雷公藤甾醇C-24甲基转移酶(TwSMT2)的cDNA克隆及表达分析

Cloning and protein expression of sterol-C-24-methyl transferase 2 in Tripterygium wilfordii

24-烷基甾醇具有构成膜结构和调节植物生长发育的作用,本研究根据雷公藤悬浮细胞转录组数据,克隆得到雷公藤甾醇生物合成途径上的一个重要限速酶:甾醇C-24甲基转移酶(SMT),其c DNA全长1 631 bp,开放阅读框1 080 bp,编码359个氨基酸。在线预测所编码蛋白的理论等电点为6.43,分子质量为40.0 k Da。生物信息学分析结果将此SMT基因归为SMT2家族。进一步构建p MAL-c2X-Tw SMT2重组质粒并转化大肠杆菌BL21(DE3),茉莉酸甲酯诱导表达分析结果显示:相比于对照组,经诱导后的Tw SMT2基因在24 h处有显著提高。SDS-PAGE及Western blot检测蛋白表达结果显示,IPTG可诱导表达出Tw SMT2蛋白。本研究首次克隆得到Tw SMT2基因,并获得重组蛋白,为进一步阐述雷公藤甾醇生物合成途径奠定基础。

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.