DISSERTATION RESEARCH: The Genetic Basis of Cytoplasmic Incompatibility

论文研究：细胞质不相容的遗传基础

• 批准号: 1501398
• 负责人: Seth Bordenstein
• 金额: $ 2.04万
• 依托单位: Vanderbilt University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1501398&HistoricalAwards=false
• 关键词: DISSERTATION; RESEARCH; Genetic; Basis; Cytoplasmic

Symbiotic bacteria are extremely widespread in nature and interest in the way that they manipulate animal evolution has risen rapidly in recent years. Discoveries have ranged from bacteria that affect nutrition, immunity, and reproduction to bacteria that cause the splitting of one species into two. An evolutionary enigma surrounds these adaptations - how do bacteria evolve to contribute to these complex animal processes? One of the preeminent bacteria involved in these interactions, Wolbachia pipientis, occurs worldwide in ~40% of all insect species and other invertebrates. Previous work has shown that Wolbachia's ability to alter animal reproduction affects animal populations by spurring the evolution of new insect species, reproductive strategies, and offspring production. Despite these evolutionary outcomes of animal-Wolbachia symbioses, we do not yet know the Wolbachia genes that evolved to control their animal hosts. This study begins the first in-depth analysis of two Wolbachia genes that are likely involved in the symbiosis.Cytoplasmic incompatibility (CI) is Wolbachia's main alteration of host sex ratios. CI drastically decreases fertility of uninfected females in crosses to Wolbachia-infected males and gives infected females a distinct advantage within the population. Preliminary work discovered two Wolbachia genes, with conserved protein and DNA binding domains, putatively involved in CI. To study their interactions, tagged versions of each gene will be expressed in Wolbachia-free Drosophila melanogaster. Immunofluorescent microscopy will then be utilized to determine whether expression of these genes in uninfected males crossed with uninfected females causes explicit CI defects in developing embryos. Next, after dissecting the larval testes, immunoprecipitation of the tagged proteins using commercially available antibodies will be used to isolate any bound host factors. Samples will be prepared for mass spectrometry to determine what host proteins may be bound. Additional samples using chromatin immunoprecipitation, coupled with next generation sequencing, will determine what regions of host DNA are targeted. Finally, candidate Drosophila pathways will be verified for their role in CI through a combination of genetic mutants and RNAi knockdown.

共生细菌在自然界中分布极其广泛，近年来，人们对它们如何操纵动物进化的兴趣迅速上升。这些发现的范围从影响营养、免疫和繁殖的细菌到导致一个物种分裂为两个物种的细菌。进化之谜围绕着这些适应性——细菌是如何进化来参与这些复杂的动物过程的？在这些相互作用中最重要的细菌之一——管状沃尔巴克氏体存在于世界范围内约40%的昆虫和其他无脊椎动物体内。先前的研究表明，沃尔巴克氏体改变动物繁殖的能力通过刺激新昆虫物种、繁殖策略和后代生产的进化来影响动物种群。尽管动物-沃尔巴克氏体共生的这些进化结果，我们还不知道沃尔巴克氏体基因进化来控制它们的动物宿主。这项研究开始了对两种可能参与共生关系的沃尔巴克氏菌基因的首次深入分析。细胞质不相容（Cytoplasmic incompatibility， CI）是沃尔巴克氏体对寄主性别比例的主要改变。在与感染沃尔巴克氏体的雄性杂交中，CI急剧降低了未感染雌性的生育力，并使感染雌性在种群中具有明显的优势。初步工作发现了两个沃尔巴克氏体基因，具有保守的蛋白质和DNA结合域，推测与CI有关。为了研究它们的相互作用，每个基因的标记版本将在无沃尔巴克氏体的黑腹果蝇中表达。然后将利用免疫荧光显微镜来确定这些基因在未感染的雄性与未感染的雌性杂交时的表达是否会导致发育中的胚胎出现明显的CI缺陷。接下来，在解剖幼虫睾丸后，使用市售抗体对标记蛋白进行免疫沉淀，以分离任何结合的宿主因子。将准备样品进行质谱分析，以确定可能结合的宿主蛋白。使用染色质免疫沉淀的额外样本，加上下一代测序，将确定宿主DNA的哪些区域是靶向的。最后，候选果蝇通路将通过基因突变和RNAi敲低的组合来验证它们在CI中的作用。