An.gambiae immune signaling gene SNPs and natural P. falciparum infection

描述（由申请人提供）：重要数据表明Toll/imd，胰岛素和TGF-¿信号通路在冈比亚按蚊中肠中调节疟疾寄生虫的发育。然而，没有数据可以证实这些信号通路在自然界中调节寄生虫的发育。我们选择使用从马里和喀麦隆实地收集的蚊子的SNP关联制图方法来解决这一问题，这两个国家是疟疾传播率高的国家，其中包括遗传分化程度最高的疟蚊的地理分布。冈比亚种群尚未被描述。我们将通过两个具体目标来实现这一目标。第一个目标包括群体遗传分析。感染和未感染的恶性疟原虫。冈比亚将从马里和喀麦隆的地点收集。这些位点经过精心挑选，包括该地区已知存在的所有遗传多样性（三种分子形式和染色体倒置多态性）。蚊子样本将根据感染地点、分子形态和核型进行分组。然后将对每只蚊子进行大约384个免疫信号基因snp的基因分型，并通过关联图谱确定这些snp与感染相关。该分析将确定与恶性疟原虫感染易感性有关的snp。在第二个目标中，将分析选定的感兴趣的snp，以确定它们对蚊子蛋白功能和对恶性疟原虫感染易感性的影响。具体来说，我们将利用永生的安。冈比亚细胞系和过表达、敲除和基因转换技术的组合，以确认从序列预测的功能，并优先考虑体内研究的snp。对于体内研究，我们将使用两种遗传上不同的An菌株。冈比亚，它们已经对感兴趣的snp进行了基因分型。我们将使用抑制剂和转染（敲除，O'nyong-nyong感染克隆介导的过表达）方案来模拟含snp等位基因对感染蚊子恶性疟原虫发展的影响。我们的工作将从我们的实验室和我们同事的实验室获得功能数据，以检查在现场收集的蚊子中选定的免疫信号通路的重要性。因此，本文提出的研究将有助于为转基因策略选择合适的基因靶点，并为安培免疫的群体遗传学提供重要的新见解。据我们所知，冈比亚目前还没有。

DESCRIPTION (provided by applicant): Significant data implicate the Toll/imd, insulin, and TGF-¿ signaling pathways in the regulation of malaria parasite development in the Anopheles gambiae midgut. However, no data are available to confirm that these signaling pathways regulate parasite development in nature. We have chosen to address this issue using a SNP association mapping approach with field-collected mosquitoes from Mali and Cameroon, countries with high malaria transmission that include the geographic distribution of the most genetically diverged An. Gambiae populations yet described. We will pursue this goal through two specific aims. The first Aim includes population genetic analyses. Plasmodium falciparum infected and uninfected An. gambiae will be collected from sites in Mali and Cameroon. These sites were carefully selected to include all of the genetic diversity known to exist in the region (three molecular forms and chromosome inversion polymorphism). Mosquito samples will be grouped by site with respect to infection, molecular form and karyotype. Each mosquito will then be genotyped for roughly 384 immune signaling gene SNPs and those SNPs correlated with infection identified by association mapping. This analysis will identify SNPs putatively involved in susceptibility to P. falciparum infection. In the second Aim, selected SNPs of interest will be analyzed to determine their effects on mosquito protein function and on susceptibility to P. falciparum infection. Specifically, we will utilize immortalized An. gambiae cell lines and a combination of over expression, knock-down, and gene conversion techniques to confirm predictions of function from sequence and to prioritize SNPs for in vivo studies. For the in vivo studies, we will use two genetically distinct strains of An. gambiae, which have been genotyped for the SNPs of interest. We will use inhibitors and transfection (knockdown, O'nyong-nyong infectious clone mediated overexpression) protocols to mimic the effects of SNP-containing alleles on P. falciparum development in infected mosquitoes. Our work will take functional data from our lab and from the labs of our colleagues to examine the importance of the selected immune signaling pathways in field-collected mosquitoes. In this light, the studies proposed herein will facilitate selection of appropriate gene targets for transgenesis strategies and provide critical new insights into the population genetics of immunity in An. gambiae that to our knowledge are not currently available.