Pollen-Pistil Interactions in Nicotiana

烟草中花粉与雌蕊的相互作用

• 批准号: 0315647
• 负责人: Bruce McClure
• 金额: --
• 依托单位: University of Missouri-Columbia
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2006-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0315647&HistoricalAwards=false
• 关键词: Pollen; Pistil; Interactions; Nicotiana

IBN-0315647McClureBecause plants are sessile, they rely on external agents such as wind or insects to disperse pollen and effect fertilization. Thus, pollen dispersal is relatively non-specific, and plants have evolved elaborate pollen-pistil interactions to control mating. The overall goal of this research is to understand the molecular basis for these interactions. The experimental system is S-RNase-based pollen recognition and rejection in Nicotiana. S-RNase determines the specificity of pollen recognition in this system. The model is that S-RNase is secreted into the extracellular matrix of the pistil where it forms complexes with glycoproteins known to interact with pollen tubes. Binding to the glycoproteins facilitates S-RNase uptake by pollen tubes. Once inside the pollen tube, a newly identified pistil factor (i.e., 4936-factor) is required for S-RNase to cause pollen rejection. Pollen factors also contribute. Some bind directly to the glycoproteins present in S-RNase complexes. Others directly determine compatibility either by inhibiting S-RNase's cytotoxic activity or by preventing such inhibition. This project has three objectives designed to test aspects of this model. First, a comprehensive screen for pistil-expressed sequences will be used to identify candidates for 4936-factor. Candidates will be sequenced, and their expression patterns will be examined by RNA blot analysis. Antibodies will be raised to test for uptake into pollen tubes. The model predicts that 4936-factor will activate S-RNase after uptake into pollen tubes. Second, a yeast two-hybrid screen for pollen and pistil factors that interact with S-RNase binding glycoproteins will be conducted. The model predicts that pollen expresses proteins that will bind to these glycoproteins. Third, immunolocalization experiments will be performed to determine whether RNase uptake occurs normally in plants with a variety of different pollen rejection responses. These experiments may reveal new steps in the model or may help define when factors act during pollen recognition and rejection. This project is linked to undergraduate teaching. Using the arrayed cDNA libraries produced to screen for 4936-factor, students will conduct an EST project to identify sequences expressed in mature pistils. Students will pick clones, analyze sequences, and pool their data to make an EST list. These activities will better prepare them for later research experiences.

因为植物是固着的，它们依靠外部因素，如风或昆虫来散布花粉和进行受精。因此，花粉传播是相对非特异性的，植物进化出复杂的花粉-雌蕊相互作用来控制交配。这项研究的总体目标是了解这些相互作用的分子基础。该实验系统是基于S核糖核酸酶的烟草花粉识别与排斥实验系统。S核糖核酸酶决定了该系统识别花粉的特异性。模型是S核糖核酸酶被分泌到雌蕊的细胞外基质中，在那里它与已知与花粉管相互作用的糖蛋白形成复合体。与糖蛋白的结合促进了花粉管摄取S核糖核酸酶。一旦进入花粉管，S核糖核酸酶需要一种新发现的雌蕊因子(即4936因子)来引起花粉排斥。花粉因素也起到了作用。有些直接与存在于S核糖核酸酶复合体中的糖蛋白结合。另一些则通过抑制S-核糖核酸酶的细胞毒活性或通过阻止这种抑制来直接决定亲和性。该项目有三个目标，旨在测试该模型的各个方面。首先，将使用雌蕊表达序列的全面筛选来确定4936因子的候选基因。将对候选基因进行测序，并通过RNA印迹分析检测它们的表达模式。抗体将被提高，以测试对花粉管的摄取。该模型预测，4936因子进入花粉管后将激活S核糖核酸酶。其次，将进行酵母双杂交筛选与S核糖核酸酶结合糖蛋白相互作用的花粉和雌蕊因子。该模型预测，花粉表达的蛋白质将与这些糖蛋白结合。第三，将进行免疫定位实验，以确定核糖核酸酶在具有各种不同花粉排斥反应的植物中是否正常发生。这些实验可能会揭示模型中的新步骤，或者可能有助于确定因素在花粉识别和排斥过程中的作用。这个项目是与本科教学相联系的。使用为筛选4936因子而产生的阵列化的cDNA文库，学生们将进行EST项目来鉴定在成熟雌蕊中表达的序列。学生们将挑选克隆，分析序列，并将他们的数据汇集在一起，形成EST列表。这些活动将使他们为以后的研究体验做好更好的准备。