and antioxidant properties. Considering the specific capabilities of S. khuzistanica to produce highly pure carvacrol, this plant is an important potential source of carvacrol that could address the abundant consumption and increasing demand for this monoterpene in current world markets. This research was performed to better understand the process of biosynthesis and accumulation of carvacrol in S. khuzistanica. Tests were performed on shoot cultures of S. khuzistanica in Linsmaier-Skoog (LS) medium treated with different concentrations of fosmidomycin (an inhibitor of the non-mevalonate pathway) and mevinolin (an inhibitor of the mevalonate pathway) for 21 days at the following concentrations: 0, 10, 25, 50, 75 and 100 μM. The present study demonstrated that the MEP pathway is the major pathway that provides IPP for the biosynthesis of carvacrol, and the expression and activity levels of the DXR enzyme have a critical effect on carvacrol biosynthesis. Surprisingly, Mevinolin at concentrations of 75 and 100 μM increased the carvacrol content and the DXR activity and gene expression in S. khuzistanica plantlets.

  13. The CTB1 gene encoding a fungal polyketide synthase is required for cercosporin biosynthesis and fungal virulence of Cercospora nicotianae.

    PubMed

    Choquer, Mathias; Dekkers, Katherine L; Chen, Hui-Qin; Cao, Lihua; Ueng, Peter P; Daub, Margaret E; Chung, Kuang-Ren

    2005-05-01

    Cercosporin is a light-activated, non-host-selective toxin produced by many Cercospora fungal species. In this study, a polyketide synthase gene (CTB1) was functionally identified and molecularly characterized to play a key role in cercosporin biosynthesis by Cercospora nicotianae. We also provide conclusive evidence to confirm the crucial role of cercosporin in fungal pathogenesis. CTB1 encoded a polypeptide with a deduced length of 2,196 amino acids containing a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and had high levels of similarity to many fungal type I polyketide synthases. Expression of a 6.8-kb CTB1 transcript was highly regulated by light and medium composition, consistent with the conditions required for cercosporin biosynthesis in cultures. Targeted disruption of CTB1 resulted in the loss of both CTB1 transcript and cercosporin biosynthesis in C. nicotianae. The ctb1-null mutants incited fewer necrotic lesions on inoculated tobacco leaves compared with the wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have demonstrated conclusively that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in C. nicotianae. The results also suggest that strategies that avoid the toxicity of cercosporin will be useful in reduction of disease incidence caused by Cercospora spp.

  14. Beyond aflatoxin: four distinct expression patterns and functional roles associated with Aspergillus flavus secondary metabolism gene clusters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis has predicted that these species have the capacity to synthesize many more compounds. It has been possible to infer the presence of 55 gene clusters associated with secondary metabolism in Aspergill...

  15. Cloning and sequencing of the kedarcidin biosynthetic gene cluster from Streptoalloteichus sp. ATCC 53650 revealing new insights into biosynthesis of the enediyne family of antitumor antibiotics†

    PubMed Central

    Lohman, Jeremy R.; Huang, Sheng-Xiong; Horsman, Geoffrey P.; Dilfer, Paul E.; Huang, Tingting; Chen, Yihua; Wendt-Pienkowski, Evelyn; Shen, Ben

    2013-01-01

    Enediyne natural product biosynthesis is characterized by a convergence of multiple pathways, generating unique peripheral moieties that are appended onto the distinctive enediyne core. Kedarcidin (KED) possesses two unique peripheral moieties, a (R)-2-aza-3-chloro-β-tyrosine and an iso-propoxy-bearing 2-naphthonate moiety, as well as two deoxysugars. The appendage pattern of these peripheral moieties to the enediyne core in KED differs from the other enediynes studied to date with respect to stereochemical configuration. To investigate the biosynthesis of these moieties and expand our understanding of enediyne core formation, the biosynthetic gene cluster for KED was cloned from Streptoalloteichus sp. ATCC 53650 and sequenced. Bioinformatics analysis of the ked cluster revealed the presence of the conserved genes encoding for enediyne core biosynthesis, type I and type II polyketide synthase loci likely responsible for 2-aza-L-tyrosine and 3,6,8-trihydroxy-2-naphthonate formation, and enzymes known for deoxysugar biosynthesis. Genes homologous to those responsible for the biosynthesis, activation, and coupling of the L-tyrosine-derived moieties from C-1027 and maduropeptin and of the naphthonate moiety from neocarzinostatin are present in the ked cluster, supporting 2-aza-L-tyrosine and 3,6,8-trihydroxy-2-naphthoic acid as precursors, respectively, for the (R)-2-aza-3-chloro-β-tyrosine and the 2-naphthonate moieties in KED biosynthesis. PMID:23360970

  16. Enhancement of heterologous production of eicosapentaenoic acid in Escherichia coli by substitution of promoter sequences within the biosynthesis gene cluster.

    PubMed

    Lee, Su-Jin; Kim, Chul Ho; Seo, Pil-Soo; Kwon, Ohsuk; Hur, Byung-Ki; Seo, Jeong-Woo

    2008-12-01

    To enhance the heterologous production of eicosapentaenoic acid (EPA) in Escherichia coli, the EPA biosynthesis gene cluster from Shewanella oneidensis MR-1 was cloned under the lacZ promoter on a high-copy number plasmid, pBluescript SK+. The production of EPA was remarkably enhanced yielding levels of up to 7.5% of the total fatty acid content in the recombinant E. coli strain by induction with IPTG, whereas the stimulation of EPA production was abolished by adding glucose into the culture medium, probably due to glucose repression acting on the promoter activity.

  17. De novo Transcriptome Analysis Revealed Genes Involved in Flavonoid and Vitamin C Biosynthesis in Phyllanthus emblica (L.)

    PubMed Central

    Kumar, Avneesh; Kumar, Sunil; Bains, Savita; Vaidya, Vanya; Singh, Baljinder; Kaur, Ravneet; Kaur, Jagdeep; Singh, Kashmir

    2016-01-01

    Phyllanthus emblica is an affluent source of various therapeutic components. A few of them like vitamin C and flavonoids are predominant bioactive compounds that are being used in immense pharmacological applications. In-spite of numerous applications, the genomic information of this plant was limited to a few expressed sequence tags (ESTs) in DNA databases. Herein, we developed in-depth transcriptome information of P. emblica using Illumina Hiseq 2000 platform and characterized. A total of 31,285,965 high-quality reads were assembled into 91,288 contigs with the N50 value 358. Out of them, 47,267 contigs were functionally annotated using BLASTX search against NCBI-non-redundant (NR) protein database. Further, 31,366 contigs showed similarity with various gene ontology (GO) terms, and 1299 were related to different enzymes and biosynthetic pathways. We identified the transcripts related to each gene involved in flavonoid and vitamin C biosynthesis. Several cytochrome P450s (CYPs) and glucosyltransferases (GTs) genes involved in flavonoid biosynthesis and various other metabolic pathways were also documented. Further, 6510 transcription factors and 4420 EST derived simple sequence repeat (SSR) markers were also predicted. The present study enlightened various characteristic features of P. emblica genome, and provided an important resource for future molecular and functional genomics studies. PMID:27833630

  18. De novo Transcriptome Analysis Revealed Genes Involved in Flavonoid and Vitamin C Biosynthesis in Phyllanthus emblica (L.).

    PubMed

    Kumar, Avneesh; Kumar, Sunil; Bains, Savita; Vaidya, Vanya; Singh, Baljinder; Kaur, Ravneet; Kaur, Jagdeep; Singh, Kashmir

    2016-01-01

    Phyllanthus emblica is an affluent source of various therapeutic components. A few of them like vitamin C and flavonoids are predominant bioactive compounds that are being used in immense pharmacological applications. In-spite of numerous applications, the genomic information of this plant was limited to a few expressed sequence tags (ESTs) in DNA databases. Herein, we developed in-depth transcriptome information of P. emblica using Illumina Hiseq 2000 platform and characterized. A total of 31,285,965 high-quality reads were assembled into 91,288 contigs with the N50 value 358. Out of them, 47,267 contigs were functionally annotated using BLASTX search against NCBI-non-redundant (NR) protein database. Further, 31,366 contigs showed similarity with various gene ontology (GO) terms, and 1299 were related to different enzymes and biosynthetic pathways. We identified the transcripts related to each gene involved in flavonoid and vitamin C biosynthesis. Several cytochrome P450s (CYPs) and glucosyltransferases (GTs) genes involved in flavonoid biosynthesis and various other metabolic pathways were also documented. Further, 6510 transcription factors and 4420 EST derived simple sequence repeat (SSR) markers were also predicted. The present study enlightened various characteristic features of P. emblica genome, and provided an important resource for future molecular and functional genomics studies.

  19. Industrial fuel ethanol yeasts contain adaptive copy number changes in genes involved in vitamin B1 and B6 biosynthesis.

    PubMed

    Stambuk, Boris U; Dunn, Barbara; Alves, Sergio L; Duval, Eduarda H; Sherlock, Gavin

    2009-12-01

    Fuel ethanol is now a global energy commodity that is competitive with gasoline. Using microarray-based comparative genome hybridization (aCGH), we have determined gene copy number variations (CNVs) common to five industrially important fuel ethanol Saccharomyces cerevisiae strains responsible for the production of billions of gallons of fuel ethanol per year from sugarcane. These strains have significant amplifications of the telomeric SNO and SNZ genes, which are involved in the biosynthesis of vitamins B6 (pyridoxine) and B1 (thiamin). We show that increased copy number of these genes confers the ability to grow more efficiently under the repressing effects of thiamin, especially in medium lacking pyridoxine and with high sugar concentrations. These genetic changes have likely been adaptive and selected for in the industrial environment, and may be required for the efficient utilization of biomass-derived sugars from other renewable feedstocks.

  20. Overexpression of an ABA biosynthesis gene using a stress inducible promoter enhances drought resistance in petunia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants respond to drought stress by closing their stomata and reducing transpirational water loss. The plant hormone abscisic acid (ABA) regulates growth and stomatal closure particularly when the plant is under environmental stresses. One of the key enzymes in the ABA biosynthesis of higher plants ...

  1. The narA Locus of Synechococcus sp. Strain PCC 7942 Consists of a Cluster of Molybdopterin Biosynthesis Genes

    PubMed Central

    Rubio, Luis M.; Flores, Enrique; Herrero, Antonia

    1998-01-01

    The narA locus required for nitrate reduction in Synechococcus sp. strain PCC 7942 is shown to consist of a cluster of genes, namely, moeA, moaC, moaD, moaE, and moaA, involved in molybdenum cofactor biosynthesis. The product of the moaC gene of strain PCC 7942 shows homology in its N-terminal half to MoaC from Escherichia coli and in its C-terminal half to MoaB or Mog. Overexpression of the Synechococcus moaC gene in E. coli resulted in the synthesis of a polypeptide of 36 kDa, a size that would conform to a protein resembling a fusion of the MoaC and MoaB or Mog polypeptides of E. coli. Insertional inactivation of the moeA, moaC, moaE, and moaA genes showed that the moeA-moa gene cluster is required for growth on nitrate and expression of nitrate reductase activity in strain PCC 7942. The moaCDEA genes constitute an operon which is transcribed divergently from the moeA gene. Expression of the moeA gene and the moa operon was little affected by the nitrogen source present in the culture medium. PMID:9495759

  2. Putative pathway of sex pheromone biosynthesis and degradation by expression patterns of genes identified from female pheromone gland and adult antenna of Sesamia inferens (Walker).

    PubMed

    Zhang, Ya-Nan; Xia, Yi-Han; Zhu, Jia-Yao; Li, Sheng-Yun; Dong, Shuang-Lin

    2014-05-01

    The general pathway of biosynthesis and degradation for Type-I sex pheromones in moths is well established, but some genes involved in this pathway remain to be characterized. The purple stem borer, Sesamia inferens, employs a pheromone blend containing components with three different terminal functional groups (Z11-16:OAc, Z11-16:OH, and Z11-16:Ald) of Type-I sex pheromones. Thus, it provides a good model to study the diversity of genes involved in pheromone biosynthesis and degradation pathways. By analyzing previously obtained transcriptomic data of the sex pheromone glands and antennae, we identified 73 novel genes that are possibly related to pheromone biosynthesis (46 genes) or degradation (27 genes). Gene expression patterns and phylogenetic analysis revealed that one desaturase (SinfDes4), one fatty acid reductase (SinfFAR2), and one fatty acid xtransport protein (SinfFATP1) genes were predominantly expressed in pheromone glands, and clustered with genes involved in pheromone synthesis in other moth species. Ten genes including five carboxylesterases (SinfCXE10, 13, 14, 18, and 20), three aldehyde oxidases (SinfAOX1, 2 and 3), and two alcohol dehydrogenases (SinfAD1 and 3) were expressed specifically or predominantly in antennae, and could be candidate genes involved in pheromone degradation. SinfAD1 and 3 are the first reported alcohol dehydrogenase genes with antennae-biased expression. Based on these results we propose a pathway involving these potential enzyme-encoding gene candidates in sex pheromone biosynthesis and degradation in S. inferens. This study provides robust background information for further elucidation of the genetic basis of sex pheromone biosynthesis and degradation, and ultimately provides potential targets to disrupt sexual communication in S. inferens for control purposes.

  3. A molecular genetic analysis of carotenoid biosynthesis and the effects of carotenoid mutations on other photosynthetic genes in Rhodobacter capsulatus

    SciTech Connect

    Armstrong, G.A.

    1989-04-01

    The nine known R. capsulatus carotenoid genes are contained within the 46 kilobase (kb) photosynthesis gene cluster. An 11 kb subcluster containing eight of these genes has been cloned and its nucleotide sequence determined. A new gene, crtK, has been located in the middle of the subcluster. The carotenoid gene cluster contains sequences homologous to Escherichia coli ..omega../sup 70/ promoters, rho-independent transcription terminators, and prokaryotic transcriptional factor binding sites. The phenotypes and genotypes of ten transposon Tn5.7 insertion mutations within the carotenoid gene cluster have been analyzed, by characterization of the carotenoids accumulated and high resolution mapping of the Tn5.7 insertions. The enzymatic blockages in previously uncharacterized early carotenoid mutants have been determined using a new in vitro synthesis system, suggesting specific roles for the CrtB and CrtE gene products. The expression of six of the eight carotenoid genes in the cluster is induced upon the shift from dark chemoheterotrophic to anaerobic photosynthetic growth. The magnitude of the induction is equivalent to that of genes encoding structural photosynthesis polypeptides, although the carotenoid genes are induced earlier after the growth shift. Different means of regulating photosynthesis genes in R. capsulatus are discussed, and a rationale for the temporal pattern of expression of the carotenoid genes during photosynthetic adaptation is presented. Comparison of the deduced amino acid sequences of the two dehydrogenases of the R. capsulatus carotenoid biosynthesis pathway reveals two regions of strong similarity. The effect of carotenoid mutations on the photosynthetic phenotype has been studied by examining growth rates, pigments, pigment-protein complexes and gene expression for a complete set of carotenoid mutants. 161 refs.

  4. Phloem-Specific Expression of Tyrosine/Dopa Decarboxylase Genes and the Biosynthesis of Isoquinoline Alkaloids in Opium Poppy.

    PubMed Central

    Facchini, P. J.; De Luca, V.

