棉花咖啡酸-O-甲基转移酶基因的克隆及特征分析

Cloning and Characterization of COMT Genes from Gossypium hirsuturm L.

【目的】克隆棉花木质素合成关键酶基因GhCOMT1、GhCOMT2和GhCOMT3全长cDNA序列,分析其在不同组织部位的表达情况并进行原核表达研究。【方法】根据棉花纤维特异表达cDNA文库分析得到的EST序列设计引物,采用RT-PCR技术克隆基因,用生物信息学方法对获得的cDNA序列及推定的氨基酸序列进行分析。采用半定量RT-PCR方法研究GhCOMT1、GhCOMT2和GhCOMT3在不同组织中的表达。构建了基因的原核表达载体,经酶切鉴定后转化到大肠杆菌BL21(DE3)中并诱导表达。【结果】从发育的棉花纤维中克隆了3个COMT基因cDNAs,GhCOMT1(GenBank登录号为FJ479708)、GhCOMT2(GenBank登录号为FJ479709)和GhCOMT3(GenBank登录号为FJ848869)分别具有1101bp、1098bp、1071bp的开放阅读框,各自编码366、365和356个氨基酸。GhCOMT1、GhCOMT2和GhCOMT3在棉花各个组织中都有表达,GhCOMT1和GhCOMT2在根部的表达量最高,GhCOMT3在茎中表达量最高。SDS-PAGE电泳分析表明,3个蛋白的最佳诱导表达条件均为0.2mmol·L-1IPTG在37℃下诱导6h。【结论】从棉花中克隆了3个木质素合成关键酶基因GhCOMT1、GhCOMT2和GhCOMT3,为进一步的生物学功能研究及其应用奠定了基础。

[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 carded 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.