Molecular genetics underlying reproductive isolation of species

本研究将使用果蝇属的果蝇来研究确保种间杂交不能繁殖的分子机制。更具体地说，我们将研究一种假说，即Vasa是一种快速进化的蛋白质，在卵子发生过程中在生殖细胞中表达，并且在后代胚胎的原始生殖细胞的母体规范中是必需的，在限制种间杂交的育性中起重要作用。包括果蝇在内的真核生物基因组含有许多转座因子（te）拷贝，为了保持染色体的完整性，必须抑制其迁移。TE转位的抑制涉及称为piwi相互作用rna （piRNAs）的小rna，其靶向TE衍生的mrna，随后使其不稳定并抑制其翻译。在种间杂交雌性果蝇中，TEs被抑制，这可能表明该系统在建立物种生殖隔离方面发挥了重要作用。如果是这样的话，那么我们就可以预料到，负责的蛋白质或蛋白质会进化得非常快，因此，来自一个物种的蛋白质会有很大的不同，而不会在不同的物种中发挥作用。最近发现的与piRNA加工有关的Vasa，其区域差异很大，甚至在关系密切的果蝇物种（如黑腹果蝇和拟象果蝇）之间也是如此。为了研究Vasa、piRNA介导的过程和生殖隔离之间的关系，我们将首先通过分析te编码基因的表达和piRNA的表达，在只表达一组位点特异性突变形式Vasa的雌性中，确定哪些Vas结构域是piRNA介导过程所必需的。接下来，我们将通过将其他物种的Vasa同源基因转基因引入Vasa突变背景，研究这些基因在黑腹龙中发挥作用的程度。最后，基于我们获得的结果，我们将用黑腹龙的对应结构域替换非救援物种的Vasa的各种结构域，以尝试恢复功能。这将使我们能够绘制Vasa的特定区域，这些区域必须从宿主物种（在这种情况下是黑腹虫）中获得功能。从长远来看，超出了目前的建议范围，我们将以类似的方式研究其他pirna效应基因。我们将结合突变分析、分子分析和其他人提供的蛋白质结构信息来了解生殖隔离背后的特定分子关联。例如，我们将测试Vasa的特定关键片段是否以物种特异性的方式与特定的伴侣蛋白相互作用。这项工作的预期影响将包括在高质量的国际科学期刊上发表文章，由赠款（以及PI）资助的学员将在国家和国际会议上展示他们的成果。学员还将完成博士论文项目，这些项目将涉及获得最先进的分子遗传学和成像技术，这将使他们能够在公共或私营部门从事研究工作。

The research in this proposal will use fruit flies of the Drosophila genus to investigate the molecular mechanisms that ensure that interspecific hybrids cannot reproduce. More specifically, we will investigate the hypothesis that Vasa, a rapidly-evolving protein that is expressed in germ cells during oogenesis, and that is required maternally for specification of primordial germ cells in progeny embryos, is an important player in limiting the fertility of interspecific hybrids.Eukaryotic genomes including those of Drosophila contain many copies of transposable elements (TEs), whose mobility has to be suppressed in order to maintain chromosome integrity. Suppression of TE transposition involves small RNAs called Piwi-interacting RNAs (piRNAs) that target TE-derived mRNAs, and subsequently destabilize them and repress their translation. In interspecific Drosophila hybrid females, TEs are derepressed, raising the possibility that this system has a fundamental role in establishing the reproductive isolation of species. If this is so, then it would be expected that the responsible protein or proteins would evolve very rapidly, so that a protein from one species would be sufficiently different to not function in a different species. Vasa, which has recently been implicated in piRNA processing, has regions that diverge extensively, even between closely related Drosophila species such as D. melanogaster and D. simulans. To examine the relationships between Vasa, piRNA mediated processes, and reproductive isolation, we will first identify which domains of Vas are required for piRNA-mediated processes by analyzing expression of TE-encoded genes, and piRNA expression, in females that only express one of a set of site-specific mutated forms of Vasa. Next, we will examine the extent to which Vasa orthologues from other species can function in D. melanogaster, by introducing these genes transgenically into a vasa mutant background. Finally, based on the results we obtain we will replace various domains of Vasa from non-rescuing species with D. melanogaster counterpart domains to attempt to restore function. This will enable us to map particular domains of Vasa that must be derived from the host species (in this case, melanogaster) for functionality. In the longer term extending beyond the scope of the present proposal we will investigate other piRNA-effector genes in a similar way. We will combine mutational analysis, molecular analysis, and protein structure information produced by others to understand particular molecular associations underlying reproductive isolation. For example, we will test whether a particular critical segment of Vasa interacts with a particular partner protein in a species-specific way.The expected impact of this work will include publications in high-quality international scientific journals, and the trainees funded by the grant (as well as the PI) will present their results at national and international conferences. The trainees will also complete PhD thesis projects that will have involved gaining facility in state-of-the-art molecular genetic and imaging techniques, which will enable them to pursue research careers in either the public or private sectors.