Evolutionary Analysis Of Isoflavone Metabolic Pathway Genes And Functional Identification Of A Transcription Factor MYB184 In Soybean

Isoflavoniods are secondary metabolic compounds generated by phenylpropanoid metabolic pathway and are predominantly found in the Papilionoideae family.Isoflavonoid metabolites play a vital role on plant-microbe interaction in plants,and are associated with health benefits of soybean consumption.Thus,increasing isoflavone content is one of the main purposes for soybean quality breeding.However,due to the complicacy of isoflavonoid sybthesis pathway,researches about single pathway genes can hardly achieve desired effect.Protein-protein interaction helps us understand more about the biosynthesis mechanism.And investigating transcription factors can promote the improvement of isoflavone content trait and make an effort to genetic engineering of soybean quality.The biosynthesis of isoflavone starts from public pathway of phenylpropanoid metabolic and phenylalanine.Then a series of multi-copy enzymes catalyze substrates;branch point enzymes lead substrates to isoflavone synthesis branch and finally generate isoflavonoids and derivatives.Phenylpropanoid metabolic pathway and isoflavone biosynthesis pathway are the most analyzed secondary pathways.However,little is known about the molecule evolution and selection of enzymes in isoflavone pathway.Thus,analysis of pathway gene evolution and selection events,together with“metabolons”hypothesis will help us understand more thoroughly about the complex regulation network of isoflavone pathway.In addition,the identification of transcription factors related to the regulation of pathway enzymes can contribute to the understanding of mechanisms about isoflavone synthesis and regulation and further lay the theoretical foundation for metabolic engineering and genetic improvement.In this study,evolution and selection patterns of isoflavone pathway genes were analyzed and it turned out that key structure enzymes experienced similar evolution and selection pattern and showed convergent evolution.Together with the“metabolons”hypothesis,we predict these enzymes may form protein-protein complex.Yeast two hybrid method was applied to investigate the interaction of part of the pathway enzymes and copies.The results turned out that IFS can interact with branch point enzymes CHS and CHR.CHI can interact with CHS and CHR.PAL can interact with C4H.These results can partly demonstrate possibility of the existence of protein-protein complex.Meanwhile,a soybean transcription factor related to abiotic stress response,GmMYB184 was cloned and the function of this transcription factor was identified.Subcellular localization confirmed the nuclear localization of GmMYB184.The expression profile of GmMYB184 was similar to IFS2 and the isoflavone accumulation pattern also showed the same way.In addition,GmMYB184 and IFS2 mainly expressed in roots and mature seeds,and expression level increased by seeds maturing.GSH induction,expression pattern showed that GmMYB184 can bind the promoters of key structure genes related to isoflavone biosynthesis.GmMYB184 can increase the expression of CHS8 and IFS2 to 5 and 7 folds respectively.Finally,to further verify the function of GmMYB184 during isoflavone biosynthesis,over expression vector and RNAi interference vetor for GmMYB184 were constructed and then transformed to soybean hairy roots respectively.RNAi silencing of GmMYB184 in hairy roots resulted in reduced levels of isoflavonoids,meaning that GmM YB184 is necessary for isoflavone biosynthesis.However,over-expression of GmMYB184 was not sufficient to increase isoflavonoid contents in hairy roots.To sum it,this study investigated soybean isoflavone biosynthesis pathway from evolution and selection events,protein-protein interaction and transcriptional regulation levels,providing theoretical foundation for understanding molecule mechanism and genetic improvement of soybean quality.