Gene Cloning And Functional Analysis Of Flavonoids Alcohol Metabolism Transcription Factors FtMYB7 And FtMYB9 In Fagopyrum Tataricum Gaertn

Buckwheat is a kind of food and medicine of the minor crops, rich in highly active flavonol compound rutin. At present, the MYB transcription factors which regulate the metabolism pathway of Arabidopsis thaliana flavonoids has been identified as the S7 subfamily. In this study, we obtained FtMYB7 and FtMYB9 gene by cloning from Jiujiang buckwheat cDNA, construction of pBI121-35S:FtMYB7 and pBI121-35S:FtMYB9 over expression vectors and genetic transformation of tobacco, quantitative expression analyze key enzyme genes of flavonoid pathway in To generation of transgenic tobacco lines and Jiujiang buckwheat total growth period, transient expression in tobacco leaves and in situ flavonoid staining technique. We identified that FtMYB7 and FtMYB9 genes are mainly involved in the regulation of the expression of key enzymes in the pathway of flavonoid metabolism. This provides a theoretical basis for further exploration of regulation mechanism of Fagopyrum tataricum with high activity of rutin compounds. The main results of this paper are as follows:?1? FtMYB7 gene has been cloned and identified:GenBank accession number is KM588379, the full length cDNA sequence is 876bp, the number of encoded amino acids was 291. FtMYB9 gene:GenBank accession number is KM588380, the full length cDNA sequence is 912bp, the number of encoded amino acids was 303. They all contain typical MYB type R2R3 domain. By constructing the phylogenetic tree, we identified that FtMYB7 belonging to the Arabidopsis S1 subfamily. NCBI-Blast protein sequence alignment results shows that the homology of SmMYB61 ?AGN52085? and SmMYB29 ?AGN52053? in Salvia miltiorrhiza is the highest, which is 62% and 61%, respectively. FtMYB9 belonging to the Arabidopsis S7 subfamily. NCBI-Blast protein sequence alignment results shows that the highest homology with VvMYBPA1 ?NP₀01268160? in vitis is 60%, and the homology of TcMYBPA?ADD51352? in cacao is 58%.?2? The real time expression analysis of various organizations in the whole growth period of Jiujiang buckwheat shows that:the expression of FtMYB7 gene was highest in the inflorescence of flowering period. The expression trend of FtUFGT1 gene in the whole growth period was positively correlated with the regulation of FtMYB7 gene. The expression of FtMYB9 gene was highest in the cotyledon of germinating stage. The expression trend of FtUFGTl gene in the whole growth period was related to the regulation of FtMYB9 gene.?3? Quantitative expression analysis of NtCHS, NtF3H and NtFLS genes in FtMYB7, FtMYB9 and FtMYB7+9 genes lines shows that:FtMYB7 and FtMYB9 genes play a negative role in the expression of NtCHS gene. FtMYB9 gene plays a positive regulatory role in the expression of NtF3H gene and NtFLS gene.?4? Through the transient expression with the micro needle injection in the wild tobacco leaf and in situ flavonoid staining, the results shows that:after the injection of 35S:FtMYB7 and 35S:FtMYB9 respectively, as well as the co-injection of 35S:FtMYB7+9, the tobacco leaves have obvious fluorescence. These results indicate that there are a large number of flavonoids synthesis.