    1995-01-01

    Tyrosine/dopa decarboxylase (TYDC) catalyzes the formation of tyramine and dopamine and represents the first steps in the biosynthesis of the large and diverse group of tetrahydroisoquinoline alkaloids. Opium poppy accumulates morphine in aerial organs and roots, whereas sanguinarine, which is derived from a distinct branch pathway, accumulates only in roots. Expression of the TYDC gene family in opium poppy was investigated in relation to the organ-specific biosynthesis of these different types of alkaloids. Members of the TYDC gene family are classified into two groups (represented by TYDC1 and TYDC2) and are differentially expressed. In the mature plant, TYDC2-like transcripts are predominant in stems and are also present in roots, whereas TYDC1-like transcripts are abundant only in roots. In situ hybridization analysis revealed that the expression of TYDC genes is developmentally regulated. TYDC transcripts are associated with vascular tissue in mature roots and stems but are also expressed in cortical tissues at earlier stages of development. Expression of TYDC genes is restricted to metaphloem and to protoxylem in the vascular bundles of mature aerial organs. Localization of TYDC transcripts in the phloem is consistent with the expected developmental origin of laticifers, which are specialized internal secretory cells that accompany vascular tissues in all organs of select species and that contain the alkaloid-rich latex in aerial organs. The differential expression of TYDC genes and the organ-dependent accumulation of different alkaloids suggest a coordinated regulation of specific alkaloid biosynthetic genes that are ultimately controlled by specific developmental programs. PMID:12242361

  5. Strengthening Triterpene Saponins Biosynthesis by Over-Expression of Farnesyl Pyrophosphate Synthase Gene and RNA Interference of Cycloartenol Synthase Gene in Panax notoginseng Cells.

    PubMed

    Yang, Yan; Ge, Feng; Sun, Ying; Liu, Diqiu; Chen, Chaoyin

    2017-04-05

    To conform to the multiple regulations of triterpene biosynthesis, the gene encoding farnesyl pyrophosphate synthase (FPS) was transformed into Panax notoginseng (P. notoginseng) cells in which RNA interference (RNAi) of the cycloartenol synthase (CAS) gene had been accomplished. Transgenic cell lines showed both higher expression levels of FPS and lower expression levels of CAS compared to the wild-type (WT) cells. In the triterpene and phytosterol analysis, transgenic cell lines provided a higher accumulation of total triterpene saponins, and a lower amount of phytosterols in comparison with the WT cells. Compared with the cells in which RNAi of the CAS gene was achieved, the cells with simultaneously over-expressed FPS and silenced CAS showed higher triterpene contents. These results demonstrate that over-expression of FPS can break the rate-limiting reaction catalyzed by FPS in the triterpene saponins biosynthetic pathway; and inhibition of CAS expression can decrease the synthesis metabolic flux of the phytosterol branch. Thus, more precursors flow in the direction of triterpene synthesis, and ultimately promote the accumulation of P. notoginseng saponins. Meanwhile, silencing and over-expressing key enzyme genes simultaneously is more effective than just manipulating one gene in the regulation of saponin biosynthesis.

  6. Two FgLEU2 Genes with Different Roles in Leucine Biosynthesis and Infection-Related Morphogenesis in Fusarium graminearum

    PubMed Central

    Liu, Xin; Han, Qi; Wang, Jian; Wang, Xin; Xu, Jianhong; Shi, Jianrong

    2016-01-01

    3-isopropylmalate dehydrogenase (IPMD) encoded by LEU2 is a key enzyme in leucine (Leu) biosynthetic pathway. Analysis of the genome sequence of Fusarium graminearum revealed two paralogous LEU2 genes (designated as FgLEU2A and FgLEU2B) in this fungus and the deduced amino acid sequences of FgLeu2A and FgLeu2B share 45% identity. Targeted disruption of individual FgLEU2A/B gene in F. graminearum assigned a more crucial role of FgLeu2A in Leu biosynthesis as disruption of FgLEU2A resulted in mutant (ΔFgLeu2A-10) that was Leu-auxotrophic and could not grow in minimal medium limited for amino acids, whereas FgLEU2B deletion mutant ΔFgLeu2B-2 was morphologically indistinguishable from the wild type strain PH-1. The growth defects of ΔFgLeu2A-10 could be overcome by exogenous addition of Leu at 0.25 mM. Double deletion of FgLEU2A and FgLEU2B (ΔFgLeu2AB-8) caused a more severe Leu-auxotrophic phenotype as the concentration of Leu exogenously added to medium to rescue the growth defect of ΔFgLeu2AB-8 should be raised to 1.25 mM, indicating a less important but nonnegligible role of FgLeu2B in Leu biosynthesis. Disturb of Leu biosynthesis caused by FgLEU2A deletion leads to slower growth rate, reduced aerial hyphal formation and red pigmentation on PDA plates and completely blocked conidial production and germination. All of the defects above could be overcome by Leu addition or complementation of the full-length FgLEU2A gene. ΔFgLeu2A-10 also showed significantly increased sensitivity to osmotic and oxidative stresses. Pathogenicity assay results showed that virulence of mutants lacking FgLEU2A were dramatically impaired on wheat heads and non-host cherry tomatoes. Additionally, a low level of deoxynivalenol (DON) production of ΔFgLeu2A-10 and ΔFgLeu2AB-8 in wheat kernels was also detected. Taken together, results of this study indicated a crucial role of FgLeu2A and a less important role of FgLeu2B in Leu biosynthesis and fungal infection-related morphogenesis in

  7. 7 CFR 983.4 - Aflatoxin.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 8 2013-01-01 2013-01-01 false Aflatoxin. 983.4 Section 983.4 Agriculture Regulations... NEW MEXICO Definitions § 983.4 Aflatoxin. Aflatoxin is one of a group of mycotoxins produced by the molds Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are naturally occurring...

  8. 7 CFR 983.4 - Aflatoxin.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 8 2014-01-01 2014-01-01 false Aflatoxin. 983.4 Section 983.4 Agriculture Regulations... NEW MEXICO Definitions § 983.4 Aflatoxin. Aflatoxin is one of a group of mycotoxins produced by the molds Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are naturally occurring...

  9. 7 CFR 983.4 - Aflatoxin.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 8 2012-01-01 2012-01-01 false Aflatoxin. 983.4 Section 983.4 Agriculture Regulations... NEW MEXICO Definitions § 983.4 Aflatoxin. Aflatoxin is one of a group of mycotoxins produced by the molds Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are naturally occurring...

  10. De Novo Sequencing and Transcriptome Analysis of Wolfiporia cocos to Reveal Genes Related to Biosynthesis of Triterpenoids

    PubMed Central

    Shu, Shaohua; Chen, Bei; Zhou, Mengchun; Zhao, Xinmei; Xia, Haiyang; Wang, Mo

    2013-01-01

    Wolfiporia cocos Ryvarden et Gilbertson is a saprophytic fungus in the Basidiomycetes. Its dried sclerotium is widely used as a traditional crude drug in East Asia. Especially in China, the dried sclerotium is regarded as the silver of the Chinese traditional drugs, not only for its white color, but also its medicinal value. Furthermore, triterpenoids from W. cocos are the main active compounds with antitumor and anti-inflammatory activity. Biosynthesis of the triterpenoids has rarely been researched. In this study, the de novo sequencing of the mycelia and sclerotia of W. cocos were carried out by Illumina HiSeq 2000. A total of 3,484,996,740 bp from 38,722,186 sequence reads of mycelia, and 3,573,921,960 bp from 39,710,244 high quality sequence reads of sclerotium were obtained. These raw data were assembled into 60,354 contigs and 40,939 singletons, and 56,938 contigs and 37,220 singletons for mycelia and sclerotia, respectively. The transcriptomic data clearly showed that terpenoid biosynthesis was only via the MVA pathwayin W. cocos. The production of total triterpenoids and pachymic acid was examined in the dry mycelia and sclerotia. The content of total triterpenoids was 5.36% and 1.43% in mycelia and sclerotia, respectively, and the content of pachymic acid was 0.458% and 0.174%. Some genes involved in the triterpenoid biosynthetic pathway were chosen to be verified by qRT-PCR. The unigenes encoding diphosphomevalonate decarboxylase (Unigene 20430), farnesyl diphosphate synthase (Unigene 14106 and 21656), hydroxymethylglutaryl-CoA reductase (NADPH) (Unigene 6395_All) and lanosterol synthase (Unigene28001_All) were upregulated in the mycelia stage. It is likely that expression of these genes influences the biosynthesis of triterpenoids in the mycelia stage. PMID:23967197

  11. A rice semi-dwarf gene, Tan-Ginbozu (D35), encodes the gibberellin biosynthesis enzyme, ent-kaurene oxidase.

    PubMed

    Itoh, Hironori; Tatsumi, Tomoko; Sakamoto, Tomoaki; Otomo, Kazuko; Toyomasu, Tomonobu; Kitano, Hidemi; Ashikari, Motoyuki; Ichihara, Shigeyuki; Matsuoka, Makoto

    2004-03-01

    A rice (Oryza sativa L.) semi-dwarf cultivar, Tan-Ginbozu (d35Tan-Ginbozu), contributed to the increase in crop productivity in Japan in the 1950s. Previous studies suggested that the semi-dwarf stature of d35Tan-Ginbozu is caused by a defective early step of gibberellin biosynthesis, which is catalyzed by ent-kaurene oxidase (KO). To study the molecular characteristics of d35Tan-Ginbozu, we isolated 5 KO-like (KOL) genes from the rice genome, which encoded proteins highly homologous to Arabidopsis and pumpkin KOs. The genes (OsKOL1 to 5) were arranged as tandem repeats in the same direction within a 120 kb sequence. Expression analysis revealed that OsKOL2 and OsKOL4 were actively transcribed in various organs, while OsKOL1 and OsKOL5 were expressed only at low levels; OsKOL3 may be a pseudogene. Sequence analysis and complementation experiments demonstrated that OsKOL2 corresponds to D35. Homozygote with null alleles of D35 showed a severe dwarf phenotype; therefore, d35Tan-Ginbozu is a weak allele of D35. Introduction of OsKOL4 into d35Tan-Ginbozu did not rescue its dwarf phenotype, indicating that OsKOL4 is not involved in GA biosynthesis. OsKOL4 and OsKOL5 are likely to take part in phytoalexin biosynthesis, because their expression was promoted by UV irradiation and/or elicitor treatment. Comparing d35Tan-Ginbozu with other high yielding cultivars, we discuss strategies to produce culm architectures suitable for high crop yield by decreasing GA levels.

  12. De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis

    DOE PAGES

    Cárdenas-Conejo, Yair; Carballo-Uicab, Víctor; Lieberman, Meric; ...

    2015-10-28

    Bixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes. In order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds frommore » B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway. In conclusion, the identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.« less

  13. De Novo RNA Sequencing and Transcriptome Analysis of Monascus purpureus and Analysis of Key Genes Involved in Monacolin K Biosynthesis

    PubMed Central

    Zhang, Chan; Liang, Jian; Yang, Le; Sun, Baoguo; Wang, Chengtao

    2017-01-01

    Monascus purpureus is an important medicinal and edible microbial resource. To facilitate biological, biochemical, and molecular research on medicinal components of M. purpureus, we investigated the M. purpureus transcriptome by RNA sequencing (RNA-seq). An RNA-seq library was created using RNA extracted from a mixed sample of M. purpureus expressing different levels of monacolin K output. In total 29,713 unigenes were assembled from more than 60 million high-quality short reads. A BLAST search revealed hits for 21,331 unigenes in at least one of the protein or nucleotide databases used in this study. The 22,365 unigenes were categorized into 48 functional groups based on Gene Ontology classification. Owing to the economic and medicinal importance of M. purpureus, most studies on this organism have focused on the pharmacological activity of chemical components and the molecular function of genes involved in their biogenesis. In this study, we performed quantitative real-time PCR to detect the expression of genes related to monacolin K (mokA-mokI) at different phases (2, 5, 8, and 12 days) of M. purpureus M1 and M1-36. Our study found that mokF modulates monacolin K biogenesis in M. purpureus. Nine genes were suggested to be associated with the monacolin K biosynthesis. Studies on these genes could provide useful information on secondary metabolic processes in M. purpureus. These results indicate a detailed resource through genetic engineering of monacolin K biosynthesis in M. purpureus and related species. PMID:28114365

  14. Exogenous GA₃ Application Enhances Xylem Development and Induces the Expression of Secondary Wall Biosynthesis Related Genes in Betula platyphylla.

    PubMed

    Guo, Huiyan; Wang, Yucheng; Liu, Huizi; Hu, Ping; Jia, Yuanyuan; Zhang, Chunrui; Wang, Yanmin; Gu, Shan; Yang, Chuanping; Wang, Chao

    2015-09-23

    Gibberellin (GA) is a key signal molecule inducing differentiation of tracheary elements, fibers, and xylogenesis. However the molecular mechanisms underlying the effect of GA on xylem elongation and secondary wall development in tree species remain to be determined. In this study, Betula platyphylla (birch) seeds were treated with 300 ppm GA₃ and/or 300 ppm paclobutrazol (PAC), seed germination was recorded, and transverse sections of hypocotyls were stained with toluidine blue; the two-month-old seedlings were treated with 50 μM GA₃ and/or 50 μM PAC, transverse sections of seedling stems were stained using phloroglucinol-HCl, and secondary wall biosynthesis related genes expression was analyzed by real-time quantitative PCR. Results indicated that germination percentage, energy and time of seeds, hypocotyl height and seedling fresh weight were enhanced by GA₃, and reduced by PAC; the xylem development was wider in GA₃-treated plants than in the control; the expression of NAC and MYB transcription factors, CESA, PAL, and GA oxidase was up-regulated during GA₃ treatment, suggesting their role in GA₃-induced xylem development in the birch. Our results suggest that GA₃ induces the expression of secondary wall biosynthesis related genes to trigger xylogenesis in the birch plants.

  15. [Determination of aflatoxins in cheeses].

    PubMed

    Bartos, J; Matyás, Z

    1979-03-01

    To investigate cheeses for the presence of aflatoxins we chose the very sensitive method of Tuinstra and Bronsgeest (1975) used for the determination of aflatoxin M1 in milk. The method was slightly modified and the presence of aflatoxins was determined in 54 samples of different cheeses. Aflatoxin M1 was found out in 24% of the investigated samples. Most of positive samples were found among the soft cheeses (53.8 3/4), then in processed cheeses (13.6%) and in hard cheeses (12.5%). Aflatoxin M1 was not found in the group of mouldy cheeses and Olomouc cake cheeses, which were investigated in a smaller range. Positive findings did not exceed concentrations of 10 ng per kg, i.e. they did not even reach the value of permissible concentration as proposed in the Czech Socialist Republic for foods (5 microgram per kg).

  16. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

    PubMed Central

    Gust, Bertolt; Challis, Greg L.; Fowler, Kay; Kieser, Tobias; Chater, Keith F.

    2003-01-01

    Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and λ-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriTRK2 for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriTRK2 to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate. PMID:12563033

  17. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin.

    PubMed

    Gust, Bertolt; Challis, Greg L; Fowler, Kay; Kieser, Tobias; Chater, Keith F

    2003-02-18

    Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and lambda-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT(RK2) for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT(RK2) to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.

  18. Natural Products as Tools for Chemogenomic Analysis of Mycotoxin Biosynthesis and Fungal Stress-Response Systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Certain phenolic compounds with antioxidant properties inhibit aflatoxin biosynthesis in the fungus Aspergillus flavus, without affecting growth. Similarly, some of the same phenolics also inhibit biosynthesis of ochratoxin by A. alliaceous. Exposing A. flavus to oxidative stress, such as hydrogen p...

