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以陆地棉‘CRI36’的叶片为材料,使用RACE技术克隆到了棉花叶绿体Cu/Zn-SOD酶基因。基因序列全长共1043bp,含有完整的开放阅读框。推导的氨基酸序列分析显示含有叶绿体信号肽,和已知植物的叶绿体Cu/Zn-SOD酶蛋白的氨基酸残基的同源性在66%～74%之间。基因的表达谱分析显示:棉花叶绿体Cu/Zn-SOD酶基因主要在叶片、茎中表达,根、花和下胚轴中没有检测到信号,即基因的表达主要在棉花的绿色组织。不同生育期的表达谱结果证实:该基因主要在苗期表达,以后表达逐渐减少。用pET-2la(+)构建了原核表达载体,在大肠杆菌BL21(DE3)的表达结果显示:表达后得到一个29.0kD的新蛋白,其分子量与预期目标一致。对SOD酶活性的分析证实,重组菌的酶活性显著增加,证明克隆的基因具有活性。
The cotton chloroplast Cu / Zn-SOD enzyme gene was cloned by RACE using the leaves of upland cotton ’CRI36’. The gene sequence of the total length of 1043bp, contains a complete open reading frame. The deduced amino acid sequence analysis showed that the amino acid residues of the chloroplast signal peptide of chloroplast and the chloroplast Cu / Zn-SOD enzyme of known plants were between 66% and 74%. Gene expression profiling showed that the Cu / Zn-SOD gene of cotton chloroplast was mainly expressed in leaves and stems, and no signal was detected in roots, flowers and hypocotyls. The gene expression was mainly in the green tissues of cotton. The expression profiles of different growth stages confirmed that the gene was mainly expressed in seedling stage and decreased gradually thereafter. The prokaryotic expression vector was constructed with pET-2la (+). The result of the expression in E. coli BL21 (DE3) showed that a new protein of 29.0kD was obtained after expression, and its molecular weight was consistent with the expected target. Analysis of SOD enzyme activity confirmed that the enzymatic activity of the recombinant bacteria was significantly increased, demonstrating that the cloned gene was active.