Molecular Basis of Self-Incompatibility in Petunia

高德惠96 - 03993 矮牵牛配子体自交不亲和性受一个多态位点S位点控制。花粉和花粉粒所携带的S等位基因的匹配导致花柱中花粉管生长的抑制。这个提议的目的是了解雌蕊如何区分自花花粉和非自花花粉。 因为到目前为止，只有雌蕊S等位基因的产物，S蛋白或S RNA酶，已被确定，本建议的前两个目标是花粉S基因及其等位基因产物的鉴定。在第一个目标中，将使用四种方法来鉴定花粉S基因的潜在候选者：S蛋白亲和层析、酵母双杂交蛋白-蛋白相互作用测定、肽展示库筛选和mRNA差异展示。将分离并检查所鉴定的蛋白质肽和PCR片段的cDNA，以确定其相应的基因是否与S等位基因共分离并显示等位基因之间的序列变异，这是花粉S基因的两个标准。第二个目标是使用功能获得和功能丧失的方法来确定是否有任何这些花粉基因，满足上述两个标准是花粉S基因。第三个目的是使用两种方法来确定等位基因特异性抑制花粉是否在于选择性摄取自身S蛋白进入花粉管。第一种方法是检查在转基因植物的S3花粉的细胞质中缺乏RNA酶活性的突变体S3蛋白的产生是否对S3花粉的自交不亲和行为具有显性负效应。第二种方法是检查S3蛋白是否被S2（非自身）和/或S3（自身）花粉管的显微镜下切片的免疫定位的S3S3雌蕊，已与S2和S3花粉授粉，分别增长出来。 使用RNA酶作为识别分子是这里研究的自交不亲和性类型所独有的，因此这项拟议的研究完成后将揭示生物系统中细胞识别的新范式，并扩大对RNA酶生物功能的理解范围。在实践方面，一旦花粉S基因被鉴定，人们可以探索将自交不亲和性状恢复到自交亲和栽培物种以促进杂交种子生产的可能性。如果成功，这将对农业产生重大影响。

Teh-hui Kao 96-03993 Petunia inflata possesses gametophytic self-incompatibility which is controlled by a polymorphic locus, the S locus. Matching of S alleles carried by the pollen and pasta results In Inhibition of pollen tube growth in the style. The goal of this proposal is to understand how a pistil distinguishes between self and non-self pollen. Because so far only the products of pistil S alleles, S proteins or S RNases, have been identified, the first two objectives of this proposal are the identification of the pollen S gene and its allelic products. In the first objective, four approaches will be used to identify potential candidates for the pollen S gene: S protein affinity chromatography, the yeast two-hybrid protein-protein interaction assay, peptide display library screening, and mRNA differential display. cDNAs for the proteins peptides, and PCR fragments identified will be isolated and examined to determine whether their corresponding genes co-segregate with S alleles and show sequence variation between alleles, two criteria for the pollen S gene. The second objective is to use gain-of-function and loss-of-function approaches to ascertain whether any of those pollen genes that meet the above two criteria is the pollen S gene. The third objective is to use two approaches to ascertain whether allele specific inhibition of pollen lies in selective uptake of self S proteins into pollen tubes. The first approach is to examine whether production of a mutant S3 protein lacking RNase activity in the cytoplasm of S3 pollen of transgenic plants has a dominant negative effect on the self-incompatibility behavior of S3 pollen. The second approach is to examine whether S3 protein is taken up by S2 (non-self) and/or S3 (self) pollen tubes by immunolocalization of microscopic sections of pollen tubes that grow out of cut ends of S3S3 pistils that have been pollinated with S2 and S3 pollen, separately. The use of RNases as recognition molecules is unique to the type of self-incompatibility studied here, thus this proposed research when completed will uncover a new paradigm for cellular recognition in biological systems and expand the scope of the understanding of the biological functions of RNases. On the practical side, once the pollen S gene is identified, one can explore the possibility of restoring the self-incompatibility trait to self-compatible cultivated species to facilitate hybrid seed production. If successful, this will have a very important impact on agriculture.