Mechanism Of Transcription Factor TT8 Regulating Proanthocyanidin Synthesis In Brassica Juncea

Brassica juncea belongs to the family of Brassica family of Brassicaceae.It has a variety of function,including providing vegetable oil for human diet,high quality animal feed and industrial raw materials.Previous studies have shown that the color difference in rapeseed seed coats is due to the difference in the accumulation of proanthocyanidins in the seed coats by the flavonoid biosynthesis pathway.In addition,proanthocyanidins belong to flavonoids,which are secondary metabolites of plants that perform a variety of functions,such as helping pollinate and spreading seeds,regulating plant growth and development,resisting biological stress and abiotic stress.Therefore,the study on the synthesis pathway of proanthocyanidins in Purpleleaf Mustard has important value significance for the cultivation of rapeseed varieties with excellent traits.The materials used in this experiment were Purple-leaf Mustard(PM,AABB,diploid hybrid black seed)and Sichuan Yellow seed(SY,aabb,genetically stable yellow seed with natural mutation).The differences in seed coat and plant color between the two materials were significant,and the content of flavonoids such as anthocyanins and proanthocyanidins were significant.In this study,two different copies of Bju A.TT8 and Bju B.TT8 genes were cloned from PM,and their expression patterns in different tissues of Brassica juncea were analyzed by combining q RT-PCR.The results showed that the expression levels of Bju A.TT8 and Bju B.TT8 genes were higher in the leaves of PM and SY than in the stems and roots.Me JA induction showed that the expression levels of Bju A.TT8 and Bju B.TT8 genes in PM and SY both peaked after 0.5 h.The biological function of the genes was verified by using the hairy root system of Brassica juncea,and the results showed that the expression levels of the key enzyme genes Bju.CHS,Bju.CHI,and Bju.BAN increased significantly from flavonoids synthesis in hairy roots of PM with Bju A.TT8 gene,while the key enzyme genes Bju.CHS and Bju.FLS increased significantly in hairy roots of SY with Bju A.TT8 gene.In addition,the expression levels of the key enzyme genes Bju.PAL,Bju.CHS,Bju.CHI and Bju.DFR were significantly increased in hairy roots of PM with Bju B.TT8 gene,while the key enzyme genes Bju.CHS and Bju.DFR were significantly increased in hairy roots of SY with Bju B.TT8 gene.Heterologous expression of Arabidopsis and by reintroducing tt8 mutants Arabidopsis showed that the leaf became purple and the seed coat color was deepened.In addition,the content of total flavonoids in plants increased,and the content of proanthocyanidins also increased significantly,proving that both Bju A.TT8 and Bju B.TT8 genes were involved in the pathway of plant flavonoids synthesis.Yeast two-hybrid experiments showed that TT8 and PAP2 proteins interacted with each other.It is speculated that the two can synergistically regulate the proanthocyanidin synthesis of Brassica juncea,but the molecular mechanism still needed further study.In summary,this study preliminarily verified the involvement of Bju A.TT8 and Bju B.TT8 genes in plant procyanidin synthesis pathway,providing theoretical basis for molecular mechanism research of procyanidin synthesis and molecular breeding improvement in Brassica juncea.