禾谷缢管蚜独特电压门控钠离子通道的功能特性

Rhopalosiphum padi (Linnaeus) is a devastating pest of wheat crops. Insecticide resistance level was increasing with the intensive application of chemical pesticides in the last decades, which caused more and more serious damages of the pest to wheat. The voltage-gated sodium channel (VGSC) is one of the most important neuro-targets of insecticides. Many insects had developed resistance to different insecticides because of mutations of VGSC genes. Traditionally, it is considered that eukaryotic organism VGSCs contain DI-DIV domains that encoded by one single gene. However, we found that the VGSC in R. padi is encoded by two genes. Both the two genes are functional, and may play role in neuro-transmission with heterodimer. Some very important amino acids of R. padi VGSCs are different with those of the other organisms, and the N ends of the two VGSCs change too much. Based the previous research results obtained, we will model the R. padi VGSC structure and analyze the contact sites of pyrethroids on the channel. Eukaryotic heterologous expression system will be used to investigate the effects of the important amino acid changes on the channel current, the effect of auxiliary subunits on the sodium channel functions, the sodium channel alternative splicing types and their functional characteristic, and the mutations of the channel in laboratory strain and field samples. The results will be of importance to the elucidation of functional characteristics of the unique R. padi VGSCs, the development of molecular markers for predicting and monitoring resistance of the pest to pyrethroids, and the developments of new insecticides based on the unique sodium channel structure.

禾谷缢管蚜是重要的小麦害虫。杀虫剂的广泛使用导致的抗药性，是其近年在我国危害逐年加重的重要原因。钠通道是最重要的杀虫剂作用靶标之一，以往的研究认为，真核生物钠通道由DI到DIV四个结构域组成，由1个基因编码。本研究组发现，禾谷缢管蚜钠通道在结构域DII和DIII间分开，由2个功能基因编码，分析认为2个基因以二聚体形式发挥作用；与其他类群昆虫相比，该虫钠通道多个关键氨基酸都不同，氨基酸序列氮末端差异很大。基于这些发现，本项目将模拟该独特钠通道的空间结构，分析其农药结合位点；利用真核异源表达研究关键氨基酸变化对其功能的影响；分析抗性品系的突变位点在钠通道上的分布，并验证其与抗性的关系；研究辅助亚基对独特钠通道的辅助特性，以及该钠通道可变剪接体的独特性及其功能；研究结果可以从多角度阐述该虫独特钠通道的功能特性、建立该虫对拟除虫菊酯抗性的分子标记、并且对以该独特钠通道为靶标的农药设计具有重要意义。

禾谷缢管蚜是重要的小麦害虫，拟除虫菊酯类杀虫剂长期应用于禾谷缢管蚜防治之中，钠离子通道是拟除虫菊酯类杀虫剂的作用靶标。本研究首次发现钠离子通道基因M918L突变导致禾谷缢管蚜田间种群对拟除虫菊酯的靶标抗性；在田间种群的基础上建立了禾谷缢管蚜对拟除虫菊酯杀虫剂的的极高抗品系，分析了该突变品系的钠离子通道突变特征及其在钠离子通道蛋白上的分布，发现M918L突变在田间种群和实验室高抗品系中皆为杂合子突变，这可能和禾谷缢管蚜钠通道结构相关；研究了禾谷缢管蚜钠通道基因与其辅助亚基基因之间的关系，分析了辅助亚基TipE基因的干扰对禾谷缢管蚜敏感性的影响；采用双电极电压钳技术研究禾谷缢管蚜5个钠通道辅助亚基的功能，发现禾谷缢管蚜辅助亚基促进了钠通道对刺激的响应；探索了在爪蟾卵母细胞中表达禾谷缢管蚜钠离子通道基因；发现禾谷缢管蚜钠通道RpNavH1基因7个选择性剪接位点和17种可变剪接体，RpNavH2基因的5个选择性剪接位点和9种可变剪接体，分析了各可变剪接体的蛋白结构；爪蟾卵母细胞中真核表达和双电极电压钳分析结果显示，M918L突变改变钠通道对拟除虫菊酯的敏感性；利用CRISPR/Cas9技术构建黑腹果蝇M918不同突变类型突变体，结合生物测定和空间结构模拟进一步分析该位点突变在昆虫对拟除虫菊酯类杀虫剂抗药性中的作用，发现M918L突变介导的拟除虫菊酯类杀虫剂抗性水平最高，M918I突变次之，M918V突变介导的抗性最低。本研究结果对于深入阐述禾谷缢管蚜钠通道的功能特性，建立基于M918L等钠通道突变的该虫抗药性分子标记检测方法，以及该虫的抗药性治理具有重要的意义。