Cloning and Prokaryotic Expression of Squalene Epoxidase Gene from Panax vietnamensis var. fuscidiscus
10.13422/j.cnki.syfjx.20192212
- VernacularTitle: 野三七鲨烯环氧酶基因的克隆及原核表达
- Author:
Bao-jie WANG
1
;
Ling-ying ZHU
1
;
Qing-qing ZHOU
1
;
Shuai ZHANG
1
;
Xiao-hui MA
1
;
Fu-rong XU
1
Author Information
1. College of Pharmaceutical Science, Yunnan University of Chinese Medicine, Kunming 650500, China
- Publication Type:Research Article
- Keywords:
Panax vietnamensis var. fuscidiscus;
squalene epoxidase;
gene cloning;
bioinformatics analysis;
prokaryotic expression
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2019;25(22):147-153
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To clone the squalene epoxidase genes of Panax vietnamensis var. fuscidiscus(PvfSE),and perform bioinformatics analysis and prokaryotic expression. Method: Total RNA was extracted from root of P. vietnamensis var. fuscidiscus by trizol method, and reverse-transcribed into first stand of cDNA. Specific primers for PvfSE cloning were designed according to the transcriptome data of P. vietnamensis var. fuscidiscus,and the cDNA sequence of PvfSE gene was isolated. Bioinformatics of PvfSE was analyzed by relevant software. The prokaryotic expression vector pMal-c2X-PvfSE was built to express recombinant protein in Escherichia coli cells. Result: The PvfSE gene contained a 1 887 bp open reading frame,encoding a predicted protein of 628 amino acids. The calculated molecular weight was 68.8 kDa,the theoretical isoelectric point was 9.28,the aliphatic index was 95.18,the grand average of hydropathicity was -0.060, and the instability index was 40.36. The protein was unstable. Bioinformatics analysis showed that PvfSE had two transmembrane domains and no signal peptide. PvfSE was most likely to be located in chloroplast or cytoplasmic membrane. PvfSE was a mixed protein with FAD/NAD(P) binding domain and squalene epoxidase domain. Sequence alignment and phylogenetic analysis demonstrated that PvfSE had a relatively close relationship with CpSE1,CpSE3,OsSE1 and OsSE2,which was involved in the biosynthesis of triterpene saponins in Cucurbita pepo and Ononis spinosa. In addition,PvfSE protein was expressed in E. coli. Conclusion: In this study,PvfSE gene was cloned and expressed in BL21(DE3),which lays a foundation for the further study on the gene functions of PvfSE and the biosynthetic pathway of triterpenoid saponins in P. vietnamensis var. fuscidiscus.
