Cloning and Analyzing of Cinnamyl Alcohol Dehydrogenase Gene from Gossypium hirsuturm L.

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.