Cloning of flavonoid 3'-hydroxylase gene from Fagopyrum tataricum and its tissue-specific expression under cold stress / 中草药

ABSTRACT

Objective: To clone and analyze the full-length of flavonoid 3'-hydroxylase (F3'H) gene from tartary buckwheat (Fagopyrum tataricum) and to express it in Escherichia coli. FtF3'H gene expression and anthocyanins accumulation is also to be analyzed in tartary buckwheat sprout under cold stress. Methods: Homology cloning and RACE method were used to obtain FtF3'H gene from flower buds of tartary buckwheat. The recombinant vector pET-30b (+)-FtF3'H was constructed and expressed in E. coli BL21 (DE3). Semi-quantitative RT-PCR was used to analyze FtF3'H gene expression when spectrophotometric method was used to determine anthocyanin content. Results: FtF3'H gene contains an open reading frame (1470 bp) encoding 489 amino acids and belongs to cytochrome P450 family. SDS-PAGE analysis of IPTG induced recombinant E. coli BL21 (DE3) showed that a predicted 54000 Da fusion protein was expressed in the culture. Cold stress significantly enhanced the expression level of FtF3'H and anthocyanin accumulation (P < 0.05). Conclusion: FtF3'H gene could be cloned from F. tataricum and efficiently expressed in E. coli. Under cold stress, FtF3'H gene may enhance its expression level to promote anthocyanin accumulation, by taking part in the process of cold-stress resistance of F. tataricum sprouts.