滇龙胆GrCMS基因的克隆与表达分析

Cloning and expression analysis of GrCMS gene in Gentiana rigescens

2-C-甲基-D-赤藓醇4-磷酸胞苷酰转移酶是赤藓糖磷酸酯(MEP)途径中的第三个催化酶。以滇龙胆转录组为基础,采用RT-PCR技术从滇龙胆幼叶克隆2-C-甲基-D-赤藓醇4-磷酸胞苷酰转移酶基因GrCMS,并进行原核表达和组织特异性表达分析。序列分析显示,GrCMS基因(登录号KJ917164)开放阅读框(ORF)长933 bp,编码310氨基酸,推测分子量为34.23 kD,等电点为7.68。蛋白质序列分析显示,GrCMS无信号肽,为亲水稳定蛋白,主要由α-螺旋和无规则卷曲构成,可能定位叶绿体;具有CMS保守结构域。进化分析结果表明,GrCMS蛋白与长春花CrCMS蛋白亲缘关系最近。原核表达结果表明,GrCMS与预期蛋白大小一致。定量PCR结果表明,GrCMS基因主要在叶中表达。结果为进一步研究该基因功能和龙胆苦苷生物合成途径奠定基础。

2-C-methyl-D-erythritol 4-phosphateeytidyltransferase（ CMS, EC 2.7.7.60） is the third enzyme in methylerythritol phosphate（MEP） pathway. The Open Reading Frame （ORF） of GrCMS gene was cloned by RT-PCR technology from young leaves of Gentiana rigescens based on the transcriptome of G. rigescens and its prokaryotie and the tissue specific expression analysis were performed. The ORF of GrCMS gene （accession number： KJ917164） was 933 bp long coding for a protein of 310 amino acids, and the predicted relative molecular weight of GrCMS was 34.23 kD with its theoretical pI of 7.68. The results of GrCMS protein analysis showed that GrCMS which possessed the conserved domains of CMS proteins and may localize in chloroplast was a hydrophilic stable protein without signal pepfide, and it was composed of mainly or-helix （ 26.45% ） and random coils （54.84%）. By phylogenetic analysis, GrCMS was close to CrCMS of Catharanthus roseus. By prokaryotic analysis, the recombinant protein of GrCMS gene in E. coli was approximately 60.23 kD （containing GST tag protein 26 kD）, which was consistent with the anticipated size. By real-time PCR analysis, GrCMS gene was primarily expressed in leaf. Our results will provide reference for further functional researches of GrCMS gene and the biosynthetic pathway of gentiopicroside.