Mechanism of Self and Non-self Recognition in Fertilizations of Higher Plant.

自交不亲和性是指雌蕊能识别自花和非自花花粉，并能与非自花花粉受精的系统。本研究旨在鉴定配子体自交不亲和性（GSI）的雌蕊和花粉因子，并阐明其分子机制。2000 - 2002年，我们利用X射线晶体分析技术，在1.5A分辨率下，对蔷薇科植物梨（Pyrus pyrifolia）的S3-RNase进行了三维结构测定，发现了其上可能存在的花粉因子识别位点。最近，一个F-box基因，在花粉特异性表达的方式，已被发现在S-位点，并强烈建议是一个花粉因子。然而，无论是F-box蛋白和S-RNase之间的相互作用，还是泛素-蛋白酶体系统在GSI中的参与，都没有被直接证明。因此，我们用纯化的S-RNase构建了悬滴法抑制花粉管生长的离体体系。接着，利用该系统，我们分析了特异性表达的蛋白质对花粉管抑制和检查S-RNase的结构变化。结果，检测到特异性表达的13种蛋白，但这些蛋白中不包括F-box蛋白。有趣的是，侵入花粉管的S-RNase不受某些翻译后修饰的影响，在2D-PAGE凝胶上分离成几个斑点。将S-RNase引入花粉管12小时后，S-RNase完全降解，这在RNase T2或RNase A中从未观察到。这些结果表明，位于花粉管中的某些系统可能正向修饰和降解入侵的S-RNase。

Self-incompatibility is a system by which pistil can recognize self and nonself pollen and can be fertilized with only nonself-pollen. The purposes of this project are to identify pistil and pollen factors responsible for gametophytic self-incompatibility (GSI), and to clarify its molecular mechanism. During the last three years, 2000-2002, we determined the three dimensional structure of S3-RNase of Pyrus pyrifolia, a member of Rosaceae, at 1.5 A resolution by X-ray crystal analysis, showing the putative recognition sites on the S-RNase to a pollen factor. Recently, an F-box gene, expressed in a pollen-specific manner, has been found within S-locus and is strongly suggested to be a pollen factor. However, neither the interaction between the F-box protein and the S-RNase nor the participation of ubiquitin-proteasome system in GSI has been directly demonstrated. Therefore, we have constructed in vitro system of pollen tube growth inhibition by hanging drop method using purified S-RNase. Next, using this system, we analyzed proteins expressed specifically on pollen tube inhibition and checked the structural changing of the S-RNase. As a result, the 13 proteins expressed specifically were detected but the F-box protein was not included in these proteins. It is interesting that the S-RNase invaded into the pollen tube is susceptive to some post-translational modifications and is separated into several spots on a 2D-PAGE gel. After 12 hrs from when the S-RNase was taken into the pollen tube, it was completely degraded, which has never been observed using RNase T2 or RNase A. These results suggest that some systems located in the pollen tube may positively modify and degrade the invading S-RNase.