Functional Characterization And Application Of Flavonoid UDP-glycosyltransferases From Three Liverworts

Flavonoid glycosides are common plant secondary metabolites,which can be synthesized by plants.They have broad pharmacological activities and mostly exist as flavonoid glycosides,the reason is that glycosylation can increase its stability,water solubility and the physiological activities.Flavonoid glycosides have extensive physiological effects and pharmacological activities with good application prospects.Glycosylations in plants are catalyzed by glycosyltransferases,mostly by uridine diphosphate-glycosyltransferases(UGTs),which catalyze flavonoid substrates to the corresponding flavonoid glycosides.Due to the rapid development of modern biotechnology,a series of studies have been conducted on the functional characteristics of glycosyltransferase genes that catalyze flavonoid glycoside compounds and their synthetic biology applications.For liverworts,there are many kinds of flavonoids and their glycosides in the plant,but the identification and research of glycosyltransferases have been slow,and only two cases have been identified.This research cloned and identified flavonoid glycosyltransferases from these three liverworts and their function identifications,enzymatic characteristics and synthetic biology characteristics were carried out.1.Cloning,prokaryotic vector construction and protein expression of UGTs in three liverwortsSeven candidate genes annotated as flavonoid:UDP-glycosyltransferase were screened from the transcriptome database of three liverworts.Four of them were screened from C.conicum and named CcUGT1,CcUGT2,CcUGT3 and CcUGT4,respectively.Two candidate genes were found in the transcriptome database of M.emarginata and named MeUGT1 and MeUGT2,respectively.A candidate gene was found in the transcriptome database of M.paleacea and named as MpUGTI.The sequence comparison revealed that the C-terminus of these seven UGTs all have a PSPG box of 44 amino acids,which is unique to UGTs.It’s phylogenetic research results indicated that they were clustered between the 7-O-glycosylation clade and the 3-O-glycosylation clade.It is speculated that these UGTs can make the 7-OH or 3-OH of flavonoids to be glycosylated.The ORFs of these UGT genes were cloned using appropriate primers.After being digested with restriction endonucleases,the amplified products were ligated into the pET32a vector and transformed into E.coli strain BL21(DE3)for protein expression.Finally,the purified proteins were obtained.2.Functional identification of UGTs in three liverwortsEnzymatic activities showed that MeUGT1,MeUGT2 and MpUGT1 all can catalyze flavonol aglycones(quercetin,kaempferol,myricetin,isorhamnetin)into their 3-O-glycosylated form.In addition to flavonols,MeUGT2 can also catalyze three flavones(apigenin,luteolin and chrysophyl)and one flavanone(naringenin)into their 7-O-glycosylated form.After determining the optimal pH and temperature,the kinetic parameters of enzyme activity showed that MeUGTl was more active to flavonols substrates than MeUGT2 and MpUGT1.And the catalytic activity of MeUGT2 on flavonols is greater than that on flavones,followed by flavanones.When MeUGT2 was expressed in E.coli,the yield of flavonol 3-O-glucosides reached to 40-60%with feeding of the substrates kaempferol or quercetin under optimal conditions.Furthermore,we constructed expression vector of MeUGTl and then it was transformed in plants.Heterologous expression of MeUGTl in Arabidopsis thaliana increased the flavonol glycoside contents in the plants.Compared with wild-type plants,the content of kaempferol 3-O-glucoside 7-O-rhamnoside(K3G7Rha)in transgenic plants has increased over 30%.Therefore,the UGTs characterized in this study could be used to synthesize flavonoid glucosides in E.coli and in plants.In conclusion,the research has been to identify some UGTs which could provide candidate genes and strategies for the biosynthesis of flavonol 3-O-glucoside.