  19. Annotation of genes involved in glycerolipid biosynthesis in Chlamydomonas reinhardtii: discovery of the betaine lipid synthase BTA1Cr.

    PubMed

    Riekhof, Wayne R; Sears, Barbara B; Benning, Christoph

    2005-02-01

    Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1Cr) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaARs and BtaBRs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaARs and BtaBRs.

  20. Transcriptional Control of Steroid Biosynthesis Genes in the Drosophila Prothoracic Gland by Ventral Veins Lacking and Knirps

    PubMed Central

    Dorry, Elad; Komura-Kawa, Tatsuya; Fujimoto, Yoshinori; Troelsen, Jesper T.; Herder, Rachel; O'Connor, Michael B.; Niwa, Ryusuke; Rewitz, Kim F.

    2014-01-01

    Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development. PMID:24945799

  1. Putative evolutionary origin of plasmids carrying the genes involved in leucine biosynthesis in Buchnera aphidicola (endosymbiont of aphids).

    PubMed Central

    van Ham, R C; Moya, A; Latorre, A

    1997-01-01

    An 8.5-kb plasmid encoding genes (leuABCD) involved in leucine biosynthesis and a small plasmid of 1.74 kb of yet unknown function were found in the intracellular symbiont, Buchnera aphidicola, of two divergent aphid species, Thelaxes suberi and Tetraneura caerulescens, respectively. The leuABCD-carrying plasmid (pBTs1) was amplified from total aphid DNA by inverse long PCR, using outwardly oriented oligonucleotide primers specific to leuA. The resulting 8.2-kb PCR fragment as well as the 1.74-kb plasmid (pBTc1) were cloned and sequenced. pBTs1 differed from a previously described B. aphidicola plasmid (pRPE) of the aphid Rhopalosiphum padi by the presence of a small heat shock gene (ibp) and in the order of the leuABCD and repA genes. Comparison of both leucine plasmids to the small plasmid pBTc1 revealed extensive similarity with respect to putative replication functions as well as in the presence of a highly conserved open reading frame that was found to be homologous to Escherichia coli YqhA and Haemophilus influenzae HI0507 and which may encode an integral membrane protein. The three B. aphidicola plasmids most likely evolved from a common ancestral replicon, which in turn may be distantly related to IncFII plasmids. Phylogenetic affiliations of the B. aphidicola strains of the two aphid species were assessed by sequencing of their 16S rRNA genes. Evaluation of the distribution of the leuABCD-encoding plasmids within a phylogenetic framework suggests independent origins for pBTs1 and pRPE from an ancestral replicon resembling pBTc1. The implications for symbiotic essential amino acid biosynthesis and provisioning are discussed. PMID:9244264

  2. The P450-4 gene of Gibberella fujikuroi encodes ent-kaurene oxidase in the gibberellin biosynthesis pathway.

    PubMed

    Tudzynski, B; Hedden, P; Carrera, E; Gaskin, P

    2001-08-01

    At least five genes of the gibberellin (GA) biosynthesis pathway are clustered on chromosome 4 of Gibberella fujikuroi; these genes encode the bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase, a GA-specific geranylgeranyl diphosphate synthase, and three cytochrome P450 monooxygenases. We now describe a fourth cytochrome P450 monooxygenase gene (P450-4). Gas chromatography-mass spectrometry analysis of extracts of mycelia and culture fluid of a P450-4 knockout mutant identified ent-kaurene as the only intermediate of the GA pathway. Incubations with radiolabeled precursors showed that the metabolism of ent-kaurene, ent-kaurenol, and ent-kaurenal was blocked in the transformants, whereas ent-kaurenoic acid was metabolized efficiently to GA(4). The GA-deficient mutant strain SG139, which lacks the 30-kb GA biosynthesis gene cluster, converted ent-kaurene to ent-kaurenoic acid after transformation with P450-4. The B1-41a mutant, described as blocked between ent-kaurenal and ent-kaurenoic acid, was fully complemented by P450-4. There is a single nucleotide difference between the sequence of the B1-41a and wild-type P450-4 alleles at the 3' consensus sequence of intron 2 in the mutant, resulting in reduced levels of active protein due to a splicing defect in the mutant. These data suggest that P450-4 encodes a multifunctional ent-kaurene oxidase catalyzing all three oxidation steps between ent-kaurene and ent-kaurenoic acid.

  3. Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis sfpo and Escherichia coli entD genes.

    PubMed Central

    Grossman, T H; Tuckman, M; Ellestad, S; Osburne, M S

    1993-01-01

    In response to iron deprivation, Bacillus subtilis secretes a catecholic siderophore, 2,3-dihydroxybenzoyl glycine, which is similar to the precursor of the Escherichia coli siderophore enterobactin. We isolated two sets of B. subtilis DNA sequences that complemented the mutations of several E. coli siderophore-deficient (ent) mutants with defective enterobactin biosynthesis enzymes. One set contained DNA sequences that complemented only an entD mutation. The second set contained DNA sequences that complemented various combinations of entB, entE, entC, and entA mutations. The two sets of DNA sequences did not appear to overlap. AB. subtilis mutant containing an insertion in the region of the entD homolog grew much more poorly in low-iron medium and with markedly different kinetics. These data indicate that (i) at least five of the siderophore biosynthesis genes of B. subtilis can function in E. coli, (ii) the genetic organization of these siderophore genes in B. subtilis is similar to that in E. coli, and (iii) the B. subtilis entD homolog is required for efficient growth in low-iron medium. The nucleotide sequence of the B. subtilis DNA contained in plasmid pENTA22, a clone expressing the B. subtilis entD homolog, revealed the presence of at least two genes. One gene was identified as sfpo, a previously reported gene involved in the production of surfactin in B. subtilis and which is highly homologous to the E. coli entD gene. We present evidence that the E. coli entD and B. subtilis sfpo genes are interchangeable and that their products are members of a new family of proteins which function in the secretion of peptide molecules. Images PMID:8407792

  4. Juvenile Hormone Biosynthesis Gene Expression in the corpora allata of Honey Bee (Apis mellifera L.) Female Castes

    PubMed Central

    Rosa, Gustavo Conrado Couto; Moda, Livia Maria; Martins, Juliana Ramos; Bitondi, Márcia Maria Gentile; Hartfelder, Klaus; Simões, Zilá Luz Paulino

    2014-01-01

    Juvenile hormone (JH) controls key events in the honey bee life cycle, viz. caste development and age polyethism. We quantified transcript abundance of 24 genes involved in the JH biosynthetic pathway in the corpora allata-corpora cardiaca (CA-CC) complex. The expression of six of these genes showing relatively high transcript abundance was contrasted with CA size, hemolymph JH titer, as well as JH degradation rates and JH esterase (jhe) transcript levels. Gene expression did not match the contrasting JH titers in queen and worker fourth instar larvae, but jhe transcript abundance and JH degradation rates were significantly lower in queen larvae. Consequently, transcriptional control of JHE is of importance in regulating larval JH titers and caste development. In contrast, the same analyses applied to adult worker bees allowed us inferring that the high JH levels in foragers are due to increased JH synthesis. Upon RNAi-mediated silencing of the methyl farnesoate epoxidase gene (mfe) encoding the enzyme that catalyzes methyl farnesoate-to-JH conversion, the JH titer was decreased, thus corroborating that JH titer regulation in adult honey bees depends on this final JH biosynthesis step. The molecular pathway differences underlying JH titer regulation in larval caste development versus adult age polyethism lead us to propose that mfe and jhe genes be assayed when addressing questions on the role(s) of JH in social evolution. PMID:24489805

  5. Juvenile hormone biosynthesis gene expression in the corpora allata of honey bee (Apis mellifera L.) female castes.

    PubMed

    Bomtorin, Ana Durvalina; Mackert, Aline; Rosa, Gustavo Conrado Couto; Moda, Livia Maria; Martins, Juliana Ramos; Bitondi, Márcia Maria Gentile; Hartfelder, Klaus; Simões, Zilá Luz Paulino

    2014-01-01

    Juvenile hormone (JH) controls key events in the honey bee life cycle, viz. caste development and age polyethism. We quantified transcript abundance of 24 genes involved in the JH biosynthetic pathway in the corpora allata-corpora cardiaca (CA-CC) complex. The expression of six of these genes showing relatively high transcript abundance was contrasted with CA size, hemolymph JH titer, as well as JH degradation rates and JH esterase (jhe) transcript levels. Gene expression did not match the contrasting JH titers in queen and worker fourth instar larvae, but jhe transcript abundance and JH degradation rates were significantly lower in queen larvae. Consequently, transcriptional control of JHE is of importance in regulating larval JH titers and caste development. In contrast, the same analyses applied to adult worker bees allowed us inferring that the high JH levels in foragers are due to increased JH synthesis. Upon RNAi-mediated silencing of the methyl farnesoate epoxidase gene (mfe) encoding the enzyme that catalyzes methyl farnesoate-to-JH conversion, the JH titer was decreased, thus corroborating that JH titer regulation in adult honey bees depends on this final JH biosynthesis step. The molecular pathway differences underlying JH titer regulation in larval caste development versus adult age polyethism lead us to propose that mfe and jhe genes be assayed when addressing questions on the role(s) of JH in social evolution.

  6. Disruption of Transporters Affiliated with Enantio-Pyochelin Biosynthesis Gene Cluster of Pseudomonas protegens Pf-5 Has Pleiotropic Effects

    PubMed Central

    Lim, Chee Kent; Penesyan, Anahit; Hassan, Karl A.; Loper, Joyce E.; Paulsen, Ian T.

    2016-01-01

    Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens) is a biocontrol bacterium that produces the siderophore enantio-pyochelin under conditions of iron starvation in a process that is often accompanied by the secretion of its biosynthesis intermediates, salicylic acid and dihydroaeruginoic acid. In this study, we investigated whether several transporters that are encoded by genes within or adjacent to the enantio-pyochelin biosynthetic cluster, serve as efflux systems for enantio-pyochelin and/or its intermediates. In addition, we determined whether these transporters have broad substrates range specificity using a Phenotype Microarray system. Intriguingly, knockouts of the pchH and fetF transporter genes resulted in mutant strains that secrete higher levels of enantio-pyochelin as well as its intermediates salicylic acid and dihydroaeruginoic acid. Analyses of these mutants did not indicate significant change in transcription of biosynthetic genes involved in enantio-pyochelin production. In contrast, the deletion mutant of PFL_3504 resulted in reduced transcription of the biosynthetic genes as well as decreased dihydroaeruginoic acid concentrations in the culture supernatant, which could either point to regulation of gene expression by the transporter or its role in dihydroaeruginoic acid transport. Disruption of each of the transporters resulted in altered stress and/or chemical resistance profile of Pf-5, which may reflect that these transporters could have specificity for rather a broad range of substrates. PMID:27442435

  7. Genes Specific for the Biosynthesis of Clavam Metabolites Antipodal to Clavulanic Acid Are Clustered with the Gene for Clavaminate Synthase 1 in Streptomyces clavuligerus

    PubMed Central

    Mosher, Roy H.; Paradkar, Ashish S.; Anders, Cecilia; Barton, Barry; Jensen, Susan E.

    1999-01-01

    Portions of the Streptomyces clavuligerus chromosome flanking cas1, which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading frame located immediately upstream of cas1, were constructed by a gene replacement procedure. Similar techniques were used to generate S. clavuligerus mutants carrying a deletion that encompassed portions of the two open reading frames, cvm4 and cvm5, located directly downstream of cas1. Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosynthesis. When a cas1 mutant of S. clavuligerus was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 and cas2 produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis. PMID:10223939

  8. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis

    PubMed Central

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  9. DNA Methylation Perturbations in Genes Involved in Polyunsaturated Fatty Acid Biosynthesis Associated with Depression and Suicide Risk

    PubMed Central

    Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-yu; Cooper, Thomas B.; Burke, Ainsley K.; Oquendo, Maria A.; Mann, J. John; Sublette, M. Elizabeth

    2015-01-01

    Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention. PMID:25972837

  10. Transcriptome Profiling of Khat (Catha edulis) and Ephedra sinica Reveals Gene Candidates Potentially Involved in Amphetamine-Type Alkaloid Biosynthesis

    PubMed Central

    Groves, Ryan A.; Hagel, Jillian M.; Zhang, Ye; Kilpatrick, Korey; Levy, Asaf; Marsolais, Frédéric; Lewinsohn, Efraim; Sensen, Christoph W.; Facchini, Peter J.

    2015-01-01

    Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for Ephedra sinica and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications. PMID:25806807

  11. De Novo Transcriptome Analysis of Warburgia ugandensis to Identify Genes Involved in Terpenoids and Unsaturated Fatty Acids Biosynthesis.

    PubMed

    Wang, Xin; Zhou, Chen; Yang, Xianpeng; Miao, Di; Zhang, Yansheng

    2015-01-01

    The bark of Warburgia ugandensis (Canellaceae family) has been used as a medicinal source for a long history in many African countries. The presence of diverse terpenoids and abundant polyunsaturated fatty acids (PUFAs) in this organ contributes to its broad range of pharmacological properties. Despite its medicinal and economic importance, the knowledge on the biosynthesis of terpenoid and unsaturated fatty acid in W. ugandensis bark remains largely unknown. Therefore, it is necessary to construct a genomic and/or transcriptomic database for the functional genomics study on W. ugandensis. The chemical profiles of terpenoids and fatty acids between the bark and leaves of W. ugandensis were compared by gas chromatography-mass spectrometry (GC-MS) analysis. Meanwhile, the transcriptome database derived from both tissues was created using Illumina sequencing technology. In total, about 17.1 G clean nucleotides were obtained, and de novo assembled into 72,591 unigenes, of which about 38.06% can be aligned to the NCBI non-redundant protein database. Many candidate genes in the biosynthetic pathways of terpenoids and unsaturated fatty acids were identified, including 14 unigenes for terpene synthases. Furthermore, 2,324 unigenes were discovered to be differentially expressed between both tissues; the functions of those differentially expressed genes (DEGs) were predicted by gene ontology enrichment and metabolic pathway enrichment analyses. In addition, the expression of 12 DEGs with putative roles in terpenoid and unsaturated fatty acid metabolic pathways was confirmed by qRT-PCRs, which was consistent with the data of the RNA-sequencing. In conclusion, we constructed a comprehensive transcriptome dataset derived from the bark and leaf of W. ugandensis, which forms the basis for functional genomics studies on this plant species. Particularly, the comparative analysis of the transcriptome data between the bark and leaf will provide critical clues to reveal the regulatory

  12. Gene targets for fungal and mycotoxin control.

    PubMed

    Kim, J H; Campbell, B C; Molyneux, R; Mahoney, N; Chan, K L; Yu, J; Wilkinson, J; Cary, J; Bhatnagar, D; Cleveland, T E

    2006-03-01

    It was initially shown that gallic acid, from hydrolysable tannins in the pelliele of walnut kernels, dramatically inhibits biosynthesis of aflatoxin byAspergillus flavus. The mechanism of this inhibition was found to take place upstream from the gene cluster, including the regulatory gene,aflR, involved in aflatoxin biosynthesis. Additional research using other antioxidant phenolics showed similar antiaflatoxigenic activity to gallic acid. Treatment ofA. flavus withtert-butyl hydroperoxide resulted in an almost doubling of aflatoxin biosynthesis compared to untreated samples. Thus, antioxidative response systems are potentially useful molecular targets for control ofA. flavus. A high throughput screening system was developed using yeast,Saccharomyces cerevisiae, as a model fungus. This screening provided an avenue to quickly identify fungal genes that were vulnerable to treatment by phenolic compounds. The assay also provided a means to quickly assess effects of combinations of phenolics and certain fungicides affecting mitochondrial respiration. For example, theS. cerevisiae sod2† mutant was highly sensitive to treatment by certain phenolics and strobilurins/antimycin A, fungicides which inhibit complex III of the mitochondrial respiratory chain. Verification of stress to this system in the target fungus,A. flavus, was shown through complementation analysis, wherein the mitochondrial superoxide dismutase (Mn-SOD) gene (sodA) ofA. flavus in the ortholog mutant,sod2†, ofS. cerevisiae, relieved phenolic-induced stress. Mitochondrial antioxidative stress systems play an important role in fungal response to antifungals. Combined treatment of fungi with phenolics and inhibitors of mitochondrial respiration can effectively suppress growth ofA. flavus in a synergistic fashion.

