Cloning And Prokaryotic Expression Analysis Of UDP-glycosyl Transferase Gene From Citrullus Colocynthis Shrad

Cucurbitacin is the important medicinal active ingredient in Citrullus colocynthis Shrad.It has so many kinds of bioactivities,it can resist cancer,eliminate inflammation,protect liver,improve immunity of body and so on.The content of cucurbitacin glycoside is similar to cucurbitacin in plants.There are some results of the studies show that the glycoside of cucurbitacin E is the main factor to decide the bitter of C.colocynthis.The UDP-glycosyl transferase is the key element for Cucurbitacin E to become the glycoside of cucurbitacin E.In this study,we cloned two cDNA sequences of UDP-glycosyl transferase genes by using the leaves of C.colocynthis Shrad WM9 as material,and proceeded the analysis of prokaryotic expression for them.The study cloned two genes with RT-PCR,designated as UDP-E1 and UDP-E2,the total length of them is 1,546 bp and 1,559 bp,respectively.Biological information analysis showed that the length of ORF of UDP-E1 is 1314 bp,encoding 437 amino acids,the molecular weight of it is 49.02 kDa,the isoelectric point is 5.99,it belongs to the stable protein;the length of ORF of UDP-E2 is 846 bp,encoding 281 amino acids,the molecular weight of it is 32.78 kDa,the isoelectric point is 5.23,it belongs to the unstable protein.The UDP-El and UDP-E2 both belong to the family of glycosyl transferase,and don't have the transmembrane domain or signal peptide for them.UDP-E1 has 37.76%random curl,40.50%a-helix and 21.74%?-fold.UDP-E2 has 44.84%random curl,35.59%a-helix and 19.57%p-fold.The UDP-E1 and UDP-E2 are similar to UDP-glycosyl transferase genes of cucumis melo and cucumis sativus by the alignment of multiple sequence and the analysis of evolutionary tree.The ORF of UDP-E1 and UDP-E2 were Cloned and connected with the pET28a to build BL21(DE3)/pET28a-UDP-E1-ORF and BL21(DE3)/pET28a-UDP-E2-ORF.The expression quantity of exogenous protein reached 19.7%and 19.7%,respectively,by the induction of IPTG.The proteins exist as inclusion body.The expression quantity of soluble protein is 5.8%and 3.1%,respectively.These results provide the theoretical basis for the next study.