Characterizing the role of the 22 up-regulated genes in mosquito resistance

描述（由申请人提供）：蚊媒疾病是全球人类的头号杀手。控制这些疾病的一个主要障碍是蚊子对杀虫剂产生了抗药性。我们的长期目标是确定控制蚊子对杀虫剂反应的基因的作用，并利用这些信息开发控制蚊子载体的新方法。大量研究表明，多种复杂的机制或基因可能是杀虫剂抗性产生的原因。然而，许多问题仍然存在，包括我们试图回答的这两个问题：1)在单个生物体（如蚊子）中，有多少解毒基因家族和个体基因参与杀虫剂抗性？2)除了代谢解毒基因外，是否还有基因的上调会带来抗性？刘实验室最近在库蚊HAmCq蚊子中发现了22种过度表达的新基因。众所周知，这些基因对细胞和分子代谢、信号转导和调节、囊泡和分子运输、蛋白质生物合成和泛素化至关重要，但其中大多数基因与杀虫剂抗性无关。本项目的目的是确定22个上调基因在蚊子杀虫剂抗性中的潜在作用。我们的假设是，在我们研究的22个上调基因中，涉及杀虫剂抗性的基因（关键基因）在杀虫剂选择后表达增加，蛋白质活性增加，蛋白质表达增加。我们的具体目标是：1)确定易感菌株S-Lab中22个上调基因的基因表达、酶活性和蛋白表达水平与抗性水平之间的关系；HAmCqG0、MAmCqG0、BAmCqG0田间抗氯菊酯品系；8代氯菊酯选择HAmCqG0子代（HAmCqG1 ~ HAmCqG8）；2)确定HAmCqG8中关键基因的沉默表达水平与其对应的杀虫剂抗性水平之间的关系。确定22个基因在杀虫剂抗性中的作用是确定抗性机制、了解这些基因在蚊子杀虫剂抗性中的功能的必要和基础的第一步，并最终为抗性基因相互作用和调控的基础研究提供重要的新信息，并为开发新的蚊虫控制方法提供应用研究。拟议的项目旨在通过确定抗性水平与以下菌株中上调基因的基因表达水平、酶活性和蛋白质表达水平之间的关系，确定上调基因在蚊子杀虫剂抗性中的作用：S-Lab敏感品系、田间抗氯菊酯品系（HAmCqG0、MAmCqG0、BAmCqG0）、8代氯菊酯选择HAmCqG0子代（hammcqg1 ~ hammcqg8）；第8代氯菊酯筛选菌株S-Lab （S-Lab- perg8）。我们将沉默（降低）HAmCqG8中关键基因的表达，以确定其在杀虫剂抗性中的作用。

DESCRIPTION (provided by applicant): Mosquito-borne diseases are the number-one killers of humans worldwide. A major obstacle in controlling these diseases is that mosquitoes have developed resistance to insecticides. Our long-term goals are to determine the roles of genes that control the mosquito's response to insecticides and to use this information to develop novel approaches to control mosquito vectors. A large number of studies has shown that multiple, complex mechanisms or genes may be responsible for insecticide resistance. However, many questions remain, including these 2 that we seek to answer: 1) how many detoxification gene families and individual genes are involved in insecticide resistance in a single organism, such as the mosquito? And 2) are there genes, in addition to metabolic detoxification genes, the up-regulation of which confers resistance? The Liu laboratory has recently identified 22 novel genes that are over-expressed in Culex HAmCq mosquitoes. These genes are known to be vital for cellular and molecular metabolism, signal transduction and regulation, vesicular and molecular transport, and protein biosynthesis and ubiquitination, but most of them have not been implicated in insecticide resistance. The objective of the proposed project is to determine the potential roles of the 22 up-regulated genes in insecticide resistance in mosquitoes. Our hypothesis is that among the 22 up-regulated genes in our study, the genes involved in insecticide resistance (the key genes) exhibit increased expression, increased protein activities, and increased protein expression following insecticide selection. Our specific aims are to: 1) Define the relationship between the levels of resistance and the levels of gene expression, enzyme activity, and protein expression in each of the 22 up-regulated genes in the susceptible S-Lab strain; the field permethrin-resistant strains of HAmCqG0, MAmCqG0, and BAmCqG0; 8 generations of permethrin-selected HAmCqG0 offspring (HAmCqG1 to HAmCqG8); and the 8th generation of the permethrin-selected S-Lab strain (S-Lab-PerG8) and 2) Determine the relationship between the silenced expression levels of key genes in HAmCqG8 and their corresponding levels of insecticide resistance. Characterizing roles of the 22 up-regulated genes in insecticide resistance is a necessary and fundamental first step in determining resistance mechanisms, understanding the function of these genes in mosquito insecticide resistance, and ultimately providing important new information that can be used for basic studies of resistance-gene interaction and regulation, and for applied studies in developing novel approaches for controlling mosquitoes. PUBLIC HEALTH RELEVANCE The proposed project seeks to identify the role of up-regulated genes in insecticide resistance in mosquitoes by defining the relationship between the levels of resistance and the levels of gene expression, enzyme activity, and protein expression of the up-regulated genes in the following strains: the susceptible S-Lab strain, the field permethrin resistant strains (HAmCqG0, MAmCqG0, and BAmCqG0), 8 generations of permethrin-selected HAmCqG0 offspring (HAmCqG1 to HAmCqG8); and the 8th generation of permethrin-selected S-Lab strain (S-Lab-PerG8). We will silence (reduce) the expression of key genes in HAmCqG8 to determine their functions in insecticide resistance.