棉花肉桂醇脱氢酶基因GhCAD3的克隆及原核表达  

CLONING AND PROKARYOTIC EXPRESSION OF GhCAD3 GENE FROM Gossypium hirsuturm L.

作　　者：倪志勇[1] 李波[1] 范玲 

机构地区：[1]新疆农业科学院核技术生物技术研究所,新疆乌鲁木齐830091

出　　处：《核农学报》2010年第5期910-916,921,共8页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金(31060173);国家自然科学基金(30660088);国家"863"项目(2006AA10Z184);新疆自治区高技术研究发展计划项目(200611101);农业部转基因重大专项(2009ZX08005-011B)

摘　　要：肉桂醇脱氢酶(CAD)是木质素生物合成过程中的一个关键酶类。本研究从棉花中克隆了一个CAD基因,命名为GhCAD3(GenBank登录号为FJ376601)。GhCAD3全长1573 bp,具有1个1080 bp的开放阅读框,5′非编码区为35 bp,3′非编码区为458 bp,编码359个氨基酸,预测分子量约为39.116kD,等电点为7.48。氨基酸同源性分析发现,GhCAD3与其他CAD一致性为64.13%。为了进一步研究GhCAD3基因的功能,构建了该基因的原核表达载体pET-28a-CAD3,经酶切鉴定后转化到大肠杆菌BL21(DE3)中。SDS-PAGE电泳分析表明,最佳诱导表达条件为0.5 mmol/L IPTG在37℃下诱导7 h。Cinnamyl alcohol dehydrogenase（CAD） is a key enzyme in the pathway of phenylpropanoid metabolism during lignin forming.A gene coding for CAD designated as GhCAD3（GenBank accession No.FJ376601） was isolated from cotton（Gossypium hirsuturm L.）.The full length GhCAD3 is 1573 bp including a 35 bp 5′-UTR,an ORF of 1080 bp and a 458 bp 3′-UTR.This cDNA sequence encoded a polypepide of 359 amino acid residues with a predicted molecular mass of 39.116 kD and a basic isoelectric point of 7.48.The deduced amino acid sequence had a 64.13% identity with CAD from other plants.To investigate the function of GhCAD3 gene,the full-length open reading frame was fused into a prokaryotic expression vector pET-28a.Double endonucleases digestion showed that the recombinant vector pET-28a-CAD3 was successfully constructed and transformed into E.coli BL21（DE3） cells.SDS-PAGE indicated that the best expression quantity was induced with 0.5 mmol/L IPTG treatment for 7 h at 37℃.

关 键 词：棉花 肉桂醇脱氢酶 基因克隆 原核表达 

分 类 号：S562[农业科学—作物学]