甜菜夜蛾拓扑异构酶I氨基酸突变对其DNA解旋活性的影响  

Effects of amino acid substitutions of topoisomerase I on its DNA relaxation activity in Spodoptera exigua( Lepidoptera: Noctuidae)

作　　者：张佩[1] 张兰[1] 张燕宁[1] 贾伟[1] 蒋红云[1] 

机构地区：[1]中国农业科学院植物保护研究所,农业部作物有害生物综合治理综合性重点实验室,北京100193

出　　处：《昆虫学报》2015年第9期933-940,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31371967,31272079)

摘　　要：【目的】为了探究甜菜夜蛾Spodoptera exigua拓扑异构酶I(topoisomerase I,Top I)氨基酸突变对其DNA解旋活性的影响。【方法】通过克隆甜菜夜蛾Top I基因,构建原核表达载体,采用完全重叠PCR定点突变技术,向甜菜夜蛾Top I的V420,L530,A653和S729(根据人Top I氨基酸序列编号)4个位点引入突变,将改造成功的重组Top I基因转化至大肠杆菌BL21(DE3)中,诱导重组蛋白表达、纯化,测定Top I突变对其解旋活性的影响。【结果】完全重叠PCR能实现甜菜夜蛾Top I定点突变。重组蛋白在体外得到稳定的表达,表达产物经SDS-PAGE电泳分析在96.0 k Da处出现特异性条带。通过对重组蛋白分离纯化并测定对质粒p BR322解旋酶活性,发现引入V420I,L530P和A653T突变后Top I的比活力显著降低,而引入S729T突变后比活力与野生型蛋白无显著差异。【结论】本研究证明在甜菜夜蛾Top I中引入V420I,L530P和A653T突变后,其对底物p BR322的解旋活性显著降低,为后期探索甜菜夜蛾Top I的定点突变与其对喜树碱及其衍生物敏感性的关系奠定了基础。【Aim】This study aims to investigate the effects of amino acid substitutions of topoisomerase I（ Top I） on its DNA relaxation activity in the beet armyworm,Spodoptera exigua（ Lepidoptera：Noctuidae）. 【Methods】The NH2-terminally truncated Top I（ Top70） from S. exigua was amplified and cloned into the Bam H I-Sal I sites of the p GEX-4T-1. Mutant constructs substituted with V420 I,L530P,A653 T and S729T（ numbered according to human Top I） were obtained by PCR-based site-directed mutagenesis using the wild construct p GEX-4T-1-Top70 as the template separately. The wild and mutated constructs were expressed in Escherichia coli strain BL21（ DE3）. The GST fusion protein was purified using GSTrap 4B,and the DNA relaxation activity of the purified protein was detected with p BR322 as substrate. 【Results 】 The wild and mutated p GEX- 4T-1-Top70 expression vector was successfully constructed,and the cell line stably expressing Top70 was established. A 96. 0 k Da specific protein band appeared when the expressed protein was analyzed with SDS-PAGE. Compared to the wild Top I,the mutated Top I with amino acid substitutions at positions V420 I,L530P and A653 T had significantly decreased DNA relaxation toward p BR322. However,the S729 T amino acid substitution showed no significant impact on the catalytic efficiency of Top I. 【Conclusion】These results suggest that V420 T,L530P and A653 T amino acid mutations in S. exigua remarkably decline the affinity of Top I to p BR322,providing important basic information for further investigating the sensitivity of Top I to the camptothecin and its analogous.

关 键 词：甜菜夜蛾 拓扑异构酶I 突变 氨基酸替换 DNA解旋活性 PBR322 

分 类 号：Q966[生物学—昆虫学]