  13. Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids

    PubMed Central

    Li, Yueqing; Liu, Xingxue; Cai, Xinquan; Shan, Xiaotong; Gao, Ruifang; Yang, Song; Han, Taotao; Wang, Shucai; Wang, Li; Gao, Xiang

    2017-01-01

    Dihydroflavonol-4-reductase (DFR) is a key enzyme in the reduction of dihydroflavonols to leucoanthocyanidins in both anthocyanin biosynthesis and proanthocyanidin accumulation. In many plant species, it is encoded by a gene family, however, how the different copies evolve either to function in different tissues or at different times or to specialize in the use of different but related substrates needs to be further investigated, especially in monocot plants. In this study, a total of eight putative DFR-like genes were firstly cloned from Freesia hybrida. Phylogenetic analysis showed that they were classified into different branches, and FhDFR1, FhDFR2, and FhDFR3 were clustered into DFR subgroup, whereas others fell into the group with cinnamoyl-CoA reductase (CCR) proteins. Then, the functions of the three FhDFR genes were further characterized. Different spatio-temporal transcription patterns and levels were observed, indicating that the duplicated FhDFR genes might function divergently. After introducing them into Arabidopsis dfr (tt3-1) mutant plants, partial complementation of the loss of cyanidin derivative synthesis was observed, implying that FhDFRs could convert dihydroquercetin to leucocyanidin in planta. Biochemical assays also showed that FhDFR1, FhDFR2, and FhDFR3 could utilize dihydromyricetin to generate leucodelphinidin, while FhDFR2 could also catalyze the formation of leucocyanidin from dihydrocyanidin. On the contrary, neither transgenic nor biochemical analysis demonstrated that FhDFR proteins could reduce dihydrokaempferol to leucopelargonidin. These results were consistent with the freesia flower anthocyanin profiles, among which delphinidin derivatives were predominant, with minor quantities of cyanidin derivatives and undetectable pelargonidin derivatives. Thus, it can be deduced that substrate specificities of DFRs were the determinant for the categories of anthocyanins aglycons accumulated in F. hybrida. Furthermore, we also found that

  14. The Phaseolus vulgaris PvTRX1h gene regulates plant hormone biosynthesis in embryogenic callus from common bean.

    PubMed

    Barraza, Aarón; Cabrera-Ponce, José L; Gamboa-Becerra, Roberto; Luna-Martínez, Francisco; Winkler, Robert; Álvarez-Venegas, Raúl

    2015-01-01

    Common bean is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants, in somatic embryo generation. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. Embryogenic calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate, by RNA interference, expression of the PvTRX1h gene. Plant hormone content was measured by mass spectrometry and gene expression was assessed by q-PCR. Detailed histological analysis was performed on selected transgenic embryogenic calli. It was determined that down-regulation of PvTRX1h gene was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was down-regulated were capable of differentiation into somatic embryos. Also, down-regulation of PvTRX1h showed increased transcript abundance of a gene coding for a second histone lysine methyltransferase, PvASHH2h. Accordingly, the PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation.

  15. The Phaseolus vulgaris PvTRX1h gene regulates plant hormone biosynthesis in embryogenic callus from common bean

    PubMed Central

    Barraza, Aarón; Cabrera-Ponce, José L.; Gamboa-Becerra, Roberto; Luna-Martínez, Francisco; Winkler, Robert; Álvarez-Venegas, Raúl

    2015-01-01

    Common bean is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants, in somatic embryo generation. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. Embryogenic calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate, by RNA interference, expression of the PvTRX1h gene. Plant hormone content was measured by mass spectrometry and gene expression was assessed by q-PCR. Detailed histological analysis was performed on selected transgenic embryogenic calli. It was determined that down-regulation of PvTRX1h gene was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was down-regulated were capable of differentiation into somatic embryos. Also, down-regulation of PvTRX1h showed increased transcript abundance of a gene coding for a second histone lysine methyltransferase, PvASHH2h. Accordingly, the PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation. PMID:26284093

  16. Some ethylene biosynthesis and AP2/ERF genes reveal a specific pattern of expression during somatic embryogenesis in Hevea brasiliensis

    PubMed Central

    2012-01-01

    Background Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. Results The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. Conclusions This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult. PMID:23268714

  17. The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630.

    PubMed

    Hernández, Martín A; Arabolaza, Ana; Rodríguez, Eduardo; Gramajo, Hugo; Alvarez, Héctor M

    2013-03-01

    Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf1 and atf2 exhibited a significant DGAT activity when expressed in E. coli, as revealed in a previous study. The contribution of atf1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al., Microbiology 154:2327-2335, 2008). In this study, we investigated the in vivo role of atf2 gene in TAG accumulation by R. opacus PD630 by using different genetic strategies. The atf2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25-30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild-type strain. Surprisingly, in contrast to single mutants, a double mutant generated by the disruption of atf1 and atf2 genes only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Overexpression of atf1 and atf2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation, while heterologous expression of atf2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf1 gene, atf2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.

  18. Stability of aflatoxins in solution.

    PubMed

    Diaz, Gonzalo J; Cepeda, Sandra M; Martos, Perry A

    2012-01-01

    The stability of aflatoxins B1, B2, G1, and G2 was studied in solutions containing different concentrations of water, acetonitrile, and/or methanol, and in autosampler vials treated with nitric acid or silanized. When stored at room temperature (20 degrees C) for 24 h, aflatoxins G1 and G2 were stable only in solutions containing 100% organic solvent, whereas aflatoxins B1 and B2 were stable in solutions of methanol-water and acetonitrile-water at greater than 60 and 40% organic content, respectively. At 5 degrees C, aflatoxins G1 and G2 showed a significant decrease in concentration only when kept in less than 20% aqueous organic solvent. Significant loss of aflatoxins was realized in standard, commercially available amber type I borosilicate autosampler vials, but chemical etching of the vials with nitric acid or with silanization prevented aflatoxin degradation. These results indicate that aflatoxins are unstable in aqueous solutions and that this instability can be counteracted by the presence of at least 20% organic solvent and keeping the solutions at 5 degrees C or by the use of treated vials.

  19. Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by some of the same strains of A. flavus that produce aflatoxins and by some Aspergillus oryzae strains. Despite its discovery 40 years ago, few reviews of its toxicity and biosynthesis have been reported. This review examines w...

  20. Tobacco Nicotine Uptake Permease Regulates the Expression of a Key Transcription Factor Gene in the Nicotine Biosynthesis Pathway1[C][W

    PubMed Central

    2014-01-01

    The down-regulation of a tobacco (Nicotiana tabacum) plasma membrane-localized nicotine uptake permease, NUP1, was previously reported to reduce total alkaloid levels in tobacco plants. However, it was unclear how this nicotine transporter affected the biosynthesis of the alkaloid nicotine. When NUP1 expression was suppressed in cultured tobacco cells treated with jasmonate, which induces nicotine biosynthesis, the NICOTINE2-locus transcription factor gene ETHYLENE RESPONSE FACTOR189 (ERF189) and its target structural genes, which function in nicotine biosynthesis and transport, were strongly suppressed, resulting in decreased total alkaloid levels. Conversely, NUP1 overexpression had the opposite effect. In these experiments, the expression levels of the MYC2 transcription factor gene and its jasmonate-inducible target gene were not altered. Inhibiting tobacco alkaloid biosynthesis by suppressing the expression of genes encoding enzymes in the nicotine pathway did not affect the expression of ERF189 and other nicotine pathway genes, indicating that ERF189 is not regulated by cellular alkaloid levels. Suppressing the expression of jasmonate signaling components in cultured tobacco cells showed that NUP1 acts downstream of the CORONATINE INSENSITIVE1 receptor and MYC2, but upstream of ERF189. These results suggest that although jasmonate-activated expression of MYC2 induces the expression of both NUP1 and ERF189, expression of ERF189 may actually be mediated by NUP1. Furthermore, NUP1 overexpression in tobacco plants inhibited the long-range transport of nicotine from the roots to the aerial parts. Thus, NUP1 not only mediates the uptake of tobacco alkaloids into root cells, but also positively controls the expression of ERF189, a key gene in the biosynthesis of these alkaloids. PMID:25344505

  1. RNAi control of aflatoxins in peanut plants, a multifactorial system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA-interference (RNAi)-mediated control of aflatoxin contamination in peanut plants is a multifactorial and hyper variable system. The use of RNAi biotechnology to silence single genes in plants has inherently high-variability among transgenic events. Also the level of expression of small interfe...

  2. Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis

    PubMed Central

    Pandey, Shiv S.; Singh, Sucheta; Babu, C. S. Vivek; Shanker, Karuna; Srivastava, N. K.; Shukla, Ashutosh K.; Kalra, Alok

    2016-01-01

    Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229–403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant. PMID:27220774

  3. Disruption of plant carotenoid biosynthesis through virus-induced gene silencing affects oviposition behaviour of the butterfly Pieris rapae.

    PubMed

    Zheng, Si-Jun; Snoeren, Tjeerd A L; Hogewoning, Sander W; van Loon, Joop J A; Dicke, Marcel

    2010-05-01

    Optical plant characteristics are important cues to plant-feeding insects. In this article, we demonstrate for the first time that silencing the phytoene desaturase (PDS) gene, encoding a key enzyme in plant carotenoid biosynthesis, affects insect oviposition site selection behaviour. Virus-induced gene silencing employing tobacco rattle virus was used to knock down endogenous PDS expression in three plant species (Arabidopsis thaliana, Brassica nigra and Nicotiana benthamiana) by its heterologous gene sequence from Brassica oleracea. We investigated the consequences of the silencing of PDS on oviposition behaviour by Pieris rapae butterflies on Arabidopsis and Brassica plants; first landing of the butterflies on Arabidopsis plants (to eliminate an effect of contact cues); first landing on Arabidopsis plants enclosed in containers (to eliminate an effect of volatiles); and caterpillar growth on Arabidopsis plants. Our results show unambiguously that P. rapae has an innate ability to visually discriminate between green and variegated green-whitish plants. Caterpillar growth was significantly lower on PDS-silenced than on empty vector control plants. This study presents the first analysis of PDS function in the interaction with an herbivorous insect. We conclude that virus-induced gene silencing is a powerful tool for investigating insect-plant interactions in model and nonmodel plants.

  4. Influences of climate on aflatoxin producing fungi and aflatoxin contamination.

    PubMed

    Cotty, Peter J; Jaime-Garcia, Ramon

    2007-10-20

    Aflatoxins are potent mycotoxins that cause developmental and immune system suppression, cancer, and death. As a result of regulations intended to reduce human exposure, crop contamination with aflatoxins causes significant economic loss for producers, marketers, and processors of diverse susceptible crops. Aflatoxin contamination occurs when specific fungi in the genus Aspergillus infect crops. Many industries frequently affected by aflatoxin contamination know from experience and anecdote that fluctuations in climate impact the extent of contamination. Climate influences contamination, in part, by direct effects on the causative fungi. As climate shifts, so do the complex communities of aflatoxin-producing fungi. This includes changes in the quantity of aflatoxin-producers in the environment and alterations to fungal community structure. Fluctuations in climate also influence predisposition of hosts to contamination by altering crop development and by affecting insects that create wounds on which aflatoxin-producers proliferate. Aflatoxin contamination is prevalent both in warm humid climates and in irrigated hot deserts. In temperate regions, contamination may be severe during drought. The contamination process is frequently broken down into two phases with the first phase occurring on the developing crop and the second phase affecting the crop after maturation. Rain and temperature influence the phases differently with dry, hot conditions favoring the first and warm, wet conditions favoring the second. Contamination varies with climate both temporally and spatially. Geostatistics and multiple regression analyses have shed light on influences of weather on contamination. Geostatistical analyses have been used to identify recurrent contamination patterns and to match these with environmental variables. In the process environmental conditions with the greatest impact on contamination are identified. Likewise, multiple regression analyses allow ranking of

  5. Systems Biology Approaches to Dissecting Plant Cell Wall Biosynthesis Genes in Poplus (JGI Seventh Annual User Meeting 2012: Genomics of Energy and Environment)

    ScienceCinema

    Glass, N Louise [UC Berkeley

    2016-07-12

    N. Louise Glass from the University of California, Berkeley, presents a talk titled "Systems Biology Approaches to Dissecting Plant Cell Wall Biosynthesis Genes in Poplus" at the JGI 7th Annual Users Meeting: Genomics of Energy & Environment Meeting on March 22, 2012 in Walnut Creek, California.

  6. Development of Methods for Determination of Aflatoxins.

    PubMed

    Xie, Lijuan; Chen, Min; Ying, Yibin

    2016-12-09

    Aflatoxins can cause damage to the health of humans and animals. Several institutions around the world have established regulations to limit the levels of aflatoxins in food, and numerous analytical methods have been extensively developed for aflatoxin determination. This review covers the currently used analytical methods for the determination of aflatoxins in different food matrices, which includes sampling and sample preparation, sample pretreatment methods including extraction methods and purification methods of aflatoxin extracts, separation and determination methods. Validation for analysis of aflatoxins and safety considerations and precautions when doing the experiments are also discussed.

  7. Effects of Exogenous Salicylic Acid on Ganoderic Acid Biosynthesis and the Expression of Key Genes in the Ganoderic Acid Biosynthesis Pathway in the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

    PubMed

    Cao, Peng-Fei; Wu, Chen-Gao; Dang, Zhi-Hao; Shi, Liang; Jiang, Ai-Liang; Ren, Ang; Zhao, Ming-Wen

    2017-01-01

    We demonstrate herein that salicylic acid (SA) can enhance ganoderic acid (GA) accumulation in the lingzhi or reishi medicinal mushroom Ganoderma lucidum. Following treatment with different concentrations of SA, the GA content was increased 22.72% to 43.04% compared with the control group. When the fungi were treated with 200 μmol/L SA at different times, the GA content was improved 10.21% to 35.24% compared with the control group. By choosing the optimum point based on response surface methodology, the GA content could be increased up to 229.03 μg/100 mg, which was improved 66.38% compared with the control group. When the fungi were treated with 200 μmol/L SA, the transcription levels of key genes in the GA biosynthesis pathway-squalene (SQ) synthase (sqs), lanosterol (Lano; osc), and hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr)-were improved 119.6-, 3.2-, and 4.2-fold, respectively. In addition, following treatment with 100 μmol/L SA, the levels of Lano and SQ, which are intermediate metabolites of GA biosynthesis, were increased 2.8- and 1.4-fold, respectively. These results indicate that SA can regulate the expression of genes related to GA biosynthesis and increases the metabolic levels of Lano and SQ, thereby resulting in the accumulation of GA.

