Cloning And Characterization Of UDP-Glycosyltransferases UGT79 Family Of Epimedium Pubescens Maxim.

1.Genome-wide analysis of UGT gene family in Epimedium pubescens Maxim.E.pubescens is one of the species included as Herba Epimedii in Pharmacopoeia of the People’s Republic of China(2020 edition)and the first species of the Epimedium genus to achieve genome sequencing.In this study,we first used the glycosyltransferase(UGT)of other plants as a query to search the genome of E.pubescens,and confirmed the complete UGT domains and conservative domains(PSPG)in Pfams and NCBI CDD databases.Finally,339 UDP-glycosyltransferase(EpUGT)family genes of E.pubescens were screened,which is one of the large UGT families identified in plants to date.The bioinformatics analysis of the gene structures,phylogenetic relationships,gene structure,and expression profile of EpUGT was analyzed.We found that EpUGT were unevenly distributed on the six chromosomes of E.pubescens,tandem repeat events and segmental repeat events are the main driving force of UGT evolution in E.pubescens.The subcellular localization prediction of EpUGT showed that most UGT(77.29%)were located in the cytoplasm,and a few UGT were located in other organelle.The analysis of tissue expression profiles shows that the UGT genes were generally highly expressed in roots,leaves,and flowers.Epimedium plants could produce a large amount of icariin after high light intensity induced treatment,and some UGT genes responsive to high light intensity were selected.2.Selecting and cloning of A Group UGT79 family UGT genes in E.pubescens.Phylogenetic analysis revealed 16 distinct groups(A-Q)of EpUGT,including 14 highly conserved groups(A-N),and two newly discovered groups O and Q.Sequence structure and motif analysis were performed on these UGT,and the results showed that reliability of this group classification was strongly supported by the conserved motif composition,gene structure,and phylogenetic analysis,which suggested that there might be functional differences among different UGT family members.Generally,UGT responsible for modifying the 3-OH position of flavonoids are from the F group,whereas the UGT glycosylating 7-OH position of flavonoids is from B,C,or D group.The UGT glycosylate glycosides(GGTs,Glycoside Glycosyltransferases)are usually clustered in Group A,producing disaccharides or even polysaccharides.Group A contained the largest number of genes in the UGT gene family of E.pubescens,accounting for 30.97%of the total UGT genes.The critical UGT genes that are responsible for transferring an additional sugar moiety to the rhamnose moiety at 3-OH of icariin to form epimedin A,epimedin B,or epimedin C remain to be determined.And a total of six prenylated flavonol glycosyltransferases in other species of the Epimedium genus,two UGT capable of adding rhamnose to 3-OH and one UGT capable of adding glucose to 7-OH were also clustered in group A,therefore,we selected UGT genes of group A to study.Group A contains the UGT79 family,UGT91 family,and UGT94 family,among which the UGT79 family genes usually recognize flavonoids as substrates.Therefore,we selected 22 UGT preferentially expressed in either roots or leaves from 59 sequences of the UGT79 family as candidate genes,together with EpGT60 that was not located on any chromosome.To further investigate the function of these candidate genes,the total cDNA of E.pubescens leaves was used as a template,and nested PCR amplification was performed to amplify these 23 UGT,only nine of them were successfully cloned.3.Enzyme characterization of A Group UGT79 family UGT genes in E.pubescens.nine UGT genes were expressed in E.coli with the production of recombinant proteins.By detecting the enzyme activity of the purified recombinant protein,we identified three functional EpUGT.The 3-O-rhamnosyltransferase EpGT60,which recognized prenylated flavonols;the xylosyltransferase EpGT2(EpF3R2"XylT),which recognized prenylated flavonol 3-O-rhamnoside,and the glucosyltransferase EpGT43,which recognized anthocyanin rhamnoside.EpGT60 could catalyze the 3-OH of 8-prenylkaempferol or icariin with UDPrhamnose as a donor,producing baohuoside II and baohuoside I,respectively.The function of the EpGT60 protein is similar to the two UGT identified in the genus Epimedium,EpPF3RT identified in E.pseudowushanense,and EkF3URhaT identified in E.koreanum.However,EpGT60 only exhibits catalytic activity for 8-prenylated flavonols,while EpPF3RT and EkF3URhaT exhibit catalytic activity for flavonols with or without prenylated modification at the C-8 site.The best temperature,pH,and time conditions were investigated for the catalytic activity of EpGT60 with 8-prenylkaempferol.Enzymatic kinetics of the recombinant EpGT60 with 8-prenylkaempferol were also calculated.The subcellular localization of EpGT60 proteins is targeted to the cytosol.Furthermore,we produced 201.60 μg·mL-1 baohuoside Ⅱ on a large scale via cell culture carrying the g EpGT60 gene.EpGT2(EpF3R2"XylT)can modify icariin,baohuoside I,baohuoside II,and epimedoside A with UDP-xylose as donor.The products were confirmed by LC-MS and NMR to be epimedin B,sagittatoside B,icarisoside F,and epimedoside E,respectively.EpF3R2"XylT could transfer xylose to the 2"-OH of the prenylated flavonol 3-Orhamnoside,which all were kaempferol or kaempferide derivatives with both C-8 prenylation and 3-O-rhamnosylation,indicating that both the C-8 prenyl group and 3-0rhamnose are essential for catalyzation of EpF3R2"XylT.EpF3R2"XylT is a key glycosyltransferase for the synthesis of epimedin B in E.pubescens.The best temperature,pH,and time conditions were investigated for the catalytic activity of EpF3R2"XylT with icariin.Enzymatic kinetics of the recombinant EpF3R2"XylT with icariin,baohuoside I,and baohuoside Ⅱ were also calculated.The subcellular localization of EpF3R2"XylT proteins is targeted to the cytosol.The function of EpF3R2"XylT in planta was explored by transient expression in tobacco leaves.The crude protein of EpF3R2"XylT extracted from the infected tobacco leaves could catalyze the xylosylation of icariin to synthesize epimedin B.EpGT43 can catalyze cyanidin-3-O-rhamnoside with UDP-glucose as a donor.Through phylogenetic tree and sequence alignment,we suggested that the product of EpGT43 with cyanidin-3-O-rhamnoside and UDP-glucose might be cyanidin 3-O-[2-O-(glucosyl)]-rhamnoside,and the chemical structure of the product needs further confirmation by experiment.This study analyzed the UGT gene family of E.pubescens,identified three functional EpUGT in the UGT79 family,preliminarily explored the function of UGT79 family genes from E.pubescens,and elucidated biosynthetic pathway of baohuoside Ⅰ,baohuoside Ⅱ,epimedin B,sagittatoside B,epimedoside E,and ikarisoside F.These results indicated that the functional diversity of UGT79 family members in Group A of E.pubescens,which includes both GGT and UGT with modified 3-OH sites.