滇龙胆1-脱氧-D-木酮糖5-磷酸合酶基因的克隆与表达分析  

Cloning and Expression Analysis of Gene Encoding 1-Deoxy-Dxylulose 5-Phosphate Synthase in Gentiana rigescens

作　　者：张海晨[1] 李彩霞[1] 王元忠[2] 张晓东[1] 

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

出　　处：《生物技术通报》2016年第4期128-136,共9页Biotechnology Bulletin

基　　金：云南省大学生创新训练项目(201411390001)

摘　　要：旨在克隆滇龙胆1-脱氧-D-木酮糖5-磷酸合酶基因GrDXS,并进行表达分析。以滇龙胆转录组为基础,采用RTPCR技术从滇龙胆幼叶克隆GrDXS基因,并进行原核表达和组织特异性表达分析。滇龙胆GrDXS基因(登录号:KJ624995)全长2145bp,编码714个氨基酸;GrDXS蛋白相对分子质量76.75kD,pI为6.93;属于DXS家族成员,可能定位于叶绿体;主要由α-螺旋和无规则卷曲构成;具有DXS蛋白的4类保守结构域:焦磷酸硫胺素结合折叠域(IPR029061,69-425、366-555、87-268、315-407、407-558)、类转酮酶嘧啶结合结构域(IPR005475,394-559、394-555)、转酮酶C端/丙酮酸铁氧还蛋白氧化还原酶结构域II(IPR009014,568-704,572-704)、转酮酶C端结构域(IPR005476,573-696)和1个转酮酶结合位点(IPR020826,500-516);与长春花CrDXS蛋白亲缘关系最近;GrDXS基因在大肠杆菌中表达的重组蛋白相对分子质量约为100kD,与预期蛋白大小一致;GrDXS基因主要在叶中表达。The aims of this study are to clone the gene GrDXS encoding 1-deoxy-D-xylulose 5-phosphate synthase from Gentiana rigescens, and to analyze its expression. Based on the transcriptome of G. rigescens, a GrDXS gene was cloned from young leaves of G. rigescens by RT-PCR technology, and its prokaryotic and tissue-specific expression were also performed. The GrDXS gene （ GenBank accession number ： KJ624995 ） had a length of 2 145 bp coding for 714 amino acids, and the relative molecular weight of GrDXS protein was 76.75 kD with its pI of 6.93. GrDXS protein belonged to the member of DXS superfamily, and may localize in chloroplast, GrDXS protein composed of mainly random coil （ 47.06% ） and a-helix （ 34.45% ）. Four kinds of conserved domains： Thiamin diphosphate-binding fold （ 1PR029061, 69-425, 366-555, 87-268, 315-407, 407-558）, Transketolase-like, pyrimidine-binding domain （IPR005475, 394-559, 394-555）, Transketolase C-terminal/Pyruvate-ferredoxin oxidoreductase, domain lI （IPR009014, 568-704, 572-704）, and Transketolase binding site （IPR020826, 500-516 ）, were all existing in GrDXS protein. GrDXS protein was the closest with CrDXS in Catharanthus roseus. The recombinant protein of GrDXS gene in Escherichia coli was approximately 100.00 kD （ containing GST tag protein 26 kD ）, which was consistent with the anticipated size. GrDXS gene was primarily expressed in leaf.

关 键 词：滇龙胆 1-脱氧-D-木酮糖5-磷酸合酶 生物信息学 表达模式 

分 类 号：S567.239[农业科学—中草药栽培] Q943.2[农业科学—作物学]