Screening And Function Identification Of Salt Stress-related Myb Transcription Factor From Tartary Buckwheat(Fagopyrum Tataricum(L.) Gaertn.)

Fagopyrum tataricum(L.)Gaertn is a minor food crop rich in flavonoids.It has the pharmacological effects of preventing and curing cardiovascular and cerebrovascular diseases and regulating blood sugar,lipid and blood pressure.As one of the transcription factors,MYBs are mainly involved in regulating plant growth and morphogenesis,responding to stress and metabolic regulation.At present,there is insufficient research on the regulation mechanism of buckwheat transcription factors on buckwheat flavonoid synthesis and metabolism,as well as the function of resistance to stress.In this study,buckwheat was used as the research material in order to screen stress-resistant MYB transcription factors and elucidate their function and regulation mechanism in buckwheat stress resistance,so as to provide a theoretical basis for buckwheat molecular breeding.The main results are as follows:1.According to the transcriptome data of buckwheat salt stress,5350 genes were significantly up-regulated and 4207 genes were significantly down-regulated.KEGG pathway enrichment analysis showed that the differential genes were mainly concentrated in phenylpropane metabolic pathway and flavonoid biosynthesis pathway.There were significant differences in 35 MYB genes screened from the transcriptome data of tartary buckwheat,19 of which were significantly up-regulated and 16 significantly down-regulated.Combined with differential expression gene screening and qRT-PCR detection,the results showed that these MYB transcription factors showed responses to abscisic acid,methyl jasmonate,sodium chloride and salicylic acid at different transcription levels.2.The metabolic profiles of buckwheat under salt stress were analyzed,including 67 compounds including primary and secondary metabolites.The contents of amino acids and organic acids in buckwheat increased,while the synthesis of rutin and quercetin,the main secondary metabolites,increased significantly.Through correlation coefficient analysis,it was found that the expression level of MYBs gene including FtMYB2 was positively correlated with the changes of rutin and other metabolites,which might be involved in the regulation of buckwheat stress response to metabolic synthesis.3.The gene cloning and functional analysis of FtMYB2 which responded significantly to buckwheat stress and showed positive response regulation were carried out in this study.The subcellular localization and qRT-PCR analysis of tobacco showed that FtMYB2 mainly distributed in the nucleus and had high gene expression in tartary buckwheat seeds and flowers.Yeast hybridization experiments showed that FtMYB2 showed strong transcriptional activation activity,and its activity of the reporter gene beta-galactosidase in yeast was 14 U.The hairy root transformation system of tartary buckwheat mediated by Agrobacterium rhizogenes showed that the total flavonoid content and anthocyanin content in FtMYB2 overexpression positive transformation lines were significantly increased by 2.6 times and 1.7 times.Five main flavonoids were identified and analyzed by HPLC.Compared with the control,the contents of ferulic acid,benzoic acid,epicatechin,gallic acid and rutin increased by 3.5,2.5,2.1,1.6 and 1.2 times,respectively.Yeast one hybridization experiments showed that FtMYB2 may partially bind with FtC4 H,FtF3H,FtANS and FtRT promoters in the flavonoid synthesis pathway,thus regulating the gene expression level,and then regulating the metabolism of flavonoid synthesis pathway in buckwheat.