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马拉硫磷是一种高效低毒的有机磷杀虫剂,分子量大且结构特殊,广泛用于农业害虫的防治。羧酸酯酶突变是昆虫对有机磷类杀虫剂产生代谢抗性的重要机制之一。本实验室前期已从棉蚜Aphis gossypii、褐飞虱Nilaparvata lugens、斜纹夜蛾Spodoptera litura、家蚕Bombyx mori、异色瓢虫Harmonia axyridis、赤拟谷盗Triboliumcastaneum和西方蜜蜂Apis mellifera中各克隆了一个非特异性羧酸酯酶基因,通过体外定点突变构建了G/A151D和W271L两种突变体,并进行了原核细胞表达和纯化。本实验在体外测定了这7种昆虫野生型和两种突变型羧酸酯酶对马拉硫磷的降解。结果显示:棉蚜、西方蜜蜂、斜纹夜蛾、赤拟谷盗的野生型羧酸酯酶能够降解马拉硫磷,两个突变并不能提高它们的降解活性,而家蚕、异色瓢虫和褐飞虱的野生型羧酸酯酶不能降解马拉硫磷,G/A151D和/或W271L突变能使这些酯酶获得马拉硫磷羧酸酯酶(MCE)的活性,有可能使这些昆虫对马拉硫磷产生抗性。不同物种的MCE活性相差较大,斜纹夜蛾的MCE活性最高,其kcat/Km值为1.8～1.9L/μmol·min,其次是赤拟谷盗,其Kcat/Km值为0.87～0.95L/μmol·min,其他昆虫的MCE活性相对较低,相差可高达10倍。
Malathion is a high efficiency and low toxicity organophosphate insecticide with large molecular weight and special structure, which is widely used in the prevention and control of agricultural pests. Carboxylesterase mutation is one of the most important mechanisms by which insects exert metabolic resistance to organophosphate insecticides. In our laboratory, a non-specific carboxyl-terminal carboxyl group was cloned from Aphis gossypii, Nilaparvata lugens, Spodoptera litura, Bombyx mori, Harmonia axyridis, Tribolium castaneum and Apis mellifera Acid esterase gene, two mutants of G / A151D and W271L were constructed by site-directed mutagenesis in vitro, and the prokaryotic cells were expressed and purified. In this experiment, the degradation of malathion in wild type and two kinds of mutant carboxylesterase of seven kinds of insects was tested in vitro. The results showed that the wild-type carboxylesterase of cotton aphid, western bee, Spodoptera frugiperda, and red moth could degrade malathion. The two mutations did not improve their biodegradability. However, silkworm, The wild-type carboxylesterase of BPH was unable to degrade malathion, and G / A151D and / or W271L mutations gave these esterases malathion-carboxylesterase (MCE) activity that made it possible for these insects to kill horses Endothionment produces resistance. The MCE activity of different species was quite different, and the MCE activity of Spodoptera litura was the highest, the kcat / Km value was 1.8-1.9 L / μmol · min, followed by the red tarragon, the Kcat / Km value was 0.87 ~ 0.95 L / μmol · min, MCE activity of other insects is relatively low, the difference can be as high as 10 times.