  8. The miR-33 gene is identified in a marine teleost: a potential role in regulation of LC-PUFA biosynthesis in Siganus canaliculatus

    PubMed Central

    Zhang, Qinghao; You, Cuihong; Wang, Shuqi; Dong, Yewei; Monroig, Óscar; Tocher, Douglas R.; Li, Yuanyou

    2016-01-01

    As the first marine teleost demonstrated to have the ability to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, rabbitfish Siganus canaliculatus provides a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. Here the potential roles of miR-33 in such regulation were investigated. The miR-33 gene was identified within intron 16 of the gene encoding sterol regulatory element-binding protein 1 (Srebp1), an activator of LC-PUFA biosynthesis. Expression of miR-33 in rabbitfish tissues correlated with that of srebp1, while its expression in liver was highly responsive to ambient salinities and PUFA components, factors affecting LC-PUFA biosynthesis. Srebp1 activation promoted the expression of Δ4 and Δ6 Δ5 fatty acyl desaturases (Fad), key enzymes for LC-PUFA biosynthesis, accompanied by elevated miR-33 abundance in rabbitfish hepatocytes. miR-33 overexpression induced the expression of the two fad, but suppressed that of insulin-induced gene 1 (insig1), which encodes a repressor blocking Srebp proteolytic activation and has targeting sites of miR-33. These results indicated that miR-33, cooperating with Srebp1, may be involved in regulation of LC-PUFA biosynthesis by facilitating fad expression, probably through targeting insig1. To our knowledge, this is the first report of the participation of miR-33 in LC-PUFA biosynthesis in vertebrates. PMID:27640649

  9. A Novel Two-Gene Requirement for the Octanoyltransfer Reaction of Bacillus subtilis Lipoic Acid Biosynthesis

    PubMed Central

    Martin, Natalia; Christensen, Quin H.; Mansilla, María C.; Cronan, John E.; de Mendoza, Diego

    2011-01-01

    SUMMARY The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM, and ywfL which we have renamed lplJ, lipM and lipL, respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to E. coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulfur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilis ΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coli ΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion paper (Christensen et al., 2011) provide evidence that LipM and LipL catalyze sequential reactions in a novel pathway for lipoic acid biosynthesis. PMID:21338420

  10. Novel Aeromonas hydrophila PPD134/91 Genes Involved in O-Antigen and Capsule Biosynthesis

    PubMed Central

    Zhang, Y. L.; Arakawa, E.; Leung, K. Y.

    2002-01-01

    The sequences of the O-antigen and capsule gene clusters of the virulent Aeromonas hydrophila strain PPD134/91 were determined. The O-antigen gene cluster is 17,296 bp long and comprises 17 genes. Seven pathway genes for the synthesis of rhamnose and mannose, six transferase genes, one O unit flippase gene, and one O-antigen chain length determinant gene were identified by amino acid sequence similarity. PCR and Southern blot analysis were performed to survey the distribution of these 17 genes among 11 A. hydrophila strains of different serotypes. A. hydrophila PPD134/91 might belong to serotype O:18, as represented by JCM3980; it contained all the same O-antigen genes as JCM3980 (97 to 100% similarity at the DNA and amino acid levels). The capsule gene cluster of A. hydrophila PPD134/91 is 17,562 bp long and includes 13 genes, which were assembled into three distinct regions similar to those of the group II capsule gene cluster of Escherichia coli and other bacteria. Regions I and III contained four and two capsule transport genes, respectively. Region II had five genes which were highly similar to capsule synthesis pathway genes found in other bacteria. Both the purified O-antigen and capsular polysaccharides increased the ability of the avirulent A. hydrophila strain PPD35/85 to survive in naïve tilapia serum. However, the purified surface polysaccharides had no inhibitory effect on the adhesion of A. hydrophila PPD134/91 to carp epithelial cells. PMID:11953367

  11. The master transcription factor Spo0A is required for poly(3-hydroxybutyrate) (PHB) accumulation and expression of genes involved in PHB biosynthesis in Bacillus thuringiensis.

    PubMed

    Chen, Hui-Ju; Tsai, Teng-Kuan; Pan, Shih-Chuan; Lin, Jer-Sheng; Tseng, Chi-Ling; Shaw, Gwo-Chyuan

    2010-03-01

    Bacillus thuringiensis is a gram-positive spore-forming bacterium that can accumulate poly(3-hydroxybutyrate) (PHB) as a carbon and energy storage substance in response to nutritional stress. The regulatory mechanism for PHB biosynthesis in B. thuringiensis and diverse Bacillus species is still poorly understood. We now report that disruption of the sigH gene or the gene encoding the master sporulation transcription factor Spo0A severely impaired PHB accumulation in B. thuringiensis. Complementation of the spo0A mutation with the spo0A gene restored PHB accumulation. We have found that the requirement of Spo0A for PHB accumulation is independent of the transition state regulator AbrB and of loss of sporulation ability. We also show that Spo0A is required for the expression of three genes involved in PHB biosynthesis. These findings have uncovered a new role of Spo0A in the regulation of stationary-phase-associated cellular events.

  12. Present and future directions of translational research on aflatoxin and hepatocellular carcinoma. A review.

    PubMed

    Wogan, Gerald N; Kensler, Thomas W; Groopman, John D

    2012-01-01

    The aflatoxins were discovered in toxic peanut meal causing "turkey X" disease, which killed large numbers of turkey poults, ducklings and chicks in the UK in the early 1960s. Extracts of toxic feed induced the symptoms in experimental animals, and purified metabolites with properties identical to aflatoxins B(1) and G(1) (AFB(1) and AFG(1)) were isolated from Aspergillus flavus cultures. Structure elucidation of aflatoxin B(1) was accomplished and confirmed by total synthesis in 1963. AFB(1) is a potent liver carcinogen in rodents, non-human primates, fish and birds, operating through a genotoxic mechanism involving metabolic activation to an epoxide, formation of DNA adducts and, in humans, modification of the p53 gene. Aflatoxins are unique among environmental carcinogens, in that elucidation of their mechanisms of action combined with molecular epidemiology provides a foundation for quantitative risk assessment; extensive evidence confirms that contamination of the food supply by AFB(1) puts an exposed population at increased risk of developing hepatocellular carcinoma (HCC). Molecular biomarkers to quantify aflatoxin exposure in individuals were essential to link aflatoxin exposure with liver cancer risk. Biomarkers were validated in populations with high HCC incidence in China and The Gambia, West Africa; urinary AFB(1)-N (7)-Guanine excretion was linearly related to aflatoxin intake, and levels of aflatoxin-serum albumin adducts also reflected aflatoxin intake. Two major cohort studies employing aflatoxin biomarkers identified their causative role in HCC etiology. Results of a study in Shanghai men strongly support a causal relationship between HCC risk and the presence of biomarkers for aflatoxin and HBV infection, and also show that the two risk factors act synergistically. Subsequent cohort studies in Taiwan confirm these results. IARC classified aflatoxin as a Group 1 human carcinogen in 1993, based on sufficient evidence in humans and experimental

  13. Characterization and Transcriptional Profile of Genes Involved in Glycoalkaloid Biosynthesis in New Varieties of Solanum tuberosum L.

    PubMed

    Mariot, Roberta Fogliatto; de Oliveira, Luisa Abruzzi; Voorhuijzen, Marleen M; Staats, Martijn; Hutten, Ronald C B; van Dijk, Jeroen P; Kok, Esther J; Frazzon, Jeverson

    2016-02-03

    Before commercial release, new potato (Solanum tuberosum) varieties must be evaluated for content of toxic compounds such as glycoalkaloids (GAs), which are potent poisons. GA biosynthesis proceeds via the cholesterol pathway to α-chaconine and α-solanine. The goal of this study was to evaluate the relationship between total glycoalkaloid (TGA) content and the expression of GAME, SGT1, and SGT3 genes in potato tubers. TGA content was measured by HPLC-MS, and reverse transcription quantitative polymerase chain reactions were performed to determine the relative expression of GAME, SGT1, and SGT3 genes. We searched for cis-elements of the transcription start site using the PlantPAN database. There was a relationship between TGA content and the relative expression of GAME, SGT1, and SGT3 genes in potato tubers. Putative promoter regions showed the presence of several cis-elements related to biotic and abiotic stresses and light. These findings provide an important step toward understanding TGA regulation and variation in potato tubers.

  14. Genome-wide gene regulation of biosynthesis and energy generation by a novel transcriptional repressor in Geobacter species.

    PubMed

    Ueki, Toshiyuki; Lovley, Derek R

    2010-01-01

    Geobacter species play important roles in bioremediation of contaminated environments and in electricity production from waste organic matter in microbial fuel cells. To better understand physiology of Geobacter species, expression and function of citrate synthase, a key enzyme in the TCA cycle that is important for organic acid oxidation in Geobacter species, was investigated. Geobacter sulfurreducens did not require citrate synthase for growth with hydrogen as the electron donor and fumarate as the electron acceptor. Expression of the citrate synthase gene, gltA, was repressed by a transcription factor under this growth condition. Functional and comparative genomics approaches, coupled with genetic and biochemical assays, identified a novel transcription factor termed HgtR that acts as a repressor for gltA. Further analysis revealed that HgtR is a global regulator for genes involved in biosynthesis and energy generation in Geobacter species. The hgtR gene was essential for growth with hydrogen, during which hgtR expression was induced. These findings provide important new insights into the mechanisms by which Geobacter species regulate their central metabolism under different environmental conditions.

  15. Enhancing the Stress Tolerance and Virulence of an Entomopathogen by Metabolic Engineering of Dihydroxynaphthalene Melanin Biosynthesis Genes ▿ †

    PubMed Central

    Tseng, Min N.; Chung, Pei C.; Tzean, Shean S.

    2011-01-01

    Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as UV radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN) melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase genes) cloned from Alternaria alternata were transformed into the amelanotic entomopathogenic fungus Metarhizium anisopliae via Agrobacterium-mediated transformation. Melanin expression in the transformant of M. anisopliae was verified by spectrophotometric methods, liquid chromatography/mass spectrometry (LC/MS), and confocal microscopy. The transformant, especially under stresses, showed notably enhanced antistress capacity and virulence, in terms of germination and survival rate, infectivity, and reduced median time to death (LT50) in killing diamondback moth (Plutella xylostella) larvae compared with the wild type. The possible mechanisms in enhancing the stress tolerance and virulence, and the significance and potential for engineering melanin biosynthesis genes in other biocontrol agents and crops to improve antistress fitness are discussed. PMID:21571888

  16. Genome-wide gene regulation of biosynthesis and energy generation by a novel transcriptional repressor in Geobacter species

    PubMed Central

    Ueki, Toshiyuki; Lovley, Derek R.

    2010-01-01

    Geobacter species play important roles in bioremediation of contaminated environments and in electricity production from waste organic matter in microbial fuel cells. To better understand physiology of Geobacter species, expression and function of citrate synthase, a key enzyme in the TCA cycle that is important for organic acid oxidation in Geobacter species, was investigated. Geobacter sulfurreducens did not require citrate synthase for growth with hydrogen as the electron donor and fumarate as the electron acceptor. Expression of the citrate synthase gene, gltA, was repressed by a transcription factor under this growth condition. Functional and comparative genomics approaches, coupled with genetic and biochemical assays, identified a novel transcription factor termed HgtR that acts as a repressor for gltA. Further analysis revealed that HgtR is a global regulator for genes involved in biosynthesis and energy generation in Geobacter species. The hgtR gene was essential for growth with hydrogen, during which hgtR expression was induced. These findings provide important new insights into the mechanisms by which Geobacter species regulate their central metabolism under different environmental conditions. PMID:19939938

  17. Bagging treatment influences production of C6 aldehydes and biosynthesis-related gene expression in peach fruit skin.

    PubMed

    Shen, Ji-Yuan; Wu, Lei; Liu, Hong-Ru; Zhang, Bo; Yin, Xue-Ren; Ge, Yi-Qiang; Chen, Kun-Song

    2014-08-29

    Bagging is a useful method to improve fruit quality by altering its exposure to light, whereas its effect on fruit volatiles production is inconsistent, and the genes responsible for the observed changes remain unknown. In the present study, single-layer yellow paper bags were used to study the effects of bagging treatment on the formation of C6 aldehydes in peach fruit (Prunus persica L. Batsch, cv. Yulu) over two succeeding seasons. Higher concentrations of n-hexanal and (E)-2-hexenal, which are characteristic aroma volatiles of peach fruit, were induced by bagging treatment. After bagging treatment, peach fruit had significantly higher LOX and HPL enzyme activities, accompanying increased contents of C6 aldehydes. The gene expression data obtained through real-time PCR showed that no consistent significant differences in transcript levels of LOX genes were observed over the two seasons, but significantly up-regulated expression was found for PpHPL1 after bagging treatment In addition, bagging-treated fruit produced more (E)-2-hexenal and had higher expression levels of PpHPL1 during postharvest ripening at room temperature. The regulatory role of the LOX-HPL pathway on the biosynthesis of n-hexanal and (E)-2-hexenal in response to bagging treatment during peach fruit development is discussed in the text.

  18. [Applications of molecular biology techniques for the control of aflatoxin contamination].

    PubMed

    Sanchis, V

    1993-02-01

    Aflatoxins are mycotoxins produced by species of Aspergillus flavus group. These toxins have received increased attention from the food industry and the general public because they shown a high toxicity against humans and animal. Different methods are applying to control the aflatoxin contamination. But these conventional methods do not seem to resolve the problem. So, new methods using techniques in biotechnology are now being developed: a) Inhibit the biosynthetic and secretory process responsible for aflatoxin contamination. b) Using biocompetitive agents that replace aflatoxigenic strains with non aflatoxigenic strains in the field. c) Using genetic engineering techniques to incorporate antifungal genes into specific plant species.

  19. Exposure measurement of aflatoxins and aflatoxin metabolites in human body fluids. A short review.

    PubMed

    Leong, Yin-Hui; Latiff, Aishah A; Ahmad, Nurul Izzah; Rosma, Ahmad

    2012-05-01

    Aflatoxins are highly toxic secondary fungal metabolites mainly produced by Aspergillus flavus and A. parasiticus. Human exposure to aflatoxins may result directly from ingestion of contaminated foods, or indirectly from consumption of foods from animals previously exposed to aflatoxins in feeds. This paper focuses on exposure measurement of aflatoxins and aflatoxin metabolites in various human body fluids. Research on different metabolites present in blood, urine, breast milk, and other human fluids or tissues including their detection techniques is reviewed. The association between dietary intake of aflatoxins and biomarker measurement is also highlighted. Finally, aspects related to the differences between aflatoxin determination in food versus the biomarker approach are discussed.

  20. 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

  1. 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.

  2. Overexpression of MusaMYB31, a R2R3 type MYB transcription factor gene indicate its role as a negative regulator of lignin biosynthesis in banana.

    PubMed

    Tak, Himanshu; Negi, Sanjana; 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.

  3. Localization of strawberry (Fragaria x ananassa) and Methylobacterium extorquens genes of strawberry flavor biosynthesis in strawberry tissue by in situ hybridization.

