Expression mechanism of insecticide-insensitive acetylcholinesterase which has no amino asid replacement

无氨基酸取代的杀虫剂不敏感乙酰胆碱酯酶的表达机制

• 批准号: 12460020
• 负责人: KONO Yoshiaki
• 金额: $ 8万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12460020/
• 关键词: organophosphorus insecticide; insecticide resistance; acetylchilinesterase; Insensitiviry; molecular mechanism; amino asid replacement; アセケルコリンエステラーゼ; アミノ配置換

The AChE of the toyama strain of Culex tritaenlorhynchus showed extremely low sensitivity to various kinds of organophosphates (OP) and carbamates compared to a susceptible strain, Taiwan. An open reading frame encoding 633 residues of AChE precursor was determined from cDNA sequence. The deduced protein sequences of AChE were identical among Toyama and Taiwan. A cloned AChE cDNA was introduced to BmNPV-BmN4 expression system and AChE sensitive to OP was produced regardless of cDNA sources; susceptible or resistant strains.To the contrary, AChE of OP resistant strains of the house fly has 2 amino acids replacement in the structure. Expression experiments of AChE gene of resistant strains confirmed mat the insensitivity to the insecticides was caused by the replacement of the amino acids.Therefore, there are two different molecular mechanism to produce insensitive AChE. The one is merely expression of the structural gene which maps insensitive AChE in it. This type of insensitive AChE was observed in D. melanogaster and M. domestica. The other is a production of two different structured AChEs from the same DNA map. The post translational modification of the protein is inevitable by other factors. According to our results, this type of insensitive AChE seems to distribute widely in insect species, C. tritaeniorhynchua, Nephotettix cincticepus, Qulema oryzae, Myzus persicae, Aphis gossipii, Tetranychtis ulticae, Plutella xyllostella. Linkage analysis of AChE atructure gene, the gene relating insensitivity of AChE and other molecular marker genes was conducted in C. tritaeniorhynchus, and indicated that the gene concerning the insensitivity located on chromosome 2 which was not the same chromosome loaded with AChE structure, gene. This result suports the existance of the second mechanism.Purification of sensitive and insensitive AChE were tried to provide the materials for furthere studies on the mechanism of insensitive AChE production in insects.

三带喙库蚊富山品系的乙酰胆碱酯酶对有机磷和氨基甲酸酯类农药的敏感性极低。从cDNA序列中确定了一个编码633个氨基酸残基的AChE前体的开放阅读框架。推导的AChE蛋白质序列在富山和台湾地区完全相同。将克隆的AChE cDNA导入BmNPV-BmN 4表达系统中，无论是敏感品系还是抗性品系，都能产生对OP敏感的AChE，而抗性品系的AChE在结构上有2个氨基酸替换。抗性品系AChE基因的表达实验证实，抗性品系对杀虫剂的不敏感性是由氨基酸的替换引起的，因此，产生不敏感AChE存在两种不同的分子机制。一种是定位不敏感AChE的结构基因的表达。melanogaster和M.南极洲另一种是从同一DNA图谱中产生两种不同结构的乙酰胆碱酯酶。蛋白质的翻译后修饰是不可避免的其他因素。根据我们的研究结果，这种不敏感的AChE似乎广泛分布在昆虫中，C。三带喙叶蝉、黑尾叶蝉、桃蚜、桃蚜、榆叶螨、小菜蛾。对AChE结构基因、与AChE不敏感相关的基因及其它分子标记基因进行了连锁分析。三带喙线虫对乙酰胆碱酯酶不敏感的基因位于第2号染色体上，与AChE结构基因不在同一条染色体上。本研究通过对敏感和不敏感AChE的纯化，为进一步研究昆虫不敏感AChE的产生机制提供了材料。