Cloning And Functional Analysis Of CsUGGT Gene In Camellia Sinensis

Catechins are a kind of important secondary metabolites in Camellia sinensis(L.)O.Kuntze.They have many beneficial effects on human health,and play an important role in the formation of tea color,aroma and taste quality.Further studies on the structural genes and regulatory genes in the biosynthetic pathway of tea catechins have great effects on tea processing and tea germplasm resources.The synthesis of Galloylated catechins is clear now.There is a research confirmed that?-glucogallin(1-O-galloyl-?-D-glucose;?G)exerts a dual role,functioning not only as an acyl acceptor but also as an efficient acyl donor.In our previous study,we obtained more than ten glycosyl transferase.Through the analysis of gene expression,it was found that the expression level of UGGT was significantly correlated with the content of EGCG,and it was speculated that UGGT is the direct enzyme for ?G biosynthesis.In this paper,we obtained the CDS sequence of UGGT useing PCR.Subsequently,the bioinformatic analysis of UGGT was performed.Moreover,the function of UGGT was identified using prokaryotic expression and over-expression.The major results of the present work were included as below.1.By designing the specific primers Cs UGGTF\Cs UGGTR,CsUGGT gene CDS sequence was successfully cloned by using No.1 Huangjin tea.Sequence analysis showed that the promoter sequence of CsUGGT gene with 1592 bp.It contained an open reading frame(ORF)of 1467 bp coding region which encoding a polypeptide of 488 amino acid residues with a predicable molecular mass of 54547.67,And the theoretical isoelectric point PI value was 5.30.A BLAST search of Genbank with the predicted amino acid protein sequence of CsUGGT was performed and results showed that the deduced CsUGGT shared83%,85%,84%,80%,79% and 75% of identity at the amino acid level with other plant UDP- glycosyl transferase from Juglans regia,Quercus robur,Vitis vinifera,Ricinus communis,Fragaria × ananassa,Citrus × paradisi,respectively.2.In order to identify the function of CsUGGT,pMAL-c5x prokaryotic expression vector was used to express CsUGGT protein in E.coli BL21.The optimal inductioncondition was IPTG with a final concentration of 0.3mM,induction temperature of 28?and induction time of 8 h,and then the soluble fusion protein was obtained.The protein band was about 54.5 KDa,which was consistent with the prediction.The recombinant CsUGGT protein was purified and concentrated in vitro.The results showed that the ?-glucogallin was biosynthesized by UGGT from gallic acid and uridine diphosphate glucose.UGGT had an optimum temperature of 30?and maximal reaction rates is pH 5.5.3.After the function of CsUGGT was verified in vitro,CsUGGT gene and plasmid pCXSN were recombined.The over expression vector CsUGGT-pCXSN and the empty vector was constructed successfully.Then the two vectors were transferred into Agrobacterium GV3101 respectively.In Agrobacterium mediated,the success of the over expression of transferred to the carrier CsUGGT-pCXSN tobacco(Nicotiana tabacum L.)fresh leaves.In resistance culture medium after induction of differentiation,rooting,transplanting,positive tobacco plants.Wild type and transgenic tobacco grass plant DNA extraction,PCR analysis,the results showed that over expression vector CsUGGT-pCXSN successfully imported transgenic tobacco.Extracting the enzymes of tabacco's fresh leaves,then verified by SDS-PAGE electrophoresis,the results showed,CsUGGT gene with fragment size 54 KDa was successfully expressed in tobacco(Nicotiana alata).The enzymatic activity of the crude enzyme was detected in vitro.It was found that the?-glucogallin was biosynthesized by UGGT from gallic acid and uridine diphosphate glucose.