    PubMed

    Nasopoulou, Constantina; Pohjanen, Johanna; Koskimäki, Janne J; Zabetakis, Ioannis; Pirttilä, Anna Maria

    2014-08-15

    Strawberry flavor is one of the most popular fruit flavors worldwide, with numerous applications in the food industry. In addition, the biosynthetic origin of the most important strawberry flavor components, such as 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF), is a challenging research area. DMHF's precursor, 2-hydroxy-propanal (or lactaldehyde), is biosynthesized by the endophytic bacterium Methylobacterium extorquens (M. extorquens). In particular, the alcohol dehydrogenase (ADH) enzymes of M. extorquens are involved in the biogenesis of DMHF precursors since they have the capacity to oxidize the strawberry-derived 1,2-propanediol to lactaldehyde. In this study, the expression of the endophytic ADH and the plant DMHF biosynthesis genes was examined in the tissues of raw and ripe strawberry receptacles by in situ hybridization. The presence of endophytic bacteria was studied in the same tissues by probes targeting bacterial 16S ribosomal ribonucleic acid. Hybridization signals of probes specific for endophytic ADH and plant DMHF biosynthesis genes, as well as bacteria-specific probes, were detected in the same locations. The probes were localized near the plasma membranes or intercellular spaces of cortical and vascular tissues of the receptacle, and intracellularly in the tissues of achenes. By localizing the expression of the endophytic methanol ADH and plant DMHF biosynthesis genes to the same tissues, we have reinforced our original hypothesis that an intimate symbiotic relationship between strawberry and endophytic cells exists and leads to the biosynthesis of DMHF.

  4. A differentially regulated AP2/ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

    PubMed

    Paul, Priyanka; Singh, Sanjay K; Patra, Barunava; Sui, Xueyi; Pattanaik, Sitakanta; Yuan, Ling

    2017-02-01

    Catharanthus roseus produces bioactive terpenoid indole alkaloids (TIAs), including the chemotherapeutics, vincristine and vinblastine. Transcriptional regulation of TIA biosynthesis is not fully understood. The jasmonic acid (JA)-responsive AP2/ERF transcription factor (TF), ORCA3, and its regulator, CrMYC2, play key roles in TIA biosynthesis. ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and 5. Here, we report that (1) the ORCA gene cluster is differentially regulated; (2) ORCA4, while overlapping functionally with ORCA3, modulates an additional set of TIA genes. Unlike ORCA3, ORCA4 overexpression resulted in dramatic increase of TIA accumulation in C. roseus hairy roots. In addition, CrMYC2 is capable of activating ORCA3 and co-regulating TIA pathway genes concomitantly with ORCA3. The ORCA gene cluster and CrMYC2 act downstream of a MAP kinase cascade that includes a previously uncharacterized MAP kinase kinase, CrMAPKK1. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulated TIA pathways genes and increased TIA accumulation. This work provides detailed characterization of a TF gene cluster and advances our understanding of the transcriptional and post-translational regulatory mechanisms that govern TIA biosynthesis in C. roseus.

  5. Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp. ATCC 31749 under simulated dissolved oxygen gradients conditions.

    PubMed

    Zhang, Hong-Tao; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Wu, Jian-Rong; Yu, Xiao-Bin; Jiang, Yun; Lin, Chi-Chung

    2011-07-01

    Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

  6. Large number of putative chemoreception and pheromone biosynthesis genes revealed by analyzing transcriptome from ovipositor-pheromone glands of Chilo suppressalis.

    PubMed

    Xia, Yi-Han; Zhang, Ya-Nan; Hou, Xiao-Qing; Li, Fei; Dong, Shuang-Lin

    2015-01-20

    The chemoreception role of moth ovipositor has long been suggested, but its molecular mechanism is mostly unknown. By transcriptomic analysis of the female ovipositor-pheromone glands (OV-PG) of Chilo suppressalis, we obtained 31 putative chemoreception genes (9 OBPs, 10 CSPs, 2 ORs, 1 SNMP, 8 CXEs and 1 AOX), in addition to 32 genes related to sex pheromone biosynthesis (1 FAS, 6 Dess, 10 FARs, 2 ACOs, 1 ACC, 4 FATPs, 3 ACBPs and 5 ELOs). Tissue expression profiles further revealed that CsupCSP2 and CsupCSP10 were OV-PG biased, while most chemoreception genes were highly and preferably expressed in antennae. This suggests that OV-PG employs mostly the same chemoreception proteins as in antennae, although the physiological roles of these proteins might be different in OV-PG. Of the 32 pheromone biosynthesis related genes, CsupDes4, CsupDes5 and CsupFAR2 are strongly OV-PG biased, and clustered with functionally validated genes from other moths, strongly indicating their involvement in specific step of the pheromone biosynthesis. Our study for the first time identified a large number of putative chemoreception genes, and provided an important basis for exploring the chemoreception mechanisms of OV-PG in C. suppressalis, as well as other moth species.

  7. Polyketide and nonribosomal peptide retro-biosynthesis and global gene cluster matching.

    PubMed

    Dejong, Chris A; Chen, Gregory M; Li, Haoxin; Johnston, Chad W; Edwards, Mclean R; Rees, Philip N; Skinnider, Michael A; Webster, Andrew L H; Magarvey, Nathan A

    2016-12-01

    Polyketides (PKs) and nonribosomal peptides (NRPs) are profoundly important natural products, forming the foundations of many therapeutic regimes. Decades of research have revealed over 11,000 PK and NRP structures, and genome sequencing is uncovering new PK and NRP gene clusters at an unprecedented rate. However, only ∼10% of PK and NRPs are currently associated with gene clusters, and it is unclear how many of these orphan gene clusters encode previously isolated molecules. Therefore, to efficiently guide the discovery of new molecules, we must first systematically de-orphan emergent gene clusters from genomes. Here we provide to our knowledge the first comprehensive retro-biosynthetic program, generalized retro-biosynthetic assembly prediction engine (GRAPE), for PK and NRP families and introduce a computational pipeline, global alignment for natural products cheminformatics (GARLIC), to uncover how observed biosynthetic gene clusters relate to known molecules, leading to the identification of gene clusters that encode new molecules.

  8. Expression of Genes Involved in Anthocyanin Biosynthesis in Relation to Anthocyanin, Proanthocyanidin, and Flavonol Levels during Bilberry Fruit Development1

    PubMed Central

    Jaakola, Laura; Määttä, Kaisu; Pirttilä, Anna Maria; Törrönen, Riitta; Kärenlampi, Sirpa; Hohtola, Anja

    2002-01-01

    The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented. PMID:12376640

  9. Botrydial and botcinins produced by Botrytis cinerea regulate the expression of Trichoderma arundinaceum genes involved in trichothecene biosynthesis.

    PubMed

    Malmierca, Mónica G; Izquierdo-Bueno, Inmaculada; Mccormick, Susan P; Cardoza, Rosa E; Alexander, Nancy J; Moraga, Javier; Gomes, Eriston V; Proctor, Robert H; Collado, Isidro G; Monte, Enrique; Gutiérrez, Santiago

    2016-09-01

    Trichoderma arundinaceum IBT 40837 (Ta37) and Botrytis cinerea produce the sesquiterpenes harzianum A (HA) and botrydial (BOT), respectively, and also the polyketides aspinolides and botcinins (Botcs), respectively. We analysed the role of BOT and Botcs in the Ta37-B. cinerea interaction, including the transcriptomic changes in the genes involved in HA (tri) and ergosterol biosynthesis, as well as changes in the level of HA and squalene-ergosterol. We found that, when confronted with B. cinerea, the tri biosynthetic genes were up-regulated in all dual cultures analysed, but at higher levels when Ta37 was confronted with the BOT non-producer mutant bcbot2Δ. The production of HA was also higher in the interaction area with this mutant. In Ta37-bcbot2Δ confrontation experiments, the expression of the hmgR gene, encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the first enzyme of the terpene biosynthetic pathway, was also up-regulated, resulting in an increase in squalene production compared with the confrontation with B. cinerea B05.10. Botcs had an up-regulatory effect on the tri biosynthetic genes, with BotcA having a stronger effect than BotcB. The results indicate that the interaction between Ta37 and B. cinerea exerts a stimulatory effect on the expression of the tri biosynthetic genes, which, in the interaction zone, can be attenuated by BOT produced by B. cinerea B05.10. The present work provides evidence for a metabolic dialogue between T. arundinaceum and B. cinerea that is mediated by sesquiterpenes and polyketides, and that affects the outcome of the interaction of these fungi with each other and their environment.

  10. Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin, proanthocyanidin, and flavonol levels during bilberry fruit development.

    PubMed

    Jaakola, Laura; Määttä, Kaisu; Pirttilä, Anna Maria; Törrönen, Riitta; Kärenlampi, Sirpa; Hohtola, Anja

    2002-10-01

    The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.

  11. Multiple Δ11-desaturase genes selectively used for sex pheromone biosynthesis are conserved in Ostrinia moth genomes.

    PubMed

    Fujii, Takeshi; Yasukochi, Yuji; Rong, Yu; Matsuo, Takashi; Ishikawa, Yukio

    2015-06-01

    Regulation of the expression of fatty acyl-CoA desaturases, which introduce a double bond into the fatty acid moiety of the substrate, is crucial for the production of species-specific sex pheromones in moths. In Ostrinia moths, two distinct Δ11-desaturases and a Δ14-desaturase are known to be selectively used in the biosynthesis of sex pheromones. Of the two Δ11-desaturases, one identified from Ostrinia nubilalis and Ostrinia scapulalis, Z/EΔ11, forms the Z and E isomers of a double bond at position 11, whereas the other identified from Ostrinia latipennis, LATPG1(=EΔ11), exclusively forms an E double bond at position 11. Since the retroposon(ezi)-fused, non-functional Δ11-desaturase gene, ezi-Δ11α, in the genomes of O. nubilalis and O. furnacalis was previously suggested to be an orthologue of latpg1, we here explored Z/EΔ11 orthologues in the genome of O. latipennis. We newly identified two Δ11-desaturase genes, latpg2 and latpg3, which were orthologous to ezi-Δ11β and Z/EΔ11, respectively. We found that an ezi-like element was integrated in intron 1 of latpg1, and confirmed that only latpg1 was expressed in the pheromone gland of O. latipennis. Thus, at least three Δ11-desaturase genes are present in the genome of O. latipennis, and latpg1 is selectively transcribed in the pheromone gland of this moth. The non-functionality of ezi-inserted desaturase genes in O. nubilalis and O. furnacalis may not be a direct consequence of the insertion of an ezi- or ezi-like element into the gene.

  12. Activation of Cryptic hop Genes from Streptomyces peucetius ATCC 27952 Involved in Hopanoid Biosynthesis.

    PubMed

    Ghimire, Gopal Prasad; Koirala, Niranjan; Sohng, Jae Kyung

    2015-05-01

    Genes encoding enzymes with sequence similarity to hopanoids biosynthetic enzymes of other organisms were cloned from the hopanoid (hop) gene cluster of Streptomyces peucetius ATCC 27952 and transformed into Streptomyces venezuelae YJ028. The cloned fragments contained four genes, all transcribed in one direction. These genes encode polypeptides that resemble polyprenyl diphosphate synthase (hopD), squalene-phytoene synthases (hopAB), and squalenehopene cyclase (hopE). These enzymes are sufficient for the formation of the pentacyclic triterpenoid lipid, hopene. The formation of hopene was verified by gas chromatography/ mass spectrometry.

  13. Cytotoxicity of aflatoxin on red blood corpuscles

    SciTech Connect

    Verma, R.J.; Raval, P.J. )

    1991-09-01

    The exact mechanism of aflatoxin action is not clearly understood. In the present investigation the authors report morphological aberrations and increased rate of hemolysis caused by aflatoxins in vitro.

  14. Reduction of aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in interaction with Streptomyces.

    PubMed

    Verheecke, C; Liboz, T; Anson, P; Diaz, R; Mathieu, F

    2015-05-01

    The aim of this study is to investigate aflatoxin gene expression during Streptomyces-Aspergillus interaction. Aflatoxins are carcinogenic compounds produced mainly by Aspergillus flavus and Aspergillus parasiticus. A previous study has shown that Streptomyces-A. flavus interaction can reduce aflatoxin content in vitro. Here, we first validated this same effect in the interaction with A. parasiticus. Moreover, we showed that growth reduction and aflatoxin content were correlated in A. parasiticus but not in A. flavus. Secondly, we investigated the mechanisms of action by reverse-transcriptase quantitative PCR. As microbial interaction can lead to variations in expression of household genes, the most stable [act1, βtub (and cox5 for A. parasiticus)] were chosen using geNorm software. To shed light on the mechanisms involved, we studied during the interaction the expression of five genes (aflD, aflM, aflP, aflR and aflS). Overall, the results of aflatoxin gene expression showed that Streptomyces repressed gene expression to a greater level in A. parasiticus than in A. flavus. Expression of aflR and aflS was generally repressed in both Aspergillus species. Expression of aflM was repressed and was correlated with aflatoxin B1 content. The results suggest that aflM expression could be a potential aflatoxin indicator in Streptomyces species interactions. Therefore, we demonstrate that Streptomyces can reduce aflatoxin production by both Aspergillus species and that this effect can be correlated with the repression of aflM expression.

  15. Impact of Oxidative Stress on Ascorbate Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a GDP-l-galactose Phosphorylase*

    PubMed Central

    Urzica, Eugen I.; Adler, Lital N.; Page, M. Dudley; Linster, Carole L.; Arbing, Mark A.; Casero, David; Pellegrini, Matteo; Merchant, Sabeeha S.; Clarke, Steven G.

    2012-01-01

    The l-galactose (Smirnoff-Wheeler) pathway represents the major route to l-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-l-galactose phosphorylases converting GDP-l-galactose to l-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of l-ascorbate. Here we report that the l-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the l-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-l-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and l-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the l-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells. PMID:22393048

  16. Differential expression of structural genes for the late phase of phytic acid biosynthesis in developing seeds of wheat (Triticum aestivum L.).

    PubMed

    Bhati, Kaushal Kumar; Aggarwal, Sipla; Sharma, Shivani; Mantri, Shrikant; Singh, Sudhir P; Bhalla, Sherry; Kaur, Jagdeep; Tiwari, Siddharth; Roy, Joy K; Tuli, Rakesh; Pandey, Ajay K

    2014-07-01

    In cereals, phytic acid (PA) or inositol hexakisphosphate (IP6) is a well-known phosphate storage compound as well as major chelator of important micronutrients (iron, zinc, calcium, etc.). Genes involved in the late phases of PA biosynthesis pathway are known in crops like maize, soybeans and barley but none have been reported from wheat. Our in silico analysis identified six wheat genes that might be involved in the biosynthesis of inositol phosphates. Four of the genes were inositol tetraphosphate kinases (TaITPK1, TaITPK2, TaITPK3, and TaITPK4), and the other two genes encode for inositol triphosphate kinase (TaIPK2) and inositol pentakisphosphate kinase (TaIPK1). Additionally, we identified a homolog of Zmlpa-1, an ABCC subclass multidrug resistance-associated transporter protein (TaMRP3) that is putatively involved in PA transport. Analyses of the mRNA expression levels of these seven genes showed that they are differentially expressed during seed development, and that some are preferentially expressed in aleurone tissue. These results suggest selective roles during PA biosynthesis, and that both lipid-independent and -dependent pathways are active in developing wheat grains. TaIPK1 and TaMRP3 were able to complement the yeast ScΔipk1 and ScΔycf1 mutants, respectively, providing evidence that the wheat genes have the expected biochemical functions. This is the first comprehensive study of the wheat genes involved in the late phase of PA biosynthesis. Knowledge generated from these studies could be utilized to develop strategies for generating low phyate wheat.

