两个单点突变对昆虫羧酸酯酶降解马拉硫磷的影响

›› 2013, Vol. 56 ›› Issue (5): 566-569.doi:

两个单点突变对昆虫羧酸酯酶降解马拉硫磷的影响

张柳平^1,2,#，姚淑敏^1,#，林哲²，崔峰^2,*

（1. 曲阜师范大学生命科学学院, 山东曲阜 273165;
2. 中国科学院动物研究所, 农业虫害鼠害综合治理研究国家重点实验室, 北京 100101）

出版日期:2013-05-20 发布日期:2013-05-20

Effect of two single mutations on malathion degradation by insect carboxylesterases

ZHANG Liu-Ping^1,2,#, YAO Shu-Min^1,#, LIN Zhe², CUI Feng^2,*

(1.College of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China; 2. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China)

Online:2013-05-20 Published:2013-05-20

摘要/Abstract

摘要： 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Tribolium castaneum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k_cat/K_m值为1.8~1.9 L/μmol·min，其次是赤拟谷盗，其K_cat/K_m值为0.87~0.95 L/μmol·min，其他昆虫的MCE活性相对较低，相差可高达10倍。

关键词: 羧酸酯酶, 抗药性, 突变, 马拉硫磷, 酶动力学

Abstract: Malathion is an efficient but low toxic organophosphate insecticide with a large molecular weight and a special structure. It is widely used in the prevention and control of various agricultural pests. Mutation in carboxylesterases is one of important metabolic resistance mechanisms to organophosphate insecticides in insects. In a previous study, seven non-specific carboxylesterase genes from Aphis gossypii, Nilaparvata lugens, Spodoptera litura, Bombyx mori, Harmonia axyridis, Tribolium castaneum and Apis mellifera, respectively, were cloned, mutated at position 151 or 271 and expressed in Escherichia coli. In this experiment, the hydrolysis of the purified recombinant proteins of the seven insects was further examined towards malathion. The results showed that the wild-type carboxylesterases from A. gossypii, A. mellifera, S. litura and T. castaneum were capable of degrading malathion and the two single mutations did not improve their hydrolysis activity. The wild-type carboxylesterases from B. mori, H. axyridis and N. lugens could not degrade malathion while the G/A151D and/or W271L mutation made these esterases acquire malathion carboxylesterase activity, implying that these two single mutations could confer resistance to malathion in the three insects. Malathion carboxylesterase activity in different species had large difference. S. litura had the highest malathion carboxylesterase activity, and its K_cat/K_m was 1.8-1.9 L/μmol·min, followed by T. castaneum, with the K_cat/K_m of 0.87-0.95 L/μmol·min. Malathion carboxylesterase activity in other insects was relatively low, with ten-fold difference.

Key words: Carboxylesterase, insecticide resistance, mutation, malathion, enzymatic dynamics

张柳平, 姚淑敏, 林哲, 崔峰. 两个单点突变对昆虫羧酸酯酶降解马拉硫磷的影响[J]. , 2013, 56(5): 566-569.

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