Cloning, sequence analysis, and prokaryotic expression vector construction of glycosyltransferase gene BcUGT8 from Bupleurum chinense / 中草药

ABSTRACT

Objective: To clone the full-length cDNA of the uridine diphosphate glycosyltransferase (UGT) gene in Bupleurum chinense (BcUGT8), which may be involved with the saikosaponin biosynthesis, and to construct the prokaryotic expression vector. The work will provide the foundation for its further function verification by in vitro expression and activity analysis of the purified protein. Methods: RACE and LD-PCR were used to clone the full-length cDNA of BcUGT8, on the basis of its partial cDNA sequence obtained from our previous high-flux sequencing by Roche (454) GS FLX system. The open reading form (ORF) was PCR cloned using primers with corresponding restriction enzymes cutting sites. The PCR products were digested with corresponding restriction enzymes and then were inserted in expression vector pET-28a (+) to construct the recombinant expression vectors. Results: The full-length cDNA of UGT gene was cloned from B. chinense, and the prokaryotic expression vector was obtained. Conclusion: The full-length cDNA cloning, sequence analysis, and prokaryotic expression vector construction provide a substantial foundation for follow-up bio-function analysis of BcUGT8 through protein expression, purification, and activity analysis in vitro.