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

Abstract: 【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 BamH I-Sal I sites of the pGEX-4T-1. Mutant constructs substituted with V420I, L530P, A653T and S729T (numbered according to human Top I) were obtained by PCR-based site-directed mutagenesis using the wild construct pGEX-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 pBR322 as substrate. 【Results】 The wild and mutated pGEX-4T-1-Top70 expression vector was successfully constructed, and the cell line stably expressing Top70 was established. A 96.0 kDa 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 V420I, L530P and A653T had significantly decreased DNA relaxation toward pBR322. However, the S729T amino acid substitution showed no significant impact on the catalytic efficiency of Top I. 【Conclusion】 These results suggest that V420T, L530P and A653T amino acid mutations in S. exigua remarkably decline the affinity of Top I to pBR322, providing important basic information for further investigating the sensitivity of Top I to the camptothecin and its analogous.

Key words: Spodoptera exigua, topoisomerase I, mutagenesis, amino acid substitution, DNA relaxation activity, pBR322