Cloning and prokaryotic expression of Gh4CL2 gene from Gossypium hirsutum L.

4-coumarate:CoA ligase (4CL) is a key enzyme in the pathway of phenylpropanoid metabolism during lignin forming.A gene coding for 4CL,designated as Gh4CL2 (GenBank accession no.FJ848870) was isolated from cotton (Gossypium hirsutum L.).The full length Gh4CL2 cDNA is 2 332 bp,including a 64 bp 5'-UTR,an ORF of 1 725 bp,and a 543 bp 3'-UTR.This cDNA sequence encoded a polypeptide of 574 amino acid residues with a predicted molecular mass of 62.106 kD and a basic isoelectric point of 5.94.The deduced amino acid sequence had a high homology with 4CL from Populus tremuloides,Glycine max and Lithospermum erythrorhizon.To investigate the function of the Gh4CL2 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-4CL2 was successfully constructed and transformed into E.coli BL21(DE3) cells.SDS-PAGE indicated that the most high expression quantity was induced by 0.5 mmol/L IPTG treatment for 4 h at 37℃,but the recombinant proteins mainly appeared as inclusion bodies.