Cloning And Function Analysis Of Flavonoid3’,5’-hydroxylase In Tea [Camellia Sinensis (L.) O. Kuntze]

Catechins, which belong to flavanol compounds, are2-phenyl-benzo pyran derivativecompounds, mainly including catechins(C), epigallocatechin (GC), epicatechin (EC),epigallocatechin (EGC) and esterification products of epicatechin, like epicatechin gallate(ECG) and epigallocatechin gallate (EGCG). In the compounds above, C, EC and ECG are3,4-position dihydroxy of the B-ring,2-phenyl-benzopyran catechins; GC, EGC andEGCG are3,4, and5-position trihydroxy of the B-ring catechins. Determination of thedata for years in our group showed that EGC and its esterification products of EGCG arethe main components of tea catechins in tea plant, accounting for65%of the total catechin.Flavonoid3’,5’-hydroxylase, which belongs to the cytochrome P450mono-oxygenasesystem, is the only enzyme catalyzing the5-position hydroxylation of the B-ring.Prokaryotic expression system, yeast expression system and transgenetic tobacco systemhave been constructed to verify the function of F3’5’H in vitro following by full-RACEcloned the tea F3’5’H gene. Real-time PCR technology was used to identify the differencesof F3’5’H expression.The main results were showed:1. The full length sequence of CsF3’5’H was gained by RACE technology.5’ UTRarea and3’ UTR area were obtained on the basis of allele logged in NCBI.2.30amino acids in the both N-terminal of the predicted CsF3’ H and CsF3’5’Hproteins are signal peptide sequences. CsF3’ H and CsF3’5’H were predicted to have theflavonoid3’-hydroxylase activity and flavonoid3’,5’-hydroxylase activity, respectively,after phylogenetic analysis with CYP75proteins in other spieces.3. Recombinant plasmid pET32a(+)-CsF3’5’H was successfully constructed, thentransformed into expression host E.coli Rosetta (DE3). The optimized condition was IPTGinduction for5h at30°C. CsF3’5’H fusion protein had been purified up to86.4%by affinityresin purification.4. Recombinant plasmids pYES-dest52-CsF3’5’H, pYES-dest52-VvF3’5’H,pYES-dest52-PhF3’5’H-1and pYES-dest52-PhF3’5’H-2were successfully constructed, which achieving F3’5’H from different species expressed in S.cerevisiae. Enzyme activityon different substrates had been detected. YES-dest52-VvF3’5’H, pYES-dest52-PhF3’5’H-1and pYES-dest52-PhF3’5’H-2had apparent catalytic activity to naringeninand eriodictyol.5. Recombinant plasmid pCB2004-CsF3’5’H was constructed, then transformed intoAgrobacterium tumefaciens EHA105by electroporation. Transgenetic tobacco wasobtaining in this paper need to be verified.6. Enzyme activity detection system of flavonoid3’-hydroxylase and flavonoid3’,5’-hydroxylase were established by optimizing the HPLC conditions. It is showed that thetea crude enzyme might have the activity of flavonoid3’,5’-hydroxylas.7. Real-time PCR was performed to identify the different experssion level of relevantB-ring-hydroxylation flavonoid genes in different matural tea leaves. The results showedthat CsF3’5’H had rare expression level in roots and belonged to induced expression.