Cloning, sequence analysis, and prokaryotic expression of GrGPPS gene in Gentiana rigescens
10.7501/j.issn.0253-2670.2014.14.020
- Author:
Cai-Yun WANG
1
Author Information
1. College of Agriculture and Biological Technology, Yunnan Agriculture University
- Publication Type:Journal Article
- Keywords:
Gene cloning;
Gentiana rigescens Franch.;
GrGPPS;
Prokaryotic expression;
Sequence analysis
- From:
Chinese Traditional and Herbal Drugs
2014;45(14):2060-2068
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To obtain the indispensable key enzyme involved in the monoterpene biosynthesis, the geranyl diphosphate synthase gene GrGPPS was cloned from Gentiana rigescens, and its sequence analysis and prokaryotic expression were performed. Methods: According to the GrGPPS gene sequence of transcriptome of triennial G. rigescens, a pair of specific primers was designed, and the full length of cDNA sequences was obtained by RT-PCR. Then TA cloning, sequencing and sequence analysing were performed. Prokaryotic expression vector pGEX-T-1-GrGPPS was constructed and transformed into Escherichia coli Rosetta for expression under 37°C and induced by 1 mmol/L IPTG. Results: The GrGPPS cDNA had a length of 1107 bp coding for 369 amino acids. Sequence analysis showed that GrGPPS was the member of "short-chain prenyltransferases" super family. Results of phylogenic analysis showed that GrGPPS was at the same evolutionary branch with AmGPPS. The SDS-PAGE results displayed that the expressed proteins were consistent with the anticipated size. Conclusion: The GrGPPS gene is cloned from G. rigescens, and the stable prokaryotic expression system of pGEX-T-1-GrGPPS is constructed. This work will provide a foundation for further purification, structure, and functional research of GrGPPS protein.
