Gene Cloning And Characterization Of Flavonoid Biosynthesis-related R2R3-MYB Transcription Factors From Fagopyrum Tataricum

Tartary buckwheat(Fagopyrum tataricum)belongs to the family of polygonaceae and genus of Fagopyrum,which is a kind of small grains with health care and medicinal function.Tartary buckwheat contains abundant flavonoids such as rutin,anthocyanins and proanthocyanidins.Because flavonoids are the main qualitative character for tartary buckwheat,it is the theoretical fundament of molecular breeding to understand the regulatory mechanism of flavonoid biosynthesis.Plant flavonoids are mainly regulated by key enzymes and transcription factors.However,many previous studies have shown that transcription factors could regulate the expression of multiple key enzyme genes in the flavonoid metabolism pathway,and transcription factors have the more extensive and obvious regulation than a single key enzyme gene.Among them,MYB transcription factor is various and complex,which is the most important transcription factor family involved in plant flavonoid metabolism."Xiqiao No.2",a cultivated verity with the high flavonoids,is used as the plant materials in this study.Firstly,we cloned the MYB transcription factor genes according to the transcriptome data of tartary buckwheat.After we screened the sequences of flavonoid related FtMYBs by bioinformatics analysis,their transcriptional activity were examined by yeast one hybrid system.Then,transgenic technique was further used to analysis the effects of FtMYBs on the flavonoids related key enzyme genes and the metabolites in Arabidopsis.So,this study not only enriched the MYB transcription factor family data in tartary buckwheat,but also help us deep understanding their roles in the flavonoid synthesis regulation mechanism.The main research results were showed as follows:1.Twenty-four cDNA and DNA sequences of R2R3-MYB transcription factors were successfully cloned from taratry buckwheat.Then bioinformatics analysis indicated that they were the typical R2R3-MYBs.Compared the sequences between cDNA and DNA,they were classified into three groups.Eleven sequences were composed of two introns and three exons.Five sequences included one intron and two exons.Their intron splicing were accorded with the GT-AG rules in the above sequences.However,there was no intron in the other eight sequences.Phylogenetic tree analysis showed that the R2R3-MYBs from taratry buckwheat were widely involved in epidermal cell fate,stress response,ligin biosynthesis and the flavonoid biosynthesis.2.The yeast one hybrid recombinant vector were constructed for the flavonoid related FtMYBs/FtMYB15 FtMYB16 FtMYB23 and FtMYB24)and they were transferred into the yeast cells of AH 109.Then,the nutrient deficient medium was used to screen the transformants with the transcriptional activity.The results implied that only FtMYB23 had the obvious transcriptional activation activity.However,it seemed that FtMYB15,FtMYB16 and FtMYB24 had weak or no independent transcriptional activation activity.3.The plant over-expression vectors were constructed for the flavonoid related FtMYBs.Then they were introduced into Arabidopsis using floral dip method mediated by the Agrobacterium cell GV3101.The positive T3 generation transgenic lines were screened by resistance medium and PCR method.FtMYB15 transgenic lines showed the increased red pigmentation in leaves and seed coats,as well as the higher accumulation of anthocyanin and proanthocyanidin in transgenic Arabidopsis.Meanwhile,the expression of both early and late biosynthesis genes,including AtCHS,AtCHI,AtF3H,AtF3'H,AtDFR,AtANS,AtBAN and AtTT12,in the anthocyanin and proanthocyanidin pathway were enhanced(P<0.01).However,the key enzyme genes of the flavonol pathway was decreased significantly(P<0.05).The expression level of anthocyanin related genes(AtCHS,AtF3'H,AtANS and AtDFR)were up-regulated significantly(P<0.01)in FtMYB16 transgenic lines,while no significant change in the key enzyme genes of the flavonols pathway were detected.And the anthocyanin was accumulated in the transgenic Arabidopsis leaves.FtMYB23 transgenic lines indicated that the expression level of proanthocyanidin related key enzyme genes(AtCHS,AtDFR and AtBAtN)were significantly increased(P<0.01),moreover,the flavonol pathway genes were up-regulated significantly(P<0.01).And the content of proanthocyanidin was increased in over-expression lines.While both anthocyanin and flavonol related genes(AtCHI,AtF3'H,AtANS AtDFR and AtFLS)were markedly boosted in FtMYB24 transgenic lines(P<0.05),thus lead the accumulation of anthocyanin in Arabidopsis.In summary,FtMYB15 could regulate both of anthocyanin and proanthocyanidin biosynthesis in plant,FtMYB16 and FtMYB24 could be involved in anthocyanin biosynthesis regulation,FtMYB23 could play a regulatory role in proanthocyanidin biosynthesis.