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℃.