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。根据棉花纤维特异表达cDNA文库分析得到的肉桂醇脱氢酶基因EST序列设计引物,采用RT-PCR技术从棉花中克隆了一个CAD基因,命名为GhCAD5(GenBank登录号为FJ848868)。研究结果表明:GhCAD5全长1488bp,具有一个1068bp的开放阅读框,5′非编码区为140bp,3′非编码区为280bp,编码355个氨基酸,预测分子量约为38.403kDa,等电点为7.67。氨基酸同源性分析发现,GhCAD5与拟南芥AtCAD1和水稻OsCAD4的同源性较高。利用PCR方法克隆了GhCAD5基因的基因组序列,长度为1695bp,包含6个外显子和5个内含子。将GhCAD5的ORF连接于原核表达载体pET-28a中,经IPTG诱导,获得了相应蛋白的表达。棉花GhCAD5基因的克隆为进一步研究其生物学功能和应用奠定了基础。
Cinnamyl alcohol dehydrogenase(CAD)is a key enzyme in the pathway of phenylpropanoid metabolism during lignin forming.According to results of cotton fibre preferentially expressed cDNA library analysis,primers of EST sequence were designed.RT-PCR method was used to clone CAD gene,A gene coding for CAD,designated as GhCAD5(GenBank accession No.FJ848868)was isolated from cotton(Gossypium hirsuturm L.).The full length GhCAD5 is 1 488 bp,including a 140 bp 5'-UTR,an ORF of 1 068 bp,and a 280 bp 3'-UTR.This cDNA sequence encoded a polypepide of 355 amino acid residues with a predicted molecular mass of 38.403 kDa and a basic isoelectric point of 7.67.The deduced amino acid sequence had a homology with AtCAD1 and OsCAD4.Furthermore,a length of 1 695 bp sequence from genomic DNA of GhCAD5 was also cloned by PCR.The genomic DNA of GhCAD5 contains six exons and five introns.The amplified ORF was ligated into the prokaryotic fusion expression vector pET-28a.E.coli BL21 was transformed with this recombinant vector and induced by IPTG for expression.The result indicated that the protein size of ORF matched the prediction.This paper report of the isolation and characterization of CAD cDNA clone involved in the lignin biosynthesis of cotton plants.Furthermore,the structure and function of this gene was analyzed and predicted.
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