  17. Cloning and characterization of the Streptomyces peucetius dnmZUV genes encoding three enzymes required for biosynthesis of the daunorubicin precursor thymidine diphospho-L-daunosamine.

    PubMed Central

    Otten, S L; Gallo, M A; Madduri, K; Liu, X; Hutchinson, C R

    1997-01-01

    Characterization of the dnmZ, dnmU, and dnmV genes from the daunorubicin-producer Streptomyces peucetius by DNA sequence analysis indicated that these genes encode a protein of unknown function plus a putative thymidine diphospho-4-keto-6-deoxyglucose-3(5)-epimerase and thymidine diphospho-4-ketodeoxyhexulose reductase, respectively. Inactivation of each of the three genes by gene disruption and replacement in the wild-type strain demonstrated that all of them are required for daunosamine biosynthesis. PMID:9209071

  18. Cloning and functional analysis of a phosphopantetheinyl transferase superfamily gene associated with jadomycin biosynthesis in Streptomyces venezuelae ISP5230.

    PubMed

    Wang, L; McVey, J; Vining, L C

    2001-06-01

    Sequence analysis of a XhoI/SacI fragment of chromosomal DNA downstream of jadL in the Streptomyces venezuelae ISP5230 gene cluster for jadomycin biosynthesis detected a partial ORF similar in its deduced amino acid sequence to the hetI product involved in synthesizing a regulator of heterocyst spacing in ANABAENA: By probing a phage library of S. venezuelae DNA with the XhoI/SacI fragment, the authors identified and isolated a hybridizing clone. The nucleotide sequence of its DNA contained three complete ORFs (jadM, N and X) and one incomplete ORF (jadO). The jadM ORF lay immediately downstream of, and partially overlapped, jadL. It contained 786 nucleotides encoding an amino acid sequence like those of enzymes in the phosphopantetheinyl transferase family. The jadN ORF contained 1794 nucleotides and encoded an amino acid sequence resembling acyl-CoA decarboxylases, thus suggesting a role in polyketide condensation reactions. The jadX ORF was not identified, but the partial jadO showed marked similarities in its deduced amino acid sequence to NDP-hexose-2,3-dehydratases, indicating a role in forming the sugar component of jadomycin B. Expression of jadM in Escherichia coli and examination of the product by SDS-PAGE established that the ORF encoded a 29.1 kDa protein, corresponding in size to the 262 amino acid polypeptide deduced from the jadM sequence. Evidence from a Northern hybridization indicated that jadM expression is correlated with jadomycin B synthesis. Cultures of S. venezuelae ISP5230 disrupted in jadM produced only 2-5% of the wild-type titre of jadomycin B, but grew well and produced chloramphenicol normally. The authors conclude that jadM encodes a holo-ACP synthase needed primarily for jadomycin B biosynthesis.

  19. Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes.

    PubMed

    Brückner, Kathleen; Božić, Dragana; Manzano, David; Papaefthimiou, Dimitra; Pateraki, Irini; Scheler, Ulschan; Ferrer, Albert; de Vos, Ric C H; Kanellis, Angelos K; Tissier, Alain

    2014-05-01

    Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split.

  20. Aflatoxin in Tunisian aleppo pine nuts.

    PubMed

    Boutrif, E; Jemmali, M; Pohland, A E; Campbell, A D

    1977-05-01

    Twenty-six of 50 Aleppo pine nuts samples collected throughout Tunisia showed relatively high levels of contamination by aflatoxin. Some samples contained as much as 2000 ppb aflatoxin B1, and very few contained less than 100 ppb. Total aflatoxins as high as 7550 ppb were found. A traditional pudding, widely consumed in Tunisia, which was prepared from contaminated nuts still contained more than 80% of the aflatoxin originally present in the nuts.

  1. Biosynthesis of Aedes aegypti lipophorin and gene expression of its apolipoproteins.

    PubMed

    van Heusden, M C; Thompson, F; Dennis, J

    1998-10-01

    The biosynthesis of lipophorin of the yellow fever mosquito, Aedes aegypti, was investigated. Fat bodies were incubated in vitro with radiolabeled methionine and cysteine, and radiolabeled proteins secreted into the medium were analyzed by density gradient ultracentrifugation, SDS-PAGE and fluorography. Lipophorin was synthesized in the fat body and secreted into the medium. Its density was 1.114 g/ml, similar to that of lipophorin circulating in hemolymph. Three peptides of a tryptic digest of apolipophorin II were sequenced and degenerate oligonucleotide primers were designed based on the amino acid sequences. With these primers, a cDNA product of 1.2 kb was amplified by RT-PCR using as template RNA extracted from adult female mosquitoes 24 h after ingestion of a blood meal. This cDNA was cloned, sequenced and used as a probe for Northern blot analysis, which revealed that the apoproteins of lipophorin were coded for by a single mRNA of approximately 10 kb. The expression of the apolipophorins was induced by blood feeding. From the data presented we concluded that Aedes aegypti lipophorin is synthesized in the fat body and that the expression of its apolipophorins is induced by blood feeding.

  2. Identification of a gene cluster in Klebsiella pneumoniae which includes citX, a gene required for biosynthesis of the citrate lyase prosthetic group.

    PubMed

    Schneider, Karin; Kästner, Christopher N; Meyer, Margareta; Wessel, Mirja; Dimroth, Peter; Bott, Michael

    2002-05-01

    The biosynthesis of the 2'-(5"-phosphoribosyl)-3'-dephospho-coenzyme A (CoA) prosthetic group of citrate lyase (EC 4.1.3.6), a key enzyme of citrate fermentation, proceeds via the initial formation of the precursor 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA and subsequent transfer to apo-citrate lyase with removal of pyrophosphate. In Escherichia coli, the two steps are catalyzed by CitG and CitX, respectively, and the corresponding genes are part of the citrate lyase gene cluster, citCDEFXG. In the homologous citCDEFG operon of Klebsiella pneumoniae, citX is missing. A search for K. pneumoniae citX led to the identification of a second genome region involved in citrate fermentation which comprised the citWX genes and the divergent citYZ genes. The citX gene was confirmed to encode holo-citrate lyase synthase, whereas citW was shown to encode a citrate carrier, the third one identified in this species. The citYZ genes were found to encode a two-component system consisting of the sensor kinase CitY and the response regulator CitZ. Remarkably, both proteins showed >or=40% sequence identity to the citrate-sensing CitA-CitB two-component system, which is essential for the induction of the citrate fermentation genes in K. pneumoniae. A citZ insertion mutant was able to grow anaerobically with citrate, indicating that CitZ is not essential for expression of citrate fermentation genes. CitX synthesis was induced to a basal level under anaerobic conditions, independent of citrate, CitB, and CitZ, and to maximal levels during anaerobic growth with citrate as the sole carbon source. Similar to the other citrate fermentation enzymes, CitX synthesis was apparently subject to catabolite repression.

  3. Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii.

    PubMed

    Deng, Xiaodong; Cai, Jiajia; Li, Yajun; Fei, Xiaowen

    2014-11-01

    The regulation of lipid biosynthesis is important in photosynthetic eukaryotic cells. This regulation is facilitated by the direct synthesis of fatty acids and triacylglycerol (TAG), and by other controls of the main carbon metabolic pathway. In this study, knockdown of the mRNA expression of the Chlamydomonas phosphoenolpyruvate carboxylase isoform 1 (CrPEPC1) gene by RNA interference increased TAG level by 20 % but decreased PEPC activities in the corresponding transgenic algae by 39-50 %. The decrease in CrPEPC1 expression increased the expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, CrPEPC1 over-expression decreased TAG level by 37 % and increased PEPC activities by 157-184 %. These observations suggest that the lipid content of algal cells can be controlled by regulating the CrPEPC1 gene.

  4. Characterization and expression of genes involved in the ethylene biosynthesis and signal transduction during ripening of mulberry fruit.

    PubMed

    Liu, Changying; Zhao, Aichun; Zhu, Panpan; Li, Jun; Han, Leng; Wang, Xiling; Fan, Wei; Lü, Ruihua; Wang, Chuanhong; Li, Zhengang; Lu, Cheng; Yu, Maode

    2015-01-01

    Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1-methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening.

  5. Characterization and Expression of Genes Involved in the Ethylene Biosynthesis and Signal Transduction during Ripening of Mulberry Fruit

    PubMed Central

    Liu, Changying; Zhao, Aichun; Zhu, Panpan; Li, Jun; Han, Leng; Wang, Xiling; Fan, Wei; Lü, Ruihua; Wang, Chuanhong; Li, Zhengang; Lu, Cheng; Yu, Maode

    2015-01-01

    Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1–methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening. PMID

  6. Transcriptional profiling of genes involved in ascorbic acid biosynthesis, recycling, and degradation during three leaf developmental stages in celery.

    PubMed

    Huang, Wei; Wang, Guang-Long; Li, Hui; Wang, Feng; Xu, Zhi-Sheng; Xiong, Ai-Sheng

    2016-12-01

    Ascorbic acid (AsA) is an important nutrient in the human body and performs various healthy functions. With considerable medicinal properties, celery (Apium graveolens L.) could be a good source of AsA for human health. However, the biosynthetic, recycling, and degradation pathways of AsA in celery have yet to be characterized. To study the metabolic pathways involved in AsA, the genes involved in AsA biosynthesis, recycling, and degradation were isolated from celery, and their expression profiles and AsA levels were analyzed in the leaf blades and petioles of two celery varieties at three different growth stages. AsA levels were higher in 'Ventura' compared with 'Liuhehuangxinqin' in both tissues possibly because of different transcription levels of genes, such as L-galactose dehydrogenase (GalDH), L-galactono-1,4-lactone dehydrogenase (GalLDH), and glutathione reductase (GR). Results revealed that the D-mannose/L-galactose pathway may be the predominant pathway in celery, and the D-galacturonic acid pathway appeared to contribute largely to AsA accumulation in petioles than in leaf blades in 'Liuhehuangxinqin.' AsA contents are regulated by complex regulatory mechanisms and vary at different growth stages, tissues, and varieties in celery. The results provide novel insights into AsA metabolic pathways in leaf during celery growth and development.

  7. Expression of essential genes for biosynthesis of antimicrobial peptides of Bacillus is modulated by inactivated cells of target microorganisms.

    PubMed

    Leães, Fernanda Leal; Velho, Renata Voltolini; Caldas, Danielle Gregório Gomes; Ritter, Ana Carolina; Tsai, Siu Mui; Brandelli, Adriano

    2016-01-01

    Certain Bacillus strains are important producers of antimicrobial peptides with great potential for biological control. Antimicrobial peptide production by Bacillus amyloliquefaciens P11 was investigated in the presence of heat-inactivated cells of bacteria and fungi. B. amyloliquefaciens P11 exhibited higher antimicrobial activity in the presence of inactivated cells of Staphylococcus aureus and Aspergillus parasiticus compared to other conditions tested. Expression of essential genes related to biosynthesis of the antimicrobial peptides surfactin (sfp), iturin A (lpa-14 and ituD), subtilosin A (sboA) and fengycin (fenA) was investigated by quantitative real-time PCR (qRT-PCR). The genes lpa-14 and ituD were highly expressed in the presence of S. aureus (inactivated cells), indicating induction of iturin A production by B. amyloliquefaciens P11. The other inducing condition (inactivated cells of A. parasiticus) suppressed expression of lpa-14, but increased expression of ituD. A twofold increase in fenA expression was observed for both conditions, while strong suppression of sboA expression was observed in the presence of inactivated cells of S. aureus. An increase in antimicrobial activity was observed, indicating that synthesis of antimicrobial peptides may be induced by target microorganisms.

  8. Expression and Anthocyanin Biosynthesis-Modulating Potential of Sweet Cherry (Prunus avium L.) MYB10 and bHLH Genes

    PubMed Central

    Starkevič, Pavel; Paukštytė, Jurgita; Kazanavičiūtė, Vaiva; Denkovskienė, Erna; Stanys, Vidmantas; Bendokas, Vidmantas; Šikšnianas, Tadeušas; Ražanskienė, Aušra; Ražanskas, Raimundas

    2015-01-01

    Anthocyanins are essential contributors to fruit coloration, an important quality feature and a breed determining trait of a sweet cherry fruit. It is well established that the biosynthesis of anthocyanins is regulated by an interplay of specific transcription factors belonging to MYB and bHLH families accompanied by a WD40 protein. In this study, we isolated and analyzed PaWD40, PabHLH3, PabHLH33, and several closely related MYB10 gene variants from different cultivars of sweet cherry, analyzed their expression in fruits with different anthocyanin levels at several developmental stages, and determined their capabilities to modulate anthocyanin synthesis in leaves of two Nicotiana species. Our results indicate that transcription level of variant PaMYB10.1-1 correlates with fruit coloration, but anthocyanin synthesis in Nicotiana was induced by another variant, PaMYB10.1-3, which is moderately expressed in fruits. The analysis of two fruit-expressed bHLH genes revealed that PabHLH3 enhances MYB-induced anthocyanin synthesis, whereas PabHLH33 has strong inhibitory properties. PMID:25978735

  9. Transcriptome Analysis to Identify the Putative Biosynthesis and Transport Genes Associated with the Medicinal Components of Achyranthes bidentata Bl.

    PubMed Central

    Li, Jinting; Wang, Can; Han, Xueping; Qi, Wanzhen; Chen, Yanqiong; Wang, Taixia; Zheng, Yi; Zhao, Xiting

    2016-01-01

    Achyranthes bidentata is a popular perennial medicine herb used for 1000s of years in China to treat various diseases. Although this herb has multiple pharmaceutical purposes in China, no transcriptomic information has been reported for this species. In addition, the understanding of several key pathways and enzymes involved in the biosynthesis of oleanolic acid and ecdysterone, two pharmacologically active classes of metabolites and major chemical constituents of A. bidentata root extracts, is limited. The aim of the present study was to characterize the transcriptome profile of the roots and leaves of A. bidentata to uncover the biosynthetic and transport mechanisms of the active components. In this study, we identified 100,987 transcripts, with an average length of 1146.8 base pairs. A total of 31,634 (31.33%) unigenes were annotated, and 12,762 unigenes were mapped to 303 pathways according to the Kyoto Encyclopedia of Genes and Genomes pathway database. Moreover, we identified a total of 260 oleanolic acid and ecdysterone genes encoding biosynthetic enzymes. Furthermore, the key enzymes involved in the oleanolic acid and ecdysterone synthesis pathways were analyzed using quantitative real-time polymerase chain reaction, revealing that the roots expressed these enzymes to a greater extent than the leaves. In addition, we identified 85 ATP-binding cassette transporters, some of which might be involved in the translocation of secondary metabolites. PMID:28018396

  10. DNA Sequence and Expression Variation of Hop (Humulus lupulus) Valerophenone Synthase (VPS), a Key Gene in Bitter Acid Biosynthesis

    PubMed Central

    Castro, Consuelo B.; Whittock, Lucy D.; Whittock, Simon P.; Leggett, Grey; Koutoulis, Anthony

    2008-01-01

    Background The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Methods Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Results Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, ‘Symphony’ and ‘Ember’, which typically have high bitter acid levels. Conclusions In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development. PMID:18519445

  11. Expression and Anthocyanin Biosynthesis-Modulating Potential of Sweet Cherry (Prunus avium L.) MYB10 and bHLH Genes.

    PubMed

    Starkevič, Pavel; Paukštytė, Jurgita; Kazanavičiūtė, Vaiva; Denkovskienė, Erna; Stanys, Vidmantas; Bendokas, Vidmantas; Šikšnianas, Tadeušas; Ražanskienė, Aušra; Ražanskas, Raimundas

    2015-01-01

    Anthocyanins are essential contributors to fruit coloration, an important quality feature and a breed determining trait of a sweet cherry fruit. It is well established that the biosynthesis of anthocyanins is regulated by an interplay of specific transcription factors belonging to MYB and bHLH families accompanied by a WD40 protein. In this study, we isolated and analyzed PaWD40, PabHLH3, PabHLH33, and several closely related MYB10 gene variants from different cultivars of sweet cherry, analyzed their expression in fruits with different anthocyanin levels at several developmental stages, and determined their capabilities to modulate anthocyanin synthesis in leaves of two Nicotiana species. Our results indicate that transcription level of variant PaMYB10.1-1 correlates with fruit coloration, but anthocyanin synthesis in Nicotiana was induced by another variant, PaMYB10.1-3, which is moderately expressed in fruits. The analysis of two fruit-expressed bHLH genes revealed that PabHLH3 enhances MYB-induced anthocyanin synthesis, whereas PabHLH33 has strong inhibitory properties.

