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

与敏感品系台湾库蚊相比，富山库蚊对各种有机磷(OP)和氨基甲酸酯类化合物的敏感性极低。从cDNA序列中确定了编码AChE前体633个残基的开放阅读框。推导出的AChE蛋白序列在富山地区和台湾地区是相同的。将克隆的乙酰胆碱酯酶基因导入BmNPV-BmN4表达系统，获得了对OP敏感的AChE，无论是敏感株还是抗性株，而抗性家蝇株的AChE在结构上有2个氨基酸的替换。抗性品系乙酰胆碱酯酶基因的表达实验证实，抗性品系对杀虫剂的不敏感是由氨基酸的替换引起的，因此，产生不敏感的乙酰胆碱酯酶有两种不同的分子机制。一种仅仅是将不敏感的AChE映射到其中的结构基因的表达。这种不敏感的疼痛在黑腹黄鼠和家蝇中观察到。另一种是来自同一DNA图谱的两种不同结构的ACH。蛋白质的翻译后修饰是由其他因素不可避免的。根据我们的结果，这种不敏感的AChE似乎广泛地分布在昆虫物种中，如三带喙小蜂、斑潜蝇、稻纵卷叶蝉、桃小实蝇、桃蚜、小菜蛾、小菜蛾等。对三带喙库蚊AChE结构基因、与AChE不敏感相关的基因与其他分子标记基因的连锁分析表明，与AChE不敏感相关的基因位于第2染色体上，与AChE结构基因不同。这一结果支持第二种机制的存在。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。

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.