Abstract:

【Objective】 In this study, full-length cDNA of a key enzyme genes GhCOMT1, GhCOMT2 and GhCOMT3 related to lignin metabolism in cotton (Gossypium hirsuturm L.) were isolated. Analysis of the expression of GhCOMT1, GhCOMT2 and GhCOMT3 genes in different tissues of cotton was carried out and the genes were also expressed in the prokaryote. 【Method】 According to the results of cotton fibre preferentially expressed cDNA library analysis, the primers of EST sequence were designed. RT-PCR method was used to clone GhCOMT1, GhCOMT2 and GhCOMT3 genes, bioinformatics methods were used to analyze the obtained cDNAs sequence and the amino acid sequences were deduced. The expression of GhCOMT1, GhCOMT2 and GhCOMT3 genes in different tissues of cotton was analyzed by semi-quantitative RT-PCR. The full-length open reading frames were fused into a prokaryotic expression vector pET-28a, and then introduced into E.coli BL21（DE3）cells. 【Result】 Three genes coding for COMT, designated as GhCOMT1 (GenBank accession no.FJ479708), GhCOMT2 (GenBank accession no.FJ479709) and GhCOMT3 (GenBank accession no.FJ848869) were isolated from the developing cotton fibres. GhCOMT1, GhCOMT2 and GhCOMT3 contain open reading frames of 1 101 bp, 1 098 bp and 1 071 bp encoding proteins of 366, 365 and 356 amino acid residues, respectively. Semi-quantitative RT-PCR analysis revealed that GhCOMT1, GhCOMT2 and GhCOMT3 were differentially expressed in different tissues, and GhCOMT1 and GhCOMT2 mRNA accumulated most abundantly in root, GhCOMT3 was highly expressed in stem. Analysis of SDS-PAGE indicated that the highest expression quantity of the three proteins were induced by 0.2 mmol•L-1 IPTG treatment for 6 h at 37℃. 【Conclusion】 This paper reported the isolation and characterization of three COMT cDNAs clones involved in the lignin biosynthesis of cotton plants. Furthermore, the structure and function of these genes were analyzed and predicted.

Key words: Gossypium hirsuturm L.')">Gossypium hirsuturm L., caffeic acid O-methyltransferase, gene cloning, expression analysis, prokaryotic expression