  12. Biotechnological advances for combating Aspergillus flavus and aflatoxin contamination in crops.

    PubMed

    Bhatnagar-Mathur, Pooja; Sunkara, Sowmini; Bhatnagar-Panwar, Madhurima; Waliyar, Farid; Sharma, Kiran Kumar

    2015-05-01

    Aflatoxins are toxic, carcinogenic, mutagenic, teratogenic and immunosuppressive byproducts of Aspergillus spp. that contaminate a wide range of crops such as maize, peanut, and cotton. Aflatoxin not only affects crop production but renders the produce unfit for consumption and harmful to human and livestock health, with stringent threshold limits of acceptability. In many crops, breeding for resistance is not a reliable option because of the limited availability of genotypes with durable resistance to Aspergillus. Understanding the fungal/crop/environment interactions involved in aflatoxin contamination is therefore essential in designing measures for its prevention and control. For a sustainable solution to aflatoxin contamination, research must be focused on identifying and improving knowledge of host-plant resistance factors to aflatoxin accumulation. Current advances in genetic transformation, proteomics, RNAi technology, and marker-assisted selection offer great potential in minimizing pre-harvest aflatoxin contamination in cultivated crop species. Moreover, developing effective phenotyping strategies for transgenic as well as precision breeding of resistance genes into commercial varieties is critical. While appropriate storage practices can generally minimize post-harvest aflatoxin contamination in crops, the use of biotechnology to interrupt the probability of pre-harvest infection and contamination has the potential to provide sustainable solution.

  13. Sucrose in bloom-forming cyanobacteria: loss and gain of genes involved in its biosynthesis.

    PubMed

    Kolman, María A; Salerno, Graciela L

    2016-02-01

    Bloom-forming cyanobacteria are widely distributed in freshwater ecosystems. To cope with salinity fluctuations, cyanobacteria synthesize compatible solutes, such as sucrose, to maintain the intracellular osmotic balance. The screening of cyanobacterial genomes revealed that homologues to sucrose metabolism-related genes only occur in few bloom-forming strains, mostly belonging to Nostocales and Stigonematales orders. Remarkably, among Chroococcales and Oscillatoriales strains, homologues were only found in M. aeruginosa PCC 7806 and Leptolyngbya boryana PCC 6306, suggesting a massive loss of sucrose metabolism in bloom-forming strains of these orders. After a complete functional characterization of sucrose genes in M. aeruginosa PCC 7806, we showed that sucrose metabolism depends on the expression of a gene cluster that defines a transcriptional unit, unique among all sucrose-containing cyanobacteria. It was also demonstrated that the expression of the encoding genes of sucrose-related proteins is stimulated by salt. In view of its ancestral origin in cyanobacteria, the fact that most bloom-forming strains lack sucrose metabolism indicates that the genes involved might have been lost during evolution. However, in a particular strain, like M. aeruginosa PCC 7806, sucrose synthesis genes were probably regained by horizontal gene transfer, which could be hypothesized as a response to salinity fluctuations.

  14. CURLY LEAF Regulates Gene Sets Coordinating Seed Size and Lipid Biosynthesis1[OPEN

    PubMed Central

    Wang, Huan; Ye, Jian; Wu, Hui-Wen; Sun, Hai-Xi; Chua, Nam-Hai

    2016-01-01

    CURLY LEAF (CLF), a histone methyltransferase of Polycomb Repressive Complex 2 (PRC2) for trimethylation of histone H3 Lys 27 (H3K27me3), has been thought as a negative regulator controlling mainly postgermination growth in Arabidopsis (Arabidopsis thaliana). Approximately 14% to 29% of genic regions are decorated by H3K27me3 in the Arabidopsis genome; however, transcriptional repression activities of PRC2 on a majority of these regions remain unclear. Here, by analysis of transcriptome profiles, we found that approximately 11.6% genes in the Arabidopsis genome were repressed by CLF in various organs. Unexpectedly, approximately 54% of these genes were preferentially repressed in siliques. Further analyses of 118 transcriptome datasets uncovered a group of genes that was preferentially expressed and repressed by CLF in embryos at the mature-green stage. This observation suggests that CLF mediates a large-scale H3K27me3 programming/reprogramming event during embryonic development. Plants of clf-28 produced bigger and heavier seeds with higher oil content, larger oil bodies, and altered long-chain fatty acid composition compared with wild type. Around 46% of CLF-repressed genes were associated with H3K27me3 marks; moreover, we verified histone modification and transcriptional repression by CLF on regulatory genes. Our results suggest that CLF silences specific gene expression modules. Genes operating within a module have various molecular functions, but they cooperate to regulate a similar physiological function during embryo development. PMID:26945048

  15. 7 CFR 983.50 - Aflatoxin regulations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 8 2012-01-01 2012-01-01 false Aflatoxin regulations. 983.50 Section 983.50..., ARIZONA, AND NEW MEXICO Regulations § 983.50 Aflatoxin regulations. The committee shall establish, with the approval of the Secretary, such aflatoxin sampling, analysis, and inspection...

  16. 7 CFR 983.150 - Aflatoxin regulations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 8 2012-01-01 2012-01-01 false Aflatoxin regulations. 983.150 Section 983.150..., ARIZONA, AND NEW MEXICO Rules and Regulations § 983.150 Aflatoxin regulations. (a) Maximum level. No handler shall ship for domestic human consumption, pistachios that exceed an aflatoxin level of 15...

  17. 7 CFR 983.50 - Aflatoxin regulations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 8 2014-01-01 2014-01-01 false Aflatoxin regulations. 983.50 Section 983.50..., ARIZONA, AND NEW MEXICO Regulations § 983.50 Aflatoxin regulations. The committee shall establish, with the approval of the Secretary, such aflatoxin sampling, analysis, and inspection...

  18. 7 CFR 983.50 - Aflatoxin regulations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 8 2013-01-01 2013-01-01 false Aflatoxin regulations. 983.50 Section 983.50..., ARIZONA, AND NEW MEXICO Regulations § 983.50 Aflatoxin regulations. The committee shall establish, with the approval of the Secretary, such aflatoxin sampling, analysis, and inspection...

  19. 7 CFR 983.150 - Aflatoxin regulations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 8 2014-01-01 2014-01-01 false Aflatoxin regulations. 983.150 Section 983.150..., ARIZONA, AND NEW MEXICO Rules and Regulations § 983.150 Aflatoxin regulations. (a) Maximum level. No handler shall ship for domestic human consumption, pistachios that exceed an aflatoxin level of 15...

  20. 7 CFR 983.150 - Aflatoxin regulations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 8 2013-01-01 2013-01-01 false Aflatoxin regulations. 983.150 Section 983.150..., ARIZONA, AND NEW MEXICO Rules and Regulations § 983.150 Aflatoxin regulations. (a) Maximum level. No handler shall ship for domestic human consumption, pistachios that exceed an aflatoxin level of 15...

  1. Aspergillus flavus: The Major Producer of Aflatoxin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus is an opportunistic pathogen of crops. It is important because it produces aflatoxin as a secondary metabolite in the seeds of a number of crops both before and after harvest. Aflatoxin is a potent carcinogen that is highly regulated in most countries. In the field, aflatoxin i...

  2. Isolation and characterization of the Z-ISO gene encoding a missing component of carotenoid biosynthesis in plants.

    PubMed

    Chen, Yu; Li, Faqiang; Wurtzel, Eleanore T

    2010-05-01

    Metabolic engineering of plant carotenoids in food crops has been a recent focus for improving human health. Pathway manipulation is predicated on comprehensive knowledge of this biosynthetic pathway, which has been extensively studied. However, there existed the possibility of an additional biosynthetic step thought to be dispensable because it could be compensated for by light. This step, mediated by a putative Z-ISO, was predicted to occur in the sequence of redox reactions that are coupled to an electron transport chain and convert the colorless 15-cis-phytoene to the red-colored all-trans-lycopene. The enigma of carotenogenesis in the absence of light (e.g. in endosperm, a target for improving nutritional content) argued for Z-ISO as a pathway requirement. Therefore, understanding of plant carotenoid biosynthesis was obviously incomplete. To prove the existence of Z-ISO, maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) mutants were isolated and the gene identified. Functional testing of the gene product in Escherichia coli showed isomerization of the 15-cis double bond in 9,15,9'-tri-cis-zeta-carotene, proving that Z-ISO encoded the missing step. Z-ISO was found to be important for both light-exposed and "dark" tissues. Comparative genomics illuminated the origin of Z-ISO found throughout higher and lower plants, algae, diatoms, and cyanobacteria. Z-ISO evolved from an ancestor related to the NnrU (for nitrite and nitric oxide reductase U) gene required for bacterial denitrification, a pathway that produces nitrogen oxides as alternate electron acceptors for anaerobic growth. Therefore, plant carotenogenesis evolved by recruitment of genes from noncarotenogenic bacteria.

  3. Molecular Characterization of Type-Specific Capsular Polysaccharide Biosynthesis Genes of Streptococcus agalactiae Type Ia

    PubMed Central

    Yamamoto, Shin; Miyake, Katsuhide; Koike, Yoichi; Watanabe, Masaki; Machida, Yuichi; Ohta, Michio; Iijima, Shinji

    1999-01-01

    The type-specific capsular polysaccharide (CP) of a group B streptococcus, Streptococcus agalactiae type Ia, is a high-molecular-weight polymer consisting of the pentasaccharide repeating unit 4)-[α-d-NeupNAc-(2→3)-β-d-Galp-(1→4)-β-d-GlcpNAc-(1→3)]-β-d-Galp-(1→4)-β-d-Glcp-(1. Here, cloning, sequencing, and transcription of the type Ia-specific capsular polysaccharide synthesis (cps) genes and functional analysis of these gene products are described. A 26-kb DNA fragment containing 18 complete open reading frames (ORFs) was cloned. These ORFs were designated cpsIaA to cpsIaL, neu (neuraminic acid synthesis gene) A to D, orf1 and ung (uracil DNA glycosylase). The cps gene products of S. agalactiae type Ia were homologous to proteins involved in CP synthesis of S. agalactiae type III and S. pneumoniae serotype 14. Unlike the cps gene cluster of S. pneumoniae serotype 14, transcription of this operon may start from cpsIaA, cpsIaE, and orf1 because putative promoter sequences were found in front of these genes. Northern hybridization, reverse transcription-PCR, and primer extension analyses supported this hypothesis. DNA sequence analysis showed that there were two transcriptional terminators in the 3′ end of this operon (downstream of orf1 and ung). The functions of CpsIaE, CpsIaG, CpsIaI, and CpsIaJ were examined by glycosyltransferase assay by using the gene products expressed in Escherichia coli JM109 harboring plasmids containing various S. agalactiae type Ia cps gene fragments. Enzyme assays suggested that the gene products of cpsIaE, cpsIaG, cpsIaI, and cpsIaJ are putative glucosyltransferase, β-1,4-galactosyltransferase, β-1,3-N-acetylglucosaminyltransferase, and β-1,4-galactosyltransferase, respectively. PMID:10464185

  4. Activation of Antibiotic Biosynthesis by Specified Mutations in the rpoB Gene (Encoding the RNA Polymerase β Subunit) of Streptomyces lividans

    PubMed Central

    Hu, Haifeng; Zhang, Qin; Ochi, Kozo

    2002-01-01

    We found that the biosynthesis of actinorhodin (Act), undecylprodigiosin (Red), and calcium-dependent antibiotic (CDA) are dramatically activated by introducing certain mutations into the rpoB gene that confer resistance to rifampin to Streptomyces lividans 66, which produces less or no antibiotics under normal growth conditions. Activation of Act and/or Red biosynthesis by inducing mutations in the rpoB gene was shown to be dependent on the mutation's position and the amino acid species substituted in the β-subunit of the RNA polymerase. Mutation analysis identified 15 different kinds of point mutations, which are located in region I, II, or III of the rpoB gene and, in addition, two novel mutations (deletion of nucleotides 1287 to 1289 and a double substitution at nucleotides 1309 and 1310) were also found. Western blot analyses and S1 mapping analyses demonstrated that the expression of actII-ORF4 and redD, which are pathway-specific regulatory genes for Act and Red, respectively, was activated in the mutants able to produce Act and Red. The ActIV-ORF1 protein (an enzyme for Act biosynthesis) and the RedD protein were produced just after the upregulation of ActII-ORF4 and RedZ, respectively. These results indicate that the mutation in the rpoB gene of S. lividans, resulting in the activation of Act and/or Red biosynthesis, functions at the transcription level by activating directly or indirectly the key regulatory genes, actII-ORF4 and redD. We propose that the mutated RNA polymerase may function by mimicking the ppGpp-bound form in activating the onset of secondary metabolism in Streptomyces. PMID:12081971

  5. Olive: a key gene required for chlorophyll biosynthesis in Antirrhinum majus.

    PubMed Central

    Hudson, A; Carpenter, R; Doyle, S; Coen, E S

    1993-01-01

    Olive (oli) is a recessive nuclear mutation of Antirrhinum majus which reduces the level of chlorophyll pigmentation and affects the ultrastructure of chloroplasts. The oli-605 allele carries a Tam3 transposon insertion which has allowed the locus to be isolated. The oli gene encodes a large putative protein of 153 kDa which shows homology to the products of two bacterial genes necessary for tetrapyrrole-metal chelation during the synthesis of bacteriochlorophyll or cobyrinic acid. We therefore propose that the product of the oli gene is necessary for a key step of chlorophyll synthesis: the chelation of magnesium by protoporphyrin IX. Somatic reversion of the oli-605 allele produces chimeric plants which indicate that the oli gene functions cell-autonomously. Expression of oli is restricted to photosynthetic cells and repressed by light, suggesting that it may be involved in regulating the rate of chlorophyll synthesis in green tissues. Images PMID:8404842

  6. Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis