Cloning, sequence analysis, and prokaryotic expression of GrCYP450-17 gene in Gentiana rigescens / 中草药

ABSTRACT

Objective: To obtain the key enzyme gene GrCYP450 involving in the terpenoid biosynthesis, a CYP450-17 gene was cloned from young leaves of Gentiana rigescens, and its sequence analysis and prokaryotic expression were performed. Methods: According to the GrCYP450-17 gene sequence of transcriptome of triennial G. rigescens, a pair of primers were designed, and the ORF (Open reading frame)of cDNA sequences was obtained by RT-PCR. Then TA cloning, sequencing, and sequence analysis were performed. Prokaryotic expression vector pGEX-4T-1-GrCYP450-17 was constructed and transformed into E. coli Rosetta (DE3) for the expression under the induction of Isopropyl β-D-1-Thiogalactopyranoside (IPTG.). Results: The ORF of GrCYP450-17 has a length of 1 545 bp coding for 514 amino acids. Sequence analysis showed that GrCYP450-17 was the member of CYP714 family. Results of phylogenic analysis showed that GrCYP450-17 was close to SlCYP450 of tomato. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size. Conclusion: The GrCYP450-17 gene is successfully cloned, and the stable prokaryotic expression system is established. This study will provide a foundation for the further purification, structural and functional researches of GrCYP450-17 protein.