Cloning and characterization of CCoAOMT gene from Gossypium hirsutum L.

According to results of cotton fibre preferentially expressed cDNA library analysis,we designed primers from EST sequences.RT-PCR method was used to clone CCoAOMT gene.A gene coding for CCoAOMT,designated as GhCCoAOMT2(GenBank accession no.FJ376606) was isolated from cotton(Gossypium hirsutum L.).The length GhCCoAOMT2 cDNA is 1 002 bp,including a 12 bp 5′-UTR,an ORF of 747 bp,and a 243 bp 3′-UTR.This cDNA sequence encoded a polypepide of 248 amino acid residues with a predicted molecular mass of 28.023 kDa and a basic isoelectric point of 5.39.Furthermore,a length of 1 442 bp sequence from genomic DNA of GhCCoAOMT2 was also cloned by PCR.The genomic DNA of GhCCoAOMT2 contains four exons and three introns.The deduced amino acid sequence of GhCCoAOMT2 had a high homology with PtCCoAOMT,NtCCoAOMT-3,and BnCCoAOMT-1.Semi-quantitative RT-PCR analysis revealed that GhCCoAOMT2 could be expressed in different kinds of cotton tissues,and its mRNA accumulated most abundantly in stem.To investigate the function of the GhCCoAOMT2 gene,the full-length open reading frame was fused into a prokaryotic expression vector pET-28a.SDS-PAGE indicated that the most high expression quantity was induced by 0.2 mmol/L IPTG treatment for 6 h at 37℃.