Cloning And Functional Characterisation Of Flavonoid Glucosyltransferase Genes From Stevia Rebaudiana Bertoni

Stevia is a perennial shrub of Asteraceae family,the leaves of stevia contain a various of flavonoids,glycosyltransferase catalyzes the glycosylation reaction,which can change the water solubility and transportability of flavonoids.At present,a variety of flavonoid glycosyltransferase genes have been cloned and applied to product flavonoid glycosides.In this study,four candidate flavonoid glycosyltransferase genes were screened,cloned and functional analyzed in leaves of stevia.The main results are as follows:1.Screening on the glycosyltransferase genes of flavonoidsSixteen glycosyltransferase genes might involved in the synthesis of flavonoid glycosides were screened out by analyzing transcriptomic data.Furthermore,the expression mode of comp63406_c0,comp72069_c0,comp74174_c0 and comp75290_c0were found similar to that of the key enzyme genes in the flavonoid synthesis pathway by using co-expression analysis.The phylogenetic tree showed that comp63406_c0,comp72069_c0,comp74174_c0 and comp75290_c0 were clustered in class III,class II,class III and class V,similar to PfUGT57,PhA5 GlcT,RhA53GlcT,Pf3 GlcT and Am7 GlcT,respectively.The above four genes were identified as putative flavonoid glycosyltransferase candidate genes.2.Cloning and bioinformatic analysis of candidate genesThe four putative glycosyltransferase genes were cloned in the leaves of stevia,named as SrUGT-634,SrUGT-720,SrUGT-741 and SrUGT-752,respectively.The DNA and cDNA sequences were compared respectively and found that all of four candidate glycosyltransferase genes contained no introns.Bioinformatic analysis indicated that all of the four genes had a conservative domain of UDP-glycosyltransferase,which belonged to the glycosyltransferase superfamily.The full-length of SrUGT-634 was 1395 bp,encoded464 amino acids,its calculated molecular mass was about 50.07 kDa,the deduced protein had a pI of 5.53.It was an unstable hydrophobin with the secondary structure of SrUGT-634 mainly containing ?-helix for 42.89%.The three-dimensional structure of the protein was constructed,with the similarity to 2vg8.1.A as 38.94%.The full-length of SrUGT-720 was 1506 bp,encoded 501 amino acids.Its calculated molecular mass wasabout 56.13 kDa.It was a stable hydrophilic protein with its secondary structure mainly contains ?-helix,which accounted for 43.91%.The three-dimensional structure of the protein was constructed,and the similarity with 5u6 n.2.A was 40.09%.The full-length of SrUGT-741 was 1395 bp,encoded 464 amino acids.Its calculated molecular mass was about 51.09 kDa,the deduced protein had a pI of 5.97.It was an unstable hydrophilic protein and its secondary structure mainly contained ?-helix,which accounted for 41.38%.The three-dimensional structure of the protein was constructed,and the similarity with5 nlm.1.A was 36.63%.The full-length of SrUGT-752 was 1476 bp,encoded 491 amino acids.Its calculated molecular mass was about 54.91 kDa with the pI of deduced protein5.20.It was an unstable hydrophilic protein.The secondary structure of the protein mainly contained ?-helix,which accounted for 41.34%.The three-dimensional structure of the protein was constructed,and the similarity of 2vg8.1.A was 28.70%.3.Recombinant expression and functional analysis of SrUGTs in Stevia rebaudianaThe above four candidate genes were inserted into the vectors pET-28a(+)respectively and then expressed in E.coli BL21(DE3),the recombinant proteins were expressed successfully under IPTG(final concentration 0.20 mmol/L)at 16?.SDS-PAGE showed that the molecular mass of SrUGT-634,SrUGT-720,SrUGT-741 and SrUGT-752 were nearly 50 kD,56 kDa,52 kDa,55 kDa,respectively.Purifing expression proteins with the Ni-NTA resin,and the biological activity were obtained by HPLC,the results showed that SrUGT-720 and SrUGT-752 could catalyze UDP-glucose to kaempferol,SrUGT-634 could catalyze UDP-glucose to apigenin,and SrUGT-741 could catalyze UDP-glucose to isoquercitrin.Furthermore,LC-MS analysis of the catalytic products revealed that no theoretical molecular weight products were found in the catalytic products of SrUGT-720,SrUGT-752 and SrUGT-634,the isoquercitrin were catalyzed to three glucoside compounds by SrUGT-741,but the products needed further validation.