Cloning And Functional Analysis Of FtMYBF Transcription Factor In Tartary Buckwheat （Fagopyrum Tataricum）

Tartary buckwheat（Fagopyrum tataricum Gaertn.）is a dicotyledonous plant of the family Polygonaceae,which is an important medicinal food crop of minor cereals.Tartary buckwheat is rich in nutrients and other bioactive flavonoids,with anti-inflammatory,antioxidant,antiatherosclerosis,reducing hypertension and other health functions.Flavonoids are the main health components of buckwheat which have a significant impact on the quality of buckwheat.Therefore,it is of great significance to explore the excellent regulatory genes associated with the synthesis of buckwheat flavonoids and analyze their functional mechanisms for the molecular improvement of high flavonoids in buckwheat.The biosynthesis of Tartary buckwheat flavonoids is an extremely complex process which involves the participation of a large number of structural genes and numerous transcriptional regulatory genes.Currently,most of the structural genes and partial transcriptional regulatory genes relevant to flavonoid biosynthesis in buckwheat have been functionally identified,but research on these genes has focused on nonseed tissues.The seed is the primary utilized part of Tartary buckwheat,so the study on the biosynthesis of flavonoids in seed is more meaningful for molecular breeding of high flavonoids.In a previous study,our team identified a transcription factor belonging to R2R3-MYB as a possible key regulatory gene for the biosynthesis of flavonoids from Tartary buckwheat seeds through combined transcriptome and metabolome analysis of Tartary buckwheat seeds at different developmental periods.In this study,the SG7R2R3-MYB transcription factor was cloned from high-flavonoid tartary buckwheat cultivar ‘Heiku 013’ and named FtMYBF.The effect on flavonoid accumulation and flavonoid synthesis-related gene expression was clarified through characterization and expression analysis of its transcription factors and functional identification of the transgene.This study initially clarified the function and regulatory mechanism of FtMYBF in the biosynthesis of flavonoids,providing a theoretical reference for future molecular breeding of high-flavonoid buckwheat.The main findings are as follows:1.FtMYBF was cloned by RT-PCR from the high-flavonoid tartary buckwheat “Heiku 013”,and the length of coding region is 1119 bp,encoding 372 amino acids.Multiple sequence alignment and phylogenetic tree analysis showed that FtMYBF is a member of the SG7 subfamily of plant R2R3-MYB transcription factors.2.The subcellular localization vector 35 S ∷ FtMYBF-GFP was constructed,and the results of subcellular localization revealed that the FtMYBF gene was mainly concentrated in the nucleus.3.The p GBKT7-FtMYBF recombinant vector and analyzed for transcriptional activation activity was constructed by using yeast system,and the results showed that FtMYBF transcription factor has strong transcriptional activation activity.4.Gene co-expression analysis demonstrated that FtMYBF was coexpressed in seeds with several structural genes related to flavonoid synthesis CHS,CHI,F3 H,F3’H,FLS,DFR,ANR and ANS.Correlation analysis between gene expression and total flavonoid content showed that the expression of FtMYBF in different tissue parts was highly positively correlated with total flavonoid content.5.The overexpression vector p K7WG2D-35S-FtMYBF,and transform Arabidopsis with Agrobacterium mediated dip in method was constructed.The contents of total flavonoids and each flavonoid compound as well as the expression of structural genes related to the flavonoid synthesis pathway were analyzed in transgenic Arabidopsis thaliana.The results showed that the total flavonoid content and the contents of 18 flavonoids were significantly increased in FtMYBF overexpressing Arabidopsis compared to wild-type Arabidopsis.Gene expression analysis showed that the expression of At CHS,At CHI,At F3 H,At F3’H and At FLS were significantly up-regulated in transgenic Arabidopsis（P<0.01）,while the expression of the DFR,ANS gene of the anthocyanin synthesis branch was not significantly changed.In conclusion,FtMYBF is a member of the R2R3-MYB SG7 subfamily and is a nuclear-localized transcriptional activator;it may promote the biosynthesis of flavonoids and flavonols in tartary buckwheat by positively regulating the expression of CHS,CHI,F3 H,F3’H and FLS genes.