滇龙胆GrSLS1基因的克隆与原核表达  

Cloning and Prokaryotic Expression of GrSLS1 Gene in Gentiana rigescens

作　　者：张晓东[1] 李彩霞[1] 王连春[1] 李涛[1] 王元忠[2] 

机构地区：[1]玉溪师范学院资源环境学院,云南玉溪653100 [2]云南省农业科学院药用植物研究所,昆明650223

出　　处：《西北植物学报》2014年第7期1311-1317,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(81260608);云南省教育厅科学研究基金重点项目(2013Z075);科技部"十二五"国家科技支撑计划(2011BAI13B02-04)

摘　　要：以滇龙胆转录组为基础,采用RT PCR技术克隆了滇龙胆幼叶裂环番木鳖苷合酶基因,命名为GrSLS1（GenBank登录号KF941191）.序列分析结果显示,GrSLS1基因开放阅读框长1 560 bp,编码519个氨基酸;GrSLS1蛋白相对分子量为59.33 kD,pI为8.96.生物信息学分析结果表明,GrSLS1蛋白属于SLS家族成员,其N端含有一跨膜螺旋区域（10～32）;二级结构分析结果表明,GrSLS1蛋白主要由α螺旋和无规则卷曲组成;系统发育分析表明,GrSLS1与帽柱木MsSLS2蛋白亲缘关系最近.构建原核表达载体pGEX 4T GrSLS1,对GrSLS1基因进行原核表达,SDS PAGE结果表明所表达蛋白与预期蛋白大小一致.该研究为进一步研究GrSLS1基因功能及通过在龙胆中过表达该基因提高龙胆苦苷含量奠定基础.Based on the transcriptome of Gentiana rigescens,a gene designated as GrSLS1 （GenBank NO.KF941191） encoding secologanin synthase was cloned from young leaves of G.rigescens by RT PCR technology.The sequence analysis results showed that the open reading frame of GrSLS1 gene has a length of 1 560 bp coding for 519 amino acids,and the relative molecular weight of GrSLS1 protein is 59.33 kD with its pI of 8.96.Bioinformatics analysis indicated that GrSLS1 protein belongs to the member of SLS family,and it has a transmembrane helix（10 32） in the N terminal.Results of secondary structure analysis suggested that GrSLS1 protein composes of mainly a helix and irregular coils.Results of phylogenic analysis showed that GrSLS1 protein is close to MsSLS2 of Mitragyna speciosa.The prokaryotic expression of GrSLS1 gene was done after construction of its prokaryotic expression vector pGEX 4T GrSLS1,and the SDSPAGE results displayed that the expressed protein was consistent with the anticipated size.This study will lay a foundation for further functional research of GrSLS1 gene and the improvement of gentiopicroside content via overexpression of this gene in gentian.

关 键 词：滇龙胆 GrSLS1基因 基因克隆 序列分析 原核表达 

分 类 号：Q785[生物学—分子生物学] Q786