Cloning And Function Analysis On Key Genes Of Flavonol Biosynthesis Pathway In Tartary Buckwheat

Flavonols,as a kind of secondary metabolites that are widely found in plants,can directly affect plant appearance and food flavor.Abundant in flavonoids such as rutin,buckwheat(Fagopyrum)belongs to Polygonaceae,which has the effects of lowering plasma cholesterol level,anti-cancer and anti-inflammation.As a kind of ancient false cereal,buckwheat has attracted more and more attention in recent years.At present,few studies have been conducted on the identification and analysis of structural genes related to the flavonoid biosynthesis of Tartary buckwheat,especially on their functional characteristics.Therefore,it is necessary to further study the key enzyme genes in the biosynthesis pathway of Tartary buckwheat.In this study,screened by jasmonic acid(JA)treated RNA-Seq database,some putative candidatekey genes of flavonoid biosynthesis in Tartary buckwheat were cloned.Taking the advantage of the agrobacterium infection,the overexpressed hairy roots of these gene were constructed,and the contents of total flavonoids and rutin in overexpressed hairy roots were measured to provide a reference for revealing the function of enzyme genes of flavonol biosynthesis in Tartary buckwheat.The main findings are as follows:1 140 glycoltransferase genes regulated by jasmonic acid,which differentially expressed in Tartary buckwheat,were screened out by RNAseq analysis.Through homologous analysis,seven genges that likely were involved in the biosynthesis of flavonoids in Tartary buckwheat were found,named FtGT1,FtGT2,FtGT3,FtGT4,FtGT5,FtGT6 and FtGT7.2 SOPMA and SWISS-MODEL software were used to predict the secondary and tertiary structures of the encoded proteins.The results showed that the seven flavonoids synthetase genes all contain four conformations,namely ?-helix,elongated chain,?-corner and random curl.Multiple sequence alignment revealed that they all contained a PSPGbox,a highly conserved amino acid sequence HCGWNS motif at the C-end,which is related to the recognition and binding of receptor molecules of glycosyltransferase.Phylogenetic analysis showed that FtGT1,FtGT2,FtGT3,FtGT4 and FtGT7 belonged to UF7 GT family.FtGT5 and FtGT6 belong to UF3 GT family.In conclusion,FtGT1,FtGT2,FtGT3,FtGT4,FtGT5,FtGT6,and FtGT7 are typical GT-B type glycolyltransferases.3 Overexpressed transgenic hairy root lines were obtained by agrobacterium rhizoides infecting,and the contents of total flavonoids and rutin in the hairy root were detected.The result showed that FtGT1,FtGT2,FtGT3,FtGT4,FtGT5,FtGT6,FtGT7 promoted the accumulation of flavonoids,and FtGT1 promoted the accumulation of rutin,but there was no significant changes in the content of rutin in the hairy roots of FtGT2,FtGT3,FtGT4,FtGT5,FtGT6,FtGT7.4 Real-time PCR showed that the expression of FtGT1 was the lowest in roots and the highest in stems.Me JA treatment can enhance the GUS activity in FtGT1pro: : GUS transgenic hairy root.