Molecular Identification Of The Specifically Inhibiting Anthocyanin Biosynthesis SG4-MYBs Transcription Factor FtMYB3 In Tartary Buckwheat

Tartary buckwheat(Fagopyrum tataricum(L)Gaertn.)is a kind of homology of medicine and food miscellaneous grain crop,which is rich with flavonoids such as rutin and anthocyanin.It has the health care functions for preventing hardening of the blood vessels,anti-aging and enhancing immunity.Both rutin and anthocyanin were derived from dihydroflavonol,the same precursor molecules,which were separated into flavonol branch and anthocyanin branch by flavonol synthase(FLS)and dihydrof Iavonol-4-reductase(DFR),respectively.However,the main flavonoids accumulated in Tartary buckwheat are rutin rather than anthocyanin,and its molecular mechanism is still unclear.Previous studies had shown that the MYB transcription factor was one of the most important transcription factor families,which was involved in the flavonoid synthesis regulation in plant.At present,some R2R3-MYB transcription factors have been functioned as activator of anthocyanin or proanthocyanidin synthesis,activator or suppressor of rutin synthesis.Previously,our research team cloned and proved a R2R3-MYB transcription factor FtMYB3 to cause albino flower in transgenic tobacco.Now,the further explores were performed from following several aspects,such as molecular characterization,gene expression patterns,biology function and protein interactions involved in MBW complex formation.In general,the above studies not only help us to clarify the down-regulation mechanism of FtMYB3 on anthocyanin synthesis,but also provide a new sight to understand the bias of rutin synthesis in the flavonoid biosynthetic pathway in Tartary buckwheat.The main results were as follows:1.Bioinformatics analysis was performed for molecular characteristics of FtMYB3 transcription factor from Tartary buckwheat.Phylogenetic tree showed that FtMYB3 was clustered with plant SG4-MYBs subfamily.Multiple sequence alignments indicated that FtMYB3 had the classical domain of SG4-MYB proteins,i.e.,its N terminal with a b HLH motif,and its C terminal with three SG4-MYB conserved motifs including C1 motif,C2/EAR motif,and C4/SID motif.2.The promoter seuqence of FtMYB3 was cloned and analyzed for its cis-acting elements,and the expression pattern of FtMYB3 in Tartary buckwheat was also detected through real-time fluorescence quantitative PCR(q RT-PCR).The results showed that the upstream sequence of FtMYB3 gene in 2000 bp length was cloned.Plant CARE prediction showed that the promoter region of FtMYB3 contained many cis-acting elements,such as meristem expression element,light responsive element,Me JA and SA hormone responsiveness element,and drought-inducibility responsive element.q RT-PCR revealed that the FtMYB3 showed the tissue-specific expression,and had the highest expression in root.The transcriptional expression of FtMYB3 was significantly increased under hormone(SA and Me JA)treatment(P < 0.01)and abiotic stress(Drought,Salt,Cold and Dark)at the seedlings stage(P < 0.01).The FtMYB3 expression pattern was consist with the cis-acting elements in its promoter region,which indicted that FtMYB3 transcription could be integratedly regulated by growth and development,hormones and stress.3.The effect of Tartary buckwheat FtMYB3 on flavonoids metabolism in Arabidopsis was analyzed by transgenic technique.The results showed that the transcriptional expressions of LBGs enzyme genes(DFR,ANS,BAN and TT13)were obviously down-regulated in FtMYB3 transgenic Arabidopsis lines(P < 0.01),while the anthocyanin content was remarkably decreased(P < 0.01).However,the rutin accumulation was significantly increased in FtMYB3 transgenic lines(P < 0.05).4.Tobacco transient transfection was performaed to verify FtMYB3 inhibiting the transcriptional activity of DFR promoters from Arabidopsis and Tartary buckwheat.The results showed that FtMYB3 inhibited the expression of reporter gene GUS individually driven by their DFR promoter of from Arabidopsis or Tartary buckwheat.5.Yeast Two Hybrid System(Y2H)was used to screen the interacting proteins of FtMYB3 transcription factor involved in the formation of MBW complex regulating anthocyanin synthesis.The results showed that FtMYB3 could interacte with GL3/EGL3/TT8/TTG1 from Tartary buckwheat and Arabidopsis,which suggested that FtMYB3 might regulate the transcriptional expression of key enzyme genes in anthocyanin synthesis pathway through competition with the activating MYB to bind with b HLHs and WD40 s for MBW complex formation.