Expression profiling and functional verification of flavonoid 3'-hydroxylase gene from leaves of Euryale ferox / 中国中药杂志

Leaves of Euryale ferox are rich in anthocyanins. Anthocyanin synthesis is one of the important branches of the flavonoid synthesis pathway, in which flavonoid 3'-hydroxylase(F3'H) can participate in the formation of important intermediate products of anthocyanin synthesis. According to the data of E. ferox transcriptome, F3'H cDNA sequence was cloned in the leaves of E. ferox and named as EfF3'H. The correlation between EfF3'H gene expression and synthesis of flavonoids was analyzed by a series of bioinforma-tics tools and qRT-PCR. Moreover, the biological function of EfF3'H was verified by the heterologous expression in yeast. Our results showed that EfF3'H comprised a 1 566 bp open reading frame which encoded a hydrophilic transmembrane protein composed of 521 amino acid residues. It was predicted to be located in the plasma membrane. Combined with predictive analysis of conserved domains, this protein belongs to the cytochrome P450(CYP450) superfamily. The qRT-PCR results revealed that the expression level of EfF3'H was significantly different among different cultivars and was highly correlated with the content of related flavonoids in the leaves. Eukaryotic expression studies showed that EfF3'H protein had the biological activity of converting kaempferol to quercetin. In this study, EfF3'H cDNA was cloned from the leaves of E. ferox for the first time, and the biological function of the protein was verified. It provi-ded a scientific basis for further utilizing the leaves of E. ferox and laid a foundation for the further analysis of the biosynthesis pathway of flavonoids in medicinal plants.

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

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.