SGER: Developing Tools for the Analysis of Reproductive Barriers

智力价值高等植物中花粉和雌蕊之间种间屏障的分子本质目前尚不清楚。我们计划研究番茄的种间障碍，番茄是一种具有显著遗传和分子优势的重要作物物种。然而，在我们开始对这些障碍进行分子分析之前，我们需要手中有几个工具。这项提议的目标是开发这些工具。首先，我们将开发一个可视化系统，用于显示雌蕊内正在生长的花粉管。其次，我们将开发一种荧光原位杂交或“鱼盒”，用于标记染色体臂和识别在生殖障碍中重要的连锁基因。这个盒将包含一个基于DS的工程转座元件，可用于诱变和增强子捕获。第三，我们将采用一种新兴的技术(“分泌组”分析)，并测试其在检测花粉和雌蕊分泌的蛋白质方面的适用性。这些细胞和分子系统将在这个项目之外的植物生物学研究中有广泛的用途。首先，科罗拉多州立大学的本科生将在整个项目期间参与工作的许多方面。如上所述，这项工作将对植物研究界产生影响，因为将要开发的工具将有广泛的应用。此外，这项工作将允许通过比较驯养和野生物种来扩大生物多样性研究的基因组方法。最后，植物繁殖的研究对整个社会来说是一个具有重要经济意义的领域，到目前为止，在基因组水平上理解生殖的努力有限。对生殖障碍的理解与水平基因转移直接相关；这既与将理想的性状从野生物种转移到驯化物种有关，也与限制从农作物向野生物种转移转基因有关。应贝丁格实验室的要求，拟南芥和番茄花粉启动子-GFP融合基因将在最初发表后提供给科学界。这些结构将对许多对花粉管生长和花粉-雌蕊相互作用感兴趣的研究人员有用。在资助期内产生的初始转基因番茄品系和绘制的鱼盒将被放大，并启动筛选过程，为将来产生完整的转基因品系做准备。一旦产生了一套完整的转基因株系(每个染色体臂上都有一个转基因盒)，这些转基因株系将在茄科基因组学网络http://www.sgn.cornell.edu/上公布，并将提供给番茄研究界。来自分泌组研究的数据将存储在乔斯林·罗斯博士正在开发的分泌组数据库中，该数据库可在他的网站http://labs.plantbio.cornell.edu/rose/上找到，序列将另外存储到基因组库中。

Intellectual MeritThe molecular nature of interspecific barriers between pollen and pistils in higher plants is not currently understood. We plan to study interspecific barriers in tomato, an important crop species with significant genetic and molecular advantages. However, before we embark on the molecular analysis of these barriers, there are several tools we need to have in hand. The objective of this proposal is the development of these tools. First, we will develop a system for the visualization of growing pollen tubes within pistils. Second, we will develop a Fluorescent In Situ Hybridization or "FISH-cassette" for the marking of chromosome arms and identifying linked genes important in reproductive barriers. This cassette will contain a Ds-based engineered transposable element that can be used for both mutagenesis and enhancer trapping. Third, we will take an emerging technology ("secretome" analysis) and test its applicability to the detection of proteins secreted from pollen and pistils. These cellular and molecular systems will have a wide utility in plant biology research beyond this project.Broader ImpactsThe work will have a broader impact at several levels. First, undergraduate students at Colorado State University will be involved with many aspects of the work throughout the duration of the project. As noted above, the work will impact the plant research community, since the tools to be developed will have wide application. In addition, the work will permit the expansion of genomic approaches to biodiversity studies with the comparison of domesticated and wild species. Finally, research in plant reproduction represents an area of significant economic importance to society at large, and efforts toward understanding of reproduction at the genomic level have been limited to date. The understanding reproductive barriers is directly relevant to horizontal gene transfer; both in terms of transferring desirable traits from wild species into domesticated ones, and in terms of limiting transfer of transgenes from crops into wild species.Project outcomes1. Arabidopsis and tomato pollen promoter-GFP fusions will be available to the scientific community after initial publications upon request from the Bedinger lab. These constructs will be of use to numerous researchers with interests in pollen tube growth and pollen-pistil interactions.2. The initial transgenic tomato lines with a mapped FISH cassette generated during the funding period will be bulked up and the curation process initiated in preparation for generating a complete series of transgenic lines in the future. Once a complete set of transgenic lines (with a transgene cassette mapped to each chromosome arm) has been generated, these will be announced on the Solanaceae Genomics Network http://www.sgn.cornell.edu/ and will be made available to the tomato research community.3. Data from the secretome studies will be deposited in Dr. Jocelyn Rose's developing secretome database, available on his website http://labs.plantbio.cornell.edu/rose/ and sequences will additionally be deposited into GenBank.