Cloning, sequence analysis, and prokaryotic expression of 1-deoxy-D-xylulose-5- phosphate reductoisomerase GrDXR gene in Gentiana rigescens / 中草药

ABSTRACT

Objective: To clone the 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene GrDXR which is the key enzyme involving in the terpenoid biosynthesis from young leaves of Gentiana rigescens, and to perform its sequence analysis and prokaryotic expression. Methods: According to the GrDXR gene sequence of G. rigescens transcriptome, a pair of primers were designed, and the ORF of cDNA sequence was obtained by RT-PCR. Then TA cloning, sequencing, and sequence analysis were performed. Prokaryotic expression vector pGEX-4T-1-GrDXR was constructed and transformed into Escherichia coli Rosetta (DE3) for expression under the induction of IPTG. Results: The ORF of GrDXR had a length of 1 425 bp coding for 474 amino acids. Sequence analysis showed that GrDXR was the member of DXR family. Results of phylogenic analysis showed that GrDXR was close to RvDXR, HbDXR, and CrDXR. The pGEX-4T-1-GrDXR recombinant plasmid was constructed and the stable prokaryotic expression system was obtained. The SDS-PAGE results displayed that the expressed proteins were consistent with the anticipated size. Conclusion: The GrDXR gene is successfully cloned, and the stable prokaryotic expression system is established. This study will provide a foundation for further purification, structural and functional research of GrDXR protein.