棉花肉桂醇脱氢酶基因GhCAD3的克隆及原核表达

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。

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

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℃.