Antimicrobial peptides as models for the evolution of gene duplication

 描述(申请人提供)：抗菌肽作为基因复制进化的模型抗菌肽(AMPs)是真核生物体液免疫反应的重要组成部分。长期以来，通过复制扩大基因家族一直被认为是产生进化新颖性的一种手段，尽管从基因复制到新功能的进化过程尚未得到很好的确立。进化的一个显著特征 在AMP基因家族中，AMP基因重复率较高。这项拟议的研究将利用AMP基因家族中的复制来更好地了解AMP的功能和基因复制的进化。使用AMPS研究基因复制可能对人类健康具有适用性，并提供果蝇家族间基因复制的内置复制。此外，在果蝇身上进行高通量实验和遗传操作的能力，以及一组参与AMP功能的预期表型的存在，使该系统易于管理。目的1是对AMP在物种内和物种间的进化进行综合分析，以解决基因复制后选择在进化中所起的作用，以及基因表达在物种和同源基因之间的差异。这项分析的一部分将在空间(跨组织)和时间上在非常精细的尺度上检查这些AMP的表达。目标1的培训部分包括最先进的种群遗传分析以及精细的基因表达研究。目的2利用工程基因敲除和使用多种病原体来研究特定AMP类似物在应对口腔感染和全身感染时的功能，因为我们有证据表明AMP免疫反应是病原体特异性的。根据本实验的结果，我们将创建具有特定AMP的多个副本的转基因系来测试基因剂量的影响，并在同源基因之间进行启动子交换实验，以测试调控序列的变化在亚功能化过程中如何影响表达。目标2的培训包括使用发展中的基因组编辑技术构建转基因系以解决这些问题。目标3将专门关注在自然种群中分离AMP复制，以解决初始复制后不久的Paralog功能的性质 事件。我们将使用与目标1和2中相同的基本框架来检查变异性、基因表达和Paralog功能的模式，但重点放在这些分离的串联复制上。我们还将创建转基因串联副本，以执行控制背景效应的功能分析。由于AMP基因在基因家族之间复制的性质，我们期望得出关于基因复制进化的结论。拟议研究的培训部分将补充申请人以前的经验，并使他能够从事富有成效的研究事业。康奈尔大学以及拉扎罗和克拉克实验室共同拥有资源和专门知识，以确保顺利完成赠款的培训阶段。

 DESCRIPTION (provided by applicant): Antimicrobial peptides as models for the evolution of gene duplication Antimicrobial peptides (AMPs) are a vital part of the humoral immune response for eukaryotes. Gene family expansion through duplication has long been recognized as a means of generating evolutionary novelty though the evolutionary processes leading from gene duplication to novel function is not well established. One striking characteristic of the evolution of AMPs is the high rate of gene duplication in AMP gene families. The proposed research will use duplications in AMP gene families to better understand both AMP function and the evolution of gene duplication. Using AMPs to study gene duplication may have applicability to human health and provides built-in replication of gene duplications across families in Drosophila. Furthermore, the ability to perform high throughput experiments and genetic manipulations in Drosophila and the existence of a set of expected phenotypes involved in AMP function make the system tractable. Aim 1 is a comprehensive analysis of AMP evolution within and between species to address the role that selection plays in evolution after gene duplication and how gene expression diverges with between species and paralogs. Part of this analysis will examine expression of these AMPs on a very fine scale both spatially (across tissues) and temporally. The training component of Aim 1 includes state-of-the art population genetic analyses as well as fine scale gene expression studies. Aim 2 consists addresses the function of specific AMP paralogs in response to both oral infection and systemic infection using engineered gene knockouts and employing a large variety of pathogens since we have evidence that AMP immune response is pathogen-specific. Based on the results from this experiment, we will create transgenic lines with multiple copies of a particular AMP to test the effects of gene dosage, perform promoter swapping experiments between paralogs to test how changes in regulatory sequence might influence expression en route to subfunctionalization. Training in Aim 2 consists of the construction of transgenic lines using developing genome editing technologies to address these questions. Aim 3 will focus specifically on AMP duplicates segregating in natural populations to address the nature of paralog function shortly after the initial duplication event. We will employ the same basic framework as in Aims 1 and 2 examining patterns of variability, gene expression and paralog function but focusing on these segregating tandem duplicates. We will additionally create transgenic tandem duplicates to perform functional assays controlling for background effects. Because of the replicated nature of AMP gene duplicates across gene families, we expect to draw conclusions about the evolution of gene duplication. The training portion of the proposed research will complement the applicant's previous experience and position him for a productive research career. Cornell University and the Lazzaro and Clark labs together have the resources and expertise to ensure the successful completion of the training phase of the grant.