Pollen-Pistil Interactions in Nicotiana

IBN-0315647 McClure由于植物是无柄的，它们依赖于外部因素，如风或昆虫来传播花粉和实现受精。 因此，花粉传播是相对非特异性的，植物已经进化出复杂的花粉-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。 实验系统是基于S-RNase的烟草花粉识别和排斥系统。 S-RNase决定了该系统识别花粉的特异性。 该模型是S-RNase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物。 与糖蛋白结合促进S-RNase被花粉管吸收。 一旦进入花粉管，一个新鉴定的雌蕊因子（即，4936-因子）是S-RNase引起花粉排斥所必需的。 花粉因素也有影响。 一些直接结合到存在于S-RNase复合物中的糖蛋白。 其他直接确定兼容性或通过抑制S-RNase的细胞毒活性或通过阻止这种抑制。 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鉴定4936因子的候选者。 将对候选物进行测序，并通过RNA印迹分析检查其表达模式。 将培养抗体以测试花粉管的吸收。 该模型预测，4936-因子将激活S-RNase后，吸收到花粉管。 其次，将进行与S-RNase结合糖蛋白相互作用的花粉和雌蕊因子的酵母双杂交筛选。 该模型预测，花粉表达的蛋白质将结合到这些糖蛋白。 第三，将进行免疫定位实验，以确定是否RNase摄取正常发生在植物与各种不同的花粉排斥反应。 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在花粉识别和排斥过程中因素何时起作用。 该项目与本科教学有关。 使用产生的阵列cDNA文库筛选4936因子，学生将进行EST项目，以确定在成熟雌蕊中表达的序列。 学生将挑选克隆，分析序列，并汇集他们的数据，使EST列表。 这些活动将使他们为以后的研究经历做好更好的准备。

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.