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

自交不亲和是雌蕊能够识别自交和非自交花粉，并且只能与非自交花粉受精的一种系统。本课题旨在鉴定配子体自交不亲和的雌蕊和花粉因子，并阐明其分子机制。在2000-2002年的三年中，我们用x射线晶体分析方法测定了蔷薇科植物Pyrus pyrifolia S3-RNase在1.5 a分辨率下的三维结构，显示了S-RNase对花粉因子的推测识别位点。最近，在s位点发现了一个以花粉特异性方式表达的F-box基因，该基因被强烈认为是花粉因子。然而，F-box蛋白与S-RNase之间的相互作用以及泛素-蛋白酶体系统在GSI中的参与都没有被直接证实。因此，我们利用纯化的S-RNase构建了挂滴法体外抑制花粉管生长的体系。接下来，我们利用该系统分析了花粉管抑制下特异性表达的蛋白，并检查了S-RNase的结构变化。结果，检测到特异性表达的13种蛋白，但F-box蛋白不包括在这些蛋白中。有趣的是，侵入花粉管的S-RNase对一些翻译后修饰很敏感，在2D-PAGE凝胶上被分成几个点。从S-RNase进入花粉管12小时后，S-RNase被完全降解，这是RNase T2或RNase a从未观察到的。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.