Gene Cloning and Prokaryotic Expression of Glycosyltransferase from Leonurus heterophyllus Sweet(英文稿)

A glycosyltransferase gene (LhsUGT) was cloned from Leonurus heterophyllus Sweet using rapid amplification of cDNA ends (RACE). The full length cDNA of LhsUGT was 1562 bp, with a 1368 bp open reading frame (ORF) encoding a 455 amino-acid protein (LhsUGT) and a molecular weight of 50.47 kD and isoelectric point of 5.52. By bioinformatics analysis, three secondary structures were discovered in LhsUGT, including 43.1% helix, 17.5% β-sheet and 39.4% random coil structural elements. There was a conserved PSPG domain at the C-terminal of LhsUGT, demonstrating that the enzyme encoded by LhsUGT belonged to the glucosyltransferase super family. Homology analysis by BLAST showed that the sequence similarities of glycosyltransferase between L. heterophyllus and other plants were 26.4% - 68.0%. Phylogenetic tree analysis indicated that LhsUGT might glycosylate simple phenolic compounds as it was found to be clustered with group L in Arabidopsis thaliana. SDS-PAGE analysis showed recombinant LhsUGT with approximate molecular weight of 69 kD was successfully expressed by a prokaryotic expression system, which contained His tags of 17.9 kD at the N-terminal and reached maximum expression level until 5 h after induction by IPTG (Isopropyl β-D-1-thiogalactopyranoside). The results in the study will lay the foundation for future studies on the biological functions of LhsUGT by enzymatic reactions in vitro.