黑松过氧化物酶cDNA的克隆及其在大肠杆菌中的表达

Cloning of cDNA Coding Peroxidase fromPinus thunbergii and Its Expression in Escherichia coli

采用RT-PCR的方法,从黑松愈伤组织中获得过氧化物酶cDNA,其开放阅读框全长981bp,编码326个氨基酸残基。将该开放读框克隆到表达载体pET-15,构建重组表达质粒pET-15b-POD,转化E.coli BL21(DE3)构建工程菌,IPTG诱导工程菌高效表达过氧化物酶,通过Ni 2+螯合亲和层析得到了电泳纯的重组过氧化物酶。生物信息学分析表明,该蛋白具有植物过氧化物酶的典型结构,以及具有过氧化物酶家族保守的活性位点。活性分析表明该蛋白的确是黑松过氧化物酶,这一结果为松萎蔫病抗性机理的研究奠定了基础。

A peroxidase cDNA containing opening reading frame of 981bp and encoding a peptide of 326 a- mino acid residues was obtained from callus cells of Pinus thunbergii using RT-PCR. The open reading frame was cloned into pET-15b to construct recombinant plasmid pET-15b-POD. This recombinant plas- mid was introduced into Escherichia coli BL21 （DE3） to construct engineer bacteria and expression of re- combinant peroxidase was achieved by IPTG induction. The recombinant protein was purified by affinity chromatography on a Ni2＋ matrix column. Bioinformation analysis show that the protein had typical struc- tures of plant peroxidase and possessed all the conserved active sites. Bioassay results show that purified recombinant protein had peroxidase activity. This study laid a good foundation for further research on the resistant mechanism of pine wilt disease.