Identification Of MYB Transcription Factors Interacted With Soybean 14-3-3 Protein And Its Function Analysis In Secondary Metabolism

14-3-3s are a class of highly conserved proteins ubiquitously found in eukaryotes,which are encoded by a multiple gene family.It can regulate the activity of target proteins through a variety of mechanisms and affect many physiological and biochemical processes of plant growth and development.Soybean is a kind of economic crop which is widely cultivated around the world.It is rich in secondary metabolites,especially soybean isoflavonoid.Previous studies have shown that soybean 14-3-3 protein negatively regulates the synthesis of isoflavonoid by regulating the subcellular localization of the isoflavonoid regulatory factor GmMYB176.As one of the largest transcription factor families in plants,MYB transcription factors also participate in the regulation of secondary metabolism of plants.Studies suggest that 14-3-3 proteins may regulate isoflavonoid biosynthesis by regulating several MYB transcription factors.Therefore,this study sought to find MYB transcription factors that interacted with 14-3-3 proteins and identify their functions in isoflavonoid synthesis and related secondary metabolic pathways.The main findings are as follows:1.Yeast two-hybrid experiments were performed and identified that GmMYB133,GmMYB138a and GmMYB52 could interact with SGF141.The interaction between GmMYB 133,GmMYB 138a and SGF141 was further confirmed by bimolecular fluorescence complementation experiment,and the interaction changed the subcellular localizations of GmMYB 133 and GmMYB 138a.In addition,the interaction between two transcription factors and GmMYB 176 also occurred.And it mainly occurred in the nucleus.Bioinformatics analysis showed that both GmMYB 133 and GmMYB 138a contained only one MYB domain and they were CCA1-like proteins.2.The expression patterns of GmMYB133 and GmMYB138a in soybean tissues and under different abiotic stresses were analyzed.The gene expression level of GmMYB133 gradually increased with the maturity of soybean embryos,and positively correlated with the accumulation of isoflavonoid.The expression level of GmMYB138a was highest in the leaves.The response patterns of two transcription factors were different when plants were subject to different abiotic stresses.GmMYB133 showed no significant change in expression under cold stress.Its expression level increased at the later stage of drought stress,and the expression level changed most significantly under salt stress,showing an upward trend.The gene expression of GmMYB138a was induced by three stresses.3.The effect of overexpression of GmMYB133 on isoflavonoid content in soybean hairy roots was clarified.Quantitative real-time PCR results indicated that overexpression of GmMYB133 could activate the expression of CHS8 and IFS,which are key genes for isoflavonoid biosynthesis.HPLC assay results demonstrated that overexpression of GmMYB133 promoted the accumulation of isoflavonoid in soybean hairy roots.ChIP-qPCR analysis showed that GmMYB133 could not recognize the binding site of GmMYB176 in CHS8 promoter region.4.Transgenic Arabidopsis thaliana plants over-expressing GmMYB133 were obtained.Compared with wild-type Arabidopsis tealiana,transgenic plants showed dwarfed statures,shortened leaf petioles,rounded and curled leaves.The results of transcriptome sequencing revealed that the genes involved in the synthesis of secondary metabolites such as anthocyanin and lignin were up-regulated.This result was verified by qRT-PCR.The results of the determination of anthocyanin and lignin content in seedlings showed that the accumulation of the two secondary metabolites in the transgenic Arabidopsis was significantly increased.In addition,ChIP-qPCR results showed that GmMYB133 bound to the promoter regions of CHS and CCOAAMT,but not to that of TT7 and PAP1.