Cloning and protein expression of sterol-C-24-methyl transferase 2 in Tripterygium wilfordii
10.16438/j.0513-4870.2016-0320
- VernacularTitle:雷公藤甾醇C-24甲基转移酶(TwSMT2)的cDNA克隆及表达分析
- Author:
Hong-yu GUAN
1
;
Ping SU
2
;
Yu-jun ZHAO
2
;
Yu-ru TONG
1
;
Yu-jia LIU
1
;
Tian-yuan HU
1
;
Yi-feng ZHANG
1
;
Xia-nan ZHANG
1
;
Xiu-juan WANG
1
;
Wei GAO
1
;
Lu-qi HUANG
2
Author Information
1. School of Traditional Chinese Mediine, Capital Medical University, Beijing 100069, China
2. State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
- Publication Type:ORIGINAL ARTICLES
- Keywords:
Tripterygium wilfordii;
24-alkyl sterols;
TwSMT2;
bioinformatics analysis;
protein expression
- From:
Acta Pharmaceutica Sinica
2016;51(11):1799-
- CountryChina
- Language:Chinese
-
Abstract:
24-Alkyl sterols are the major players in the control of membrane component and plant growth. In this paper, we cloned an important rate-limiting enzyme:sterol-C-24-methyl transferase (SMT) in the sterol biosynthetic pathway according to the transcriptome data of Tripterygium wilfordii. suspension cells, whose full-length cDNA was 1 631 bp with an open reading frame of 1 080 bp, encoding a protein of 359 amino acids. It was estimated that theoretical isoelectric point (pI) was 6.43 and the molecular mass was 40.0 kDa. Bioinformatics analysis attributed the SMT gene to SMT2 family. The expression vector was constructed as the pMAL-c2x-TwSMT2 plasmid and the recombinant protein was expressed in E. coil BL21 (DE3) competent cells. After methyl jasmonate treatment, the relative expression level of TwSMT2 has improved significantly in 24 h. SDS-PAGE electrophoresis and Western Blot showed that protein of TwSMT2 in BL21 (DE3) strain was expressed after induction by IPTG. In this study, TwSMT2 was cloned for the first time and the recombinant protein was expressed, which lay the foundation for elucidation of the sterol biosynthetic pathway of Tripterygium wilfordii in the future.
