比利时杜鹃花类黄酮3'-羟化酶(F3'H)基因克隆及功能分析OA北大核心CSTPCD

[Objective]The flavonoid 3'-hydroxylase(F3'H)is a key enzyme in the synthesis of anthocyanins in plants,and the function and expression characteristics of F3'H gene in Rhododendron hybridum Hort were investigated.[Method]The petals and roots,stems and leaves of R.hybridum Hort at different developmental stages were used as experimental materials.The flavonoid 3'-hydroxylase gene sequence information of flavonoid biosynthesis pathway was screened from the R.hybridum Hort transcript database and bioinformatics analysis was performed,and the RhF3'H gene was cloned by reverse transcription(RT-PCR)technology.The anthocyanin content in the petals of R.hybridum Hort at different stages was determined by ultraviolet spectrophotometer,and the expression of RhF3'H gene was analyzed by RT-qPCR technology on different tissues of petals at different development stages and maturity stages.The overexpression vector 35S:RhF3'H-GFP recombinant vector was constructed by Gateway technology for subcellular localization verification,and p1302-RhF3'H overexpression vector was constructed to infect the petals of R.hybridum Hort.[Result]The RhF3'H gene was 1 557 bp in length and encoded 518 amino acids,and the protein encoded by RhF3'H belonged to the P450 superfamily with a conserved F3'H domain.Phylogenetic tree analysis showed that RhF3'H was the most closely related to Dimocarpus longan and Litchi chinensis F3'H proteins.The results of RT-qPCR showed that RhF3'H was expressed in different petal stages and roots,stems,and leaf tissues of R.hybridum Hort.The expressions of RhF3'H gene were higher in the blooming and decaying stages of different petal development stages,which was consistent with the anthocyanin content,and subcellular localization analysis showed that RhF3'H mainly existed on the cell membrane.Compared with CK and p1302,p1302-RhF3'H was significantly higher in the petals of R.hybridum Hort and its anthocyanin content significantly increased.[Conclusion]The RhF3'H gene was expressed in the petal cell membrane,and the expression pattern was consistent with the accumulation trend of anthocyanins.Overexpression of the RhF3'H gene promotes the synthesis of anthocyanins.