Pollen-pistil Interactions in the Genus Nicotiana

本项目的目的是鉴定和表征有助于烟叶s等位基因特异性花粉识别的因素。在这个物种中，s位点控制着种内相容性。当花粉中的s等位基因与雌蕊中表达的s等位基因中的一个相同时，花粉被拒斥。S-RNases控制着花粉排斥的特异性，但其他因素也以其他方式促进s -等位基因特异性花粉识别。我们的主要目标是识别和表征这些非s - rnase因子。ht蛋白就是这样一个因素，它似乎是柱状细胞外基质的不稳定成分。ht2的反义抑制可防止s等位基因特异性花粉排斥，但其确切功能尚不清楚。我们建议通过实验来确定样式发育过程中ht蛋白的丰度，以及为什么它如此不稳定。我们已经在体外发现了两种特异性的柱状蛋白与S-RNase结合。一种是先前被发现的蛋白质NaTTS，它与相容花粉管的生长有关。第二种称为p11，尚未被很好地表征。NaTTS和p11被认为是非s - rnase因子，可能与花粉排斥和支持相容花粉管生长有关。提出凝胶过滤和蛋白印迹分析来确定NaTTS/S-RNase/p11复合物的分子量和组成。NaTTS和p11也将在细菌中表达，然后用于体外相互作用的测试。反义方法将用于确定p11在体内是否影响亲和性或非亲和性授粉。为了鉴定新的非s - rnase因子，我们产生了具有s等位基因特异性花粉排斥能力的群体。cDNA AFLP将用于筛选与花粉排斥表型相关的新序列。本研究将有助于更好地理解花粉识别和排斥的遗传机制。这对希望操纵植物育种行为的生物技术专家具有实际意义。

The goal of this project is to identify and characterize factorsthat contribute to S-allele-specific pollen recognition in Nicotiana alata.In this species, the S-locus controls intraspecific compatibility. Pollenis rejected when the S-allele in the pollen is the same as one of theS-alleles expressed in the pistil. S-RNases control the specificity ofpollen rejection, but other factors contribute to S-allele-specific pollenrecognition in other ways. Our main objective is to identify andcharacterize these non-S-RNase factors. HT-protein is one such factor that appears to be an unstablecomponent of the stylar extracellular matrix. Antisense suppression of HTprevents S-allele-specific pollen rejection, but its precise function isunknown. We propose experiments to determine the abundance of HT-proteinduring style development, and why it is so unstable. We have found that twospecific stylar proteins bind to S-RNase in vitro. One is a previouslycharacterized protein, NaTTS, that is implicated in growth of compatiblepollen tubes. The second, designated p11, has not been well characterized.NaTTS and p11 are putative non-S-RNase factors that may be implicated inboth pollen rejection and in supporting compatible pollen tube growth. Gelfiltration and protein blot analysis are proposed to determine the molecularweight and composition of NaTTS/S-RNase/p11 complexes. NaTTS and p11 willbe also expressed in bacteria and then used to test for interactions invitro. Antisense methods will be used to determine whether p11 affectscompatible or incompatible pollinations in vivo. To identify newnon-S-RNase factors, we have generated populations that segregate for theability to perform S-allele-specific pollen rejection. cDNA AFLP will beused to screen for new sequences associated with pollen rejection phenotype. The impact of this research will be to provide a betterunderstanding of the genetic mechanisms for pollen recognition andrejection. This is of practical significance to biotechnologists who wishto manipulate plant breeding behavior.