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

描述(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媒介的新方法。大量研究表明，多种复杂的机制或基因可能与抗药性有关。然而，仍然存在许多问题，包括我们试图回答的这两个问题：1)在单个有机体(如蚊子)中，有多少解毒基因家族和单个基因参与了抗药性？2)除了代谢解毒基因外，是否还有基因上调会产生抵抗力？刘实验室最近发现了22个在库蚊HAmCq蚊子中过度表达的新基因。已知这些基因在细胞和分子代谢、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等方面起重要作用，但它们中的大多数尚未与抗药性有关。拟议项目的目标是确定22个上调基因在蚊子抗药性中的潜在作用。我们的假设是，在我们研究的22个上调基因中，与抗药性相关的基因(关键基因)在杀虫剂选择后表达增加，蛋白活性增加，蛋白表达增加。我们的具体目标是：1)确定敏感的S实验室品系、田间抗氯菊酯品系HAMCqG0、MAMCqG0和BAMCqG0、8代氯菊酯选择后代(HAMCqG1到HAMCqG8)和第八代氯菊酯选择S实验室品系(S-Lab-PerG8)关键基因沉默表达水平与相应抗药性水平之间的关系。确定这22个上调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抗药性中的功能，并最终提供重要的新信息，这些信息可用于抗性-基因相互作用和调控的基础研究，以及开发控制蚊子的新方法的应用研究。公共卫生相关性拟议的项目旨在通过确定以下品系中上调基因的抗药性水平与基因表达水平、酶活性和蛋白质表达之间的关系来确定上调基因在蚊子抗药性中的作用：敏感的S实验室品系、野外抗氯菊酯品系(HAmCqG0、MAmCqG0和BAmCqG0)、8代氯菊酯选择的HAmCqG0后代(HAmCqG1至HAmCqG8)；以及第八代氯氰菊酯选择的S实验室品系(S-Lab-PerG8)。我们将沉默(降低)HAmCqG8中关键基因的表达，以确定它们在抗药性中的功能。

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.