棉铃虫伤害性感受相关离子通道的功能特性及其与害虫抗药性的关系研究

For insecticide resistant and sensitive insects, are there any differences in the input nociceptive signals generated from the nociceptor and primary sensory neurons when stimulated by neurotoxic insecticides? How many types of ion channels are expressed in cotton bollworm, Helicoverpa armigera (Hübner)? And what kinds of ion channels are involoved in the conduction and regulation of the input noxcions signals? Compared with central neurons inside the ganglion, the functions of sensory neurons outside the ganglion are more easily impacted by neurotoxic insecticides. The differences in expression variation and the alteration of kinetic characteristics of ion channels in nociceptive neurons may be the basic reasons of insecticide resistance for cotton bollworm. In other words, the differences in input signals before encoding procession in central nervous system are the original reasons for pesticide resistance. The transient receptor potential channel (TRP channel) are closely related to the conduction of nociceptive signals in vertibrate. Patch clamp technique is a powerful tool in evaluating the electrophysiological properties of ion channels. And this technique is widely used to analyze the action mechanisms of insecticides and pesticides resistance. Based on the hypothesis that the basic reasons of insecticide resistance are originated from the changing of characteristics of ion channels in neurons outside the central nerve system, and considering the fact that pyrethroid insecticides are used worldwide to control agricultural pests, the proposed research will investigate the types of ion channels expressed in cotton bollworm first employing the patch clamp and RT-PCR techniques. Then, the electrophysiological characteristics of ion channels, especially the kinetic properties of TRP channel will be analyzed in detail. The effects of three kinds of pyrethroids insecticides on TRP channels will be compared, and the expression rhythm of TRP channels in neuronal membrane of different stage and different instar larval Helicoverpa armigera will be mapped. The results will help us to elucidate the basic origination points of insecticide resistance and the roles of TRP channels in the development of insecticide resistance. It will also provide us new insights into the scientific application of pyrethroids.

抗性和敏感昆虫的感受器/感觉神经元被以离子通道为作用靶标的神经毒性杀虫剂刺激时，传入中枢神经系统的信号有无区别？棉铃虫究竟表达多少种离子通道，哪些通道参与伤害性刺激信号的传导与调控？由于神经节外的感觉神经元较节内的中枢神经元更易受杀虫剂影响，因此抗性和敏感昆虫的感觉神经元膜上参与形成传入信号的离子通道在门控特性或表达量上的差异，极有可能是抗性形成的最根本原因之一。瞬时受体电位通道（TRP通道）与人及多种动物的伤害性信息的传导密切相关。本项目拟用膜片钳技术和RT-PCR技术，检测棉铃虫表达的离子通道类型，重点对比敏感及抗性棉铃虫感觉和中枢神经元膜上TRP通道的门控特性，并分析杀虫剂对TRP通道的影响，以及不同龄期幼虫/虫态TRP通道的表达规律。以确定TRP通道是否也是拟除虫菊酯类杀虫剂的作用靶标，明确TRP通道与害虫抗药性的相关性，以进一步解释拟除虫菊酯类杀虫剂的作用机理和棉铃虫的抗性机理。

棉铃虫Helicoverpa armigera为重要农业害虫，其神经系统对外界伤害性刺激信息的感受与传导对棉铃虫的生存及抗药性的形成至关重要。据通道的药理学和电生理特性，结合分子生物学方法，对棉铃虫幼虫神经系统表达的电压敏感通道的类型进行了鉴定，并用膜片钳技术分析了通道的电生理特性，检测了七氟菊酯和溴氰菊酯对这些通道的影响。基于这两种菊酯修饰钠通道后均能诱发标志性的晚钠电流和尾电流，将二者用作工具药阐明了两种萜类化合物的神经毒性机理。利用分子生物学和电生理方法分析了低电压激活T型钙通道的失活机制。棉铃虫至少表达两种TRP通道。另外对室内饲养的棉铃虫感染不明来源病毒后很快死亡的现象进行了研究。.总体结果表明棉铃虫至少表达TTX敏感型电压门控钠通道、对Cd2+敏感的高电压激活钙通道、对Cd2+不敏感的高电压激活钙通道、大电导钙激活钾通道、延迟整流钾通道、瞬时外向钾通道及多种TRP通道。延迟整流钾通道和瞬时外向钾通道可同时表达于同一个神经元，或分别表达于不同类型的神经元。菊酯类杀虫剂对棉铃虫的电压门控钠、钾、钙及钙激活钾通道均有修饰作用，这几种通道均参与伤害性信息的传导。T型钙通道的结构域Ⅱ是调控其电压依赖性失活的关键因素，结构域I不参与该通道失活过程的调控。七氟菊酯和溴氰菊酯对敏感及抗性棉铃虫神经细胞钠通道的作用均具有状态依赖性，二者均可诱发晚电流和尾电流，七氟菊酯作用于钠通道的静息状态，溴氰菊酯对静息和开放状态均有影响，但对开放状态的钠通道具有更高亲和力，其诱发的晚电流和尾电流几乎不失活，有使用依赖性。对比七氟菊酯和溴氰菊酯对通道的修饰作用表明，萜类化合物通过结合于钠通道的静息状态抑制其失活而产生神经毒性。七氟菊酯和溴氰菊酯对BKCa的激活和快速失活均有影响，可使峰值电流减小，激活曲线右移，失活时间缩短。高效氯氰菊酯使Cd2+敏感的高电压激活钙通道电流幅值减小，激活曲线左移。棉铃虫疑似核型多角体病毒对棉铃虫的致死效果检测发现致死源不止一种病毒。