| Isoflavones is one of the flavonoids, which mainly presented in legumes.Isoflavones is not only as a legume secondary metabolites involving in plant diseaseresistance, stress tolerance and other regulatory mechanisms, but also has potentialefficacy for human health. Meanwhile, as a female hormone analogues, improper useof it can also cause adverse effects. Breeders need to improve soybean varieties thatare high or low content of isoflavones according to our demands. Therefore, it isimportant to understand the pattern and process of isoflavones accumulation duringseed development period. Molecular biology and genetic analysis showed that thesoybean genome belongs to ancient sources tetraploid, with more non-homologoussingle copy gene, and the genes involved in the regulation of isoflavones arenumerous. In this study, we acquired some genes involved in the regulation of soyisoflavone synthesis through bioinformatics methods and expression analysis of thesegenes were conducted, in combination with the results of expression profiling ofsoybean embryos at different developmental stages. Meanwhile, one newbHLH-MYC transcription factors, named GmbHLH3a, was cloned. Whereafter, theroles of the gene were studied and in order to validate the accuracy of expressionprofile data. This study further elaborated the expression changes of genes inisoflavone synthesis-related pathway, which not only enriched soy MYC transcriptionfactor gene resources, but also provide a theoretical basis for its further application,the main findings are as follows:1. Determination of isoflavone content of soybean Jilin32embryo using HPLCtechniques at different developmental period showed that the isoflavone level at50dwas significantly higher than these of20d and30d. The result indicated the relatedpathways regulated isoflavone synthesis were activated from30d to50d.2. Functional annotations for soybean genes and phylogenetic analysis ofrelevant gene family were conducted using multiple databases, such as GO, KEGG,etc. Combined with the expression profiles of soybean Jilin32embryo developmental period of20d,30d and50d, the expression patterns of related genes were acquired.Further research was made to verify genes regulated the isoflavone synthesis viaqRT-PCR and obtained these genes.3. Using soybean bHLH transcription factor released in planttfdb, thesub-family of MYC transcription factor was gained through phylogenetic analysis.Combined with the gene functional annotation and expression analysis, two MYCtranscription factor genes were cloned.4. Two fused expression vectors of GmMYC2a∷eGFP and GmbHLH3a∷eGFPwere constructed by overlapping PCR, and then subcellular localization resultsshowed that the two genes are localized in the nucleus.The prokaryotic expression of GmbHLH3a and SDS-PAGE electrophoresis showedGmbHLH3a protein can highly expresse in prokaryotic systems. And the highestexpression of GmbHLH3a recombinant protein is presented at10h after induction byIPTG5. The transcriptional activation activity of GmMYC2a and GmbHLH3a wereanalyzed via yeast one-hybrid, the results show that these two genes does not have anindependent transactivation function in yeast.6. Constructing yeast two-hybrid libraries of the soybean entire embryodevelopmental period, two potential proteins AD1-1(Gene Bank No. KP297803) andAD5-1(Gene Bank No. KP297802) interacted with GmbHLH3a were gained usingY2H. The results indicated GmbHLH3a may formed protein complex with othertranscription factor to involve in the transcriptional regulation.7. The GmbHLH3a plant expression vector was introduced into Agrobacteriumrhizogenes and then transformed into soybean seed to generate transgenic hairy roots.Subsequently, the isoflavones of transgenic hairy roots were determined, the resultsshowed that isoflavones content was significantly higher in transgenic roots than thatin control. This result suggests that GmbHLH3a involved in the accumulation ofisoflavone. |
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