Transcription Factor FtMYB6/FtMYB8 Of Tartary Buckwheat Regulates The Biosynthesis Of Flavonol/anthocyanin In Plant

Tartary buckwheat（Fagopyrum tataricum）,as a mountain crop belonging to polygonaceae family,is rich in flavonoids,mainly flavonols represented by rutin,followed by anthocyanins and proanthocyanidins.It is a kind of characteristic coarse cereal with important health value.Rutin content is an important quality trait of Tartary buckwheat seeds,and anthocyanin and proanthocyanidin content give it additional ornamental and economic value.Therefore,it is important to reveal the regulation mechanism of their biosynthesis for improving quality traits and economic value of Tartary buckwheat.A large number of studies have shown that MYB transcription factors play an irreplaceable role in the regulation of plant flavonoid metabolism.They have many members and complex regulatory mechanisms.Here,Tartary buckwheat cultivar"Xiqiao No.2"bred in Liangshan Prefecture of Sichuan Province was used as experimental material.Based on the genome and transcriptome data,FtMYB6 and FtMYB8 genes were screened and cloned,and they belong to SG7-MYB subgroup and SG4-Like-MYB subgroup,respectively.Their transcriptional activation activities were analyzed by transcriptional activation experiments.The effects of FtMYB6 and FtMYB8on flavonoid synthase gene expression and flavonoid composition types and accumulation were analyzed by transgenic tobacco experiment and transgenic Arabidopsis experiment,respectively.The spatiotemporal transcriptional activity of P_FtMYB6 and P_FtMYB8 promoter sequences,and their responed to environmental factors and hormone response were analyzed by transgenic Arabidopsis thaliana experiment.Finally,the MBW interaction members of FtMYB8 protein were identified by yeast two-hybrid assay.This study preliminarily elucidated the transcriptional regulation mechanism of FtMYB6 and FtMYB8 of Tartary buckwheat and their molecular mechanism of regulating flavonoid metabolism and the fundamental characteristics of their response to internal and external factors,and improved the understanding of the regulation mechanism of rutin,anthocyanin and proanthocyanidin metabolism in Tartary buckwheat.It also provided theoretical support for the future use of assistant molecular breeding technology to improve the flavonoid species and content of Tartary buckwheat and increase its economic added value.The main results are as follows:1.Based on the genome and transcriptome data of Tartary buckwheat,two SG7-MYB transcription factors and four SG4-Like-MYB transcription factors were screened out.Combined with the content of flavonoids and gene expression level,FtMYB6 belonging to SG7-MYB subfamily gene and FtMYB8 belonging to SG4-Like-MYB subfamily gene were isolated and cloned.The g DNA sequence analysis showed that FtMYB6 and FtMYB8 both contained three exons and two introns.Multi-sequence alignment and phylogenetic analysis showed that FtMYB6 and FtMYB8may be involved in the biosynthesis of flavonol,anthocyanin and proanthocyanidin,respectively.2.The yeast expression transcription activation vectors of FtMYB6 and FtMYB8were constructed and introduced into transcription-activated yeast cell AH109.The positive transformants were screened and identified by acid defective plate.The analysis ofβ-galactosidase filter paper displayed that FtMYB6 and FtMYB8 had individual transcription activity and did not have individual transcription activity,respectively.3.The 1891 b P promoter of P_FtMYB6 and 2313 b P promoter of P_FtMYB8 were cloned and constructed plant expression vectors p BI101-P_FtMYB6-GUS and p BI101-P_FtMYB8-GUS.Agrobacterium tumefaciens GV3101 was used to transfect Arabidopsis thaliana and positive plants were obtained.Gus staining analysis showed that P_FtMYB6 promoter had significant transcriptional activity only in flower buds during the whole growth period,and its transcriptional activity was induced significantly by UV-B,cold and light（P<0.05）;While the P_FtMYB8 promoter has obvious transcriptional activity in the roots of the true leaf stage,root primordia and stem trichomes of the flowering stage and all bud trichomes,and its transcriptional activity increased significantly under Me JA,ABA and UV-B treatments（P<0.05）and was repressed by dark treatment,but not significantly（P>0.05）.4.Plant expression vectors p CHF3-FtMYB6-YFP and p CHF3-FtMYB8-YFP were constructed.Agrobacterium tumefaciens GV3101 was used to transfect tobacco and Arabidopsis thaliana by leaf disc method and floral-dip,respectively,and positive plants were obtained by PCR.Nuclear localization staining with DAPI showed that FtMYB6and FtMYB8 were may located in the nucleus.Overexpression of FtMYB6 tobacco showed that the total flavonoid and rutin contents of transgenic tobacco were significantly higher than of wild type tobacco,and the anthocyanin content of transgenic tobacco was significantly lower than that of wild type tobacco;the flowers of transgenic tobacco were pink,and showing less pigmentation accumulation.The expression levels of Nt C4H、Nt CHI and Nt F3’H genes in flavonoid metabolism pathway of transgenic tobacco were up-regulated significantly（P<0.05）,and the expression levels of Nt FLS and Nt CHS genes were up-regulated extremely significant（P<0.01）,while the expression levels of Nt PAL,Nt4CL and Nt DFR genes were down-regulated significantly（P<0.05）.Overexpression of FtMYB8 in Arabidopsis thaliana showed that the content of proanthocyanidin and anthocyanidin in wild type plants was significantly higher than that of in transgenic FtMYB8 plants（P<0.05）.Transgenic seedlings showed less pigmentation and transgenic seeds showed lighter seed coat color.There was no significantly difference in the expression of At EBGs and At LBGs genes in flavonoid metabolism pathway（P>0.05）,but the expression of At TT12 gene was down-regulated extremely significantly（P<0.01）.The percentage of marginal trichomes in young leaves of wild type Arabidopsis thaliana was significantly higher than that of transgenic FtMYB8 Arabidopsis thaliana（P<0.05）.5.The potential MBW ternary complex genes,Ft GL3,Ft EGL3,Ft TT8 and Ft TTG1,which are involved in transcriptional regulation of anthocyanin synthesis,were screened and cloned.The yeast expression vectors of p GADT7-FtMYB8,p GBKT7-At GL3/At EGL3/At TT8/At TTG1/Ft GL3/Ft EGL3/Ft TT8/Ft TTG1 were constructed and introduced into yeast two-hybrid cell AH109,respectively.The results showed that FtMYB8 could interact strongly with At TT8 and Ft GL3,and FtMYB8 could also interact with Ft TT8,but the interaction intensity was weak.In conclusion,FtMYB6 is a unique SG7-MYB transcription factor,its transcription level is regulated by specific developmental stages and environmental factors,and is regulating the biosynthesis of plant flavonol（mainly rutin）;FtMYB8 is a unique SG4-Like-MYB transcription factor,its transcription level is regulated by specific developmental stages,environmental factors and hormone signals,and inhibits accumulation of plant proanthocyanidin and anthocyanidin,and probably regulate marginal trichome initiation in the buds.