Molecular genetics underlying reproductive isolation of species

这项提案中的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，我们将研究这样一个假设，即Vasa是一种在卵子发生期间在生殖细胞中表达的快速进化的蛋白质，并且是子代胚胎中原始生殖细胞特化所必需的母体蛋白质，是限制种间杂种生育力的重要因素。真核基因组包括果蝇的基因组包含许多转座因子（TE）拷贝，其移动性必须被抑制以保持染色体的完整性。 TE转座的抑制涉及称为Piwi相互作用RNA（piRNA）的小RNA，其靶向TE衍生的mRNA，随后使其不稳定并抑制其翻译。 在种间果蝇杂交雌性中，TE是去抑制的，提高了这种系统在建立物种生殖隔离中起根本作用的可能性。 如果是这样的话，那么可以预期，负责的蛋白质或蛋白质将非常迅速地进化，使得来自一个物种的蛋白质将足够不同，而不会在不同的物种中起作用。**Vasa最近被认为与皮尔纳加工有关，它的区域差异很大，甚至在密切相关的果蝇物种之间也是如此，如D。melanogaster和D. simulans。** 为了检查Vasa、皮尔纳介导的过程和生殖隔离之间的关系，我们将首先通过分析仅表达一种的女性中TE编码基因的表达和皮尔纳表达来确定piRNA介导的过程需要Vas的哪些结构域。一组位点特异性突变形式的Vasa。 接下来，我们将研究来自其他物种的Vasa直系同源物在D.通过将这些基因转基因导入vasa突变体背景中， 最后，基于我们得到的结果，我们将用D.黑腹菌对应结构域试图恢复功能。 这将使我们能够映射Vasa的特定结构域，这些结构域必须来自宿主物种（在这种情况下，黑腹）的功能。 ** 在超出本提案范围的长期内，我们将以类似的方式研究其他piRNA效应基因。 我们将结合联合收割机的突变分析，分子分析，和蛋白质结构的信息产生的其他人，以了解特定的分子协会生殖隔离。 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，由赠款资助的受训人员（以及PI）将在国家和国际会议上介绍他们的成果。 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和成像技术，这将使他们能够在公共或私营部门从事研究事业。

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.