有机磷抗性致倦库蚊种群中酯酶基因扩增的定量分析

Quantitative differences between populations of Culex quinquefasciatus in both the esterases α and β involved in insecticide resistance

致倦库蚊Culexqinquefasciatus是丝虫病的主要传染媒介。通过生物测定、单个蚊虫酯酶α2和 β2基因拷贝数分析和酯酶 β基因序列比较 ,分析了抗性水平、抗性相关基因在种群中的分布及其基因拷贝数等的抗性分子特征。应用快速PCR仪(real timequantitativePCRs)直接检测库蚊中酯酶基因和mRNA拷贝数。结果显示 :上海致倦库蚊对对硫磷的抗性LC50 为8 12 ,酯酶活性升高是上海致倦库蚊种群对有机磷杀虫药剂产生抗性的主要机理。编码致倦库蚊酯酶 β的氨基酸序列同编码尖音库蚊酯酶B1的氨基酸序列相比同源性为 98% ;同致倦库蚊酯酶B2氨基酸序列相比同源性为 10 0 % ,同环蹶库蚊酯酶B3氨基酸序列相比同源性为 90 % ,上海致倦库蚊中酯酶α和 β基因均扩增。有机磷抗性的上海和PellRR蚊虫种群中单个蚊虫酯酶α2和β2定量基因拷贝数均不同 ,其同一蚊虫个体的酯酶α2比酯酶 β2基因的拷贝数高 ,但没有明显的规律性 ,酯酶结构基因的扩增是上海致倦库蚊种群对有机磷杀虫药剂抗性的主要机理 ,估计在野外种群的杂合个体中存在多种调控机制。

Culex quinquefasciatus, the major vector of the disease filariasis in China, is resistant to organophosphorus insecticides with an 8 .12 fold resistance ratio at the LC 50 for parathion, and a high heterogeneity factor in wild populations. The main mechanism of resistance in this species is amplification of non specific esterases. Most resistant Culex quinquefasciatus mosquitoes have co amplified est α2 and est β2 genes. Southern and dot blot analysis can be used to roughly quantify the est gene copy number in mosquitoes using probe hybridization and auoradiography. In addition, we measured gene and mRNA copy numbers using realtime PCRs. This accurate quantitative approach (fluorescent microvolume PCR) was performed to assess the specific expression profiles of est α and est β genes. The cDNA product of the PCR reaction was 500 base pairs long for the esterase α gene and 418 base pairs for the β gene. The predicted protein alignment for the esterase β fragment had high homology with Culex pipiens esterase β1 (98%), Culex quinquefasciatus β2 (100%) and Culex tarsalis esterase β3 (90%). The gene copy number differed between individuals for the α and β genes, as well as between populations of Shanghai and PellRR mosquitoes from Sri Lanka, in which both the esterase α2 and β2 are involved in insecticide resistance.