The Identification Of A Flavonoids Glycosyltransferase With Multiple Regioselective Glucosylation From Camellia Sinensis

Glycosylation of flavonoid derivatives,not only affects the astringent quality of tea beverages,but also related to resistance characteristics with tea plant.In our study,a gene encoding a flavonoids glycosyltransferase was isolated from Camellia sinensis was responsed to plant resistance stress.The recombinant protein showed the activity of glucosylation toward diverse substrates including naringenin,apigenin,kaempferol,quercetin,myricetin,kaempferide and kaempferol monoglucosides.These glycosylation reactions was found taking place on multiple sites such as 3-OH,7-OH.Also the accumulation of flavonol glycosidic product was found increased than control group in the overexpressed transgenic tobacoo.The main research results are as follows:1.The 3' and 5 ' RACE cloning technology was apply to obtained the full-length sequence of the flavonoid glycosyltransferase gene.The full-length cDNA of the gene is 1789 bp,a 236 bp 3' UTR(un-translation region)and a 128 bp length 5' UTR(un-translation region).The ORF(open reading frame)of this gene is 1428 bp,which encoding a protein of 475 amino acid residues(the theoretical molecular mass,53.72Da),with isoelectric point of 5.62.Signal peptide analysis showed that no peptide was found in the N or C terminal of this protein.Phylogenetic analysis revealed the flavonoid glycosyltransferase was classified in Cluster III group,which may function as a 7-OH flavonoid glycosyltransferase.A protein crystal structure model was constructed according to the VvGT1,this protein showed 31% similarity of this protein compared to VvGT1,molecular docking analysis indicated this protein may glycosylated at 3-OH and 7-OH sites of flavonoids substrates.We submitted this gene to UGTs Nomenclature Committee and was named CsUGT73A20.2.The ORF(open read frame)of CsUGT73A20 was cloned into recombinant plasmid pMAL-C2 X for prokaryotic expression and then transferred into Escherichia-coli Novablue.The fusion protein of CsUGT73A20 was purified by affinity chromatography.The reaction products of rCsUGT73A20 was identified by HPLC and UPLC-MS,it was showed that flavanones(naringenin),flavonoids(apigenin),flavonols(quercetin,kaempferol,kaempferide,myricetin)and flavonol glycosides(kaempferol 3-O-glucoside and kaempferol 7-O-glucoside)all could be catalyzed by CsUGT73A20.More interesting,multiple glycosylation products were detected in the reaction system,suggesting that glycosylation reaction of CsUGT73A20 occurred in multiple hydroxyl groups points.3.Enzyme kinetics were measured when at conditions of 35? and pH8,the KM value of naringenin,apigenin,quercetin,kaempferol,kaempferide,kaempferol 3-O-glucose glycosides and kaempferol 7-O-glucose glycoside substrates were 13.6 uM,18.2 uM,37.7 uM,4.9 u M,19.0 uM,27.5 uM and 13.1uM respectively,indicated the optimal substrate in vivo could be kaempferol.We also found when using kaempferol as substrate,the product glycosylation was affected by reaction pH.kaempferol 3-7-O-diglucoside were the highest product at pH 8.0,while under the conditions of pH 9.0,it was accumulated the most kaempferol 3-O-glucoside product.The following experiments showed whether at pH 8.0 or 9.0 conditions,the KM value of kaempferol 7-Oglucoside as substrate was less than kaempferol 3-O-glucoside as substrate,indicating that 3-OH glycosylation was the main enzyme reaction of rCsUGT73A20,also showing the activity of glucosylation at 7-OH were greatly damaged than 3-OH at pH 9.0.4.The overexpression vector pCB2004-CsUGT73A20 was constructed for tobacco and arabidopsis transformation.The positive transgenic tobacco and arabidopsis plants were detected by PCR analysis.UPLC-MS analysis of transgenic tobacco plants shown CsUGT73A20 also has glycosyltransferase activity in plants.