Functional Analysis Of Flavonoid 3',5'-hydroxylase (F3'5'H) From Tea Plant [Camellia Sinensis(L.)]

Flavonoid 3'5'-hydroxylase(F3'5'H)is an important branch point enzyme of flavan-3-ols'synthesis in tea plant.F3'5'Hs belong to CYP75A subfamily and catalyze the conversion of flavones,flavanones,dihydroflavonols and flavonols into 3',4',5'-hydroxylated derivatives.In this study,F3'5'H(CsF3'5'H)gene and its promoter were isolated from tea cDNA and genomic DNA libraries Camellia sinensis.31 amino acids(MALDTVFLLRELSFATLVILITHIFMRSILT)in the N-terminal of the predicted CsF3'5'H protein are a signal peptide sequences.CsF3'5'H was predicted to have the flavonoid 3',5'-hydroxylase activity,after phylogenetic analysis with CYP75 proteins in other spieces.The analysis of CsF3'5'H gene promoter indicated that MYB proteins and light signals possibly control CsF3'5'H transcription.In addition,CSF3'5'H promoter contained a pathogen and salt-responsive promoter element.Real-time PCR was performed to identify the different experssion levels of relevant B-ring-hydroxylation flavonoid genes in different matural tea leaves.The expression analysis showed that CsF3'5'H gene expression was tissue specific,very high in mature leaves and extremely low in the root.Interestingly,CsF3'5'H relative expression was enhanced by light and sucrose,which was consistent with the promoter analysis of this gene.Over-expression of CsF3'5'H produced new-delphinidin derivatives,and increased the content of cyanidin derivatives in the corollas of transgenic tobacco plants,resulting in deeper and redder flowers in transgenic plants compared with wild type controls.Sequence optimization and signal peptide modification were carried out for constructing recombinant plasmids pYES-dest52-VvF3'5'H?pYES-dest52-CzyF3'5'H1(FSI)?pYES-dest52-CzyF3'5'H2(FSII)?pYES-dest52-CzyF3'5'H(FSIII),which achieving F3'5'H from different species expressed in S.cerevisiae.Heterologous expression of the modified CsF3'5'H(CsF3'5'H gene fused with Vitis vinifera signal peptide,FSI)revealed that 4'-hydroxylated flavanone(naringenin,N)was the optimum substrate for the CsF3'5'H enzyme,and was effectively converted into both 3'4'-and 3'4'5'-forms.Of note,the ratio of 3'4'5'-to 3'4'-hydroxylated products in FSI transgenic cells was remarkably higher than in VvF3'5'H cells.These findings further explain the B-ring hydroxyl pattern of flavonoids in tea plants and provide an important basis for further studies of flavonoid biosynthesis in tea plants.Such studies would help accelerate flavonoid metabolic engineering in order to increase B-ring tri-hydroxyl product yields.