P糖蛋白介导昆虫表皮穿透抗性及其作用机理研究

Decrease in cutuliar penetration is an important cause for the resistance of insects to insecticides; however, it is unclear for the mechanism of the resistance. The goal of this proposal is to study the role of p-glycoprotein (P-gp) in development of non-specific resistance of insects to insecticides and its mechanism by applying the drosophia and avermectins (AVM) as the testing insect and testing chemical respectively. By comparing with the wild type and deficient in P-gp insects, and the AVM-resistance and sensitive strains, the role of P-gp in the resistance of insects to insecticides will be studied. We will investigate the expression of P-gp in the insects with resistance induced by low catuliar penetration, or briefly penetration resistance, and the expression of catuliar proteins, skeleton proteins, and link proteins, as well as the relationship between the P-gp and the other proteins. We will study the change of the siganls in the process which is relative to the regulation of skeleton proteins, and understand that how the skeleton system was affected through the pathway of the regulatory siganls of skeleton proteins and then the decrease of catuliar penetration was induced and the resistance was finally developed. The mechanism of the catuliar penetration resistance of insects to the insecticides will be revealed. It is expected that the study not only makes us understand the role of P-gp in the resistance of insect to AVMs, but also provide us with a new view for understanding the mechanism of non-specific resistance of insects to insecticides, which will be applied in the control of pest insect resistance to the insecticides.

表皮穿透性降低是昆虫抗药性形成的一种重要机制，但其机理并不清楚。本申请项目分别以果蝇和阿维菌素（AVM）作为测试昆虫和测试药物，研究P糖蛋白（P-gp）在昆虫对杀虫剂非特异性抗性中的作用及机理。通过比较P-gp基因缺失型/野生型果蝇、AVM抗性品系/敏感品系果蝇对AVM的敏感性，以确定P-gp在昆虫抗药性中的作用。进一步深入研究表皮穿透性降低导致的抗性（简称穿透抗性）形成过程中昆虫表皮细胞中的P-gp以及表皮蛋白、骨架蛋白和连接蛋白的表达及其相关性，研究在这一过程中与骨架蛋白调控有关的信号通路的变化，以确定P-gp如何通过对骨架蛋白调控的信号通路影响骨架系统导致昆虫表皮通透性降低而致抗性形成，以期揭示昆虫表皮穿透抗性形成的机理。预期此项研究不仅揭示P-gp在果蝇对阿维菌素抗药性中的作用，而且将为理解害虫非特异性抗性的分子机制提供新视点，也可能为生产实践中害虫抗药性的治理提供科学依据。

阿维菌素药物（AVM）是一类广泛应用于杀虫、杀螨和杀线虫的十六环大环内酯类药物。随着AVM的广泛使用，部分害虫已产生严重的抗药性，但相关的抗药性机理尚未完全阐明。本研究首先在实验室内通过药物压力筛选实验，获得AVM抗性果蝇品系。随后对其进行P糖蛋白（P-gp）的蛋白表达及活性检测发现，P-gp与果蝇对AVM的抗性产生有关。通过相关信号通路蛋白检测发现，抗性品系果蝇可同时通过dEGFR/dAkt及dEGFR/dERK等信号通路上调P-gp表达。另外，在果蝇S2细胞上对AVM诱导P-gp表达机制开展了相关研究，发现AVM可诱导细胞内钙离子浓度和氯离子浓度升高，这种变化与AVM诱导细胞内P-gp的表达水平增加有关，AVM通过CaM/Relish(NF-κB)通路诱导P-gp表达。通过对比敏感品系与抗性品系有虫发现，还发现，阿维菌素穿透抗性的产生与幼虫表皮中的几丁质层的增厚和P-gp的表达上调有关。EGFR及其相关信号通路EGFR/AKT/ERK的激活介导了转录因子Relish 的激活，近而调控了几丁质合成酶DmeCHS1/2和P-gp的上调表达；EGFR信号通路的激活由阿维菌素与EGFR相互作用调控。此外还发现，抗性品系幼虫中粘附连接和间隔连接相关蛋白DE-cadherin、Dα-catenin、Armadillo和Coracle的表达高于敏感品系幼虫。阿维菌素可以通过某种机制（初步发现可能是上调细胞内钙离子浓度激活PKC信号）导致连接蛋白的表达上调，使得细胞间的连接更紧密、细胞间阿维菌素穿透屏障功能增强。.综上所述，P-gp低导致了果蝇幼虫对阿维菌素比成虫更敏感。阿维菌素通过激活EGFR信号通路上调DmeCHS1/2、P-gp和连接蛋白的表达，使表皮的穿透屏障功能增强，进入体内的阿维菌素减少，细胞外排阿维菌素能力增加，由此形成了对阿维菌素的穿透抗性。这些发现不仅为深入揭示昆虫对AVM抗性形成机理提供科学依据，也为发展新的手段解决杀虫剂抗性的问题提供重要的参考。