Molecular Basis of Self-Incompatibility in Petunia

膨胀矮牵牛花具有配子体自交不亲和性，这种自交不亲和性受多态位点S控制。花粉携带的S等位基因与面食携带的S等位基因匹配，抑制花柱花粉管生长。这个建议的目的是了解雌蕊如何区分自我和非自我花粉。由于目前只鉴定了雌蕊S等位基因的产物，即S蛋白或S rna酶，因此本课题的前两个目标是鉴定花粉S基因及其等位基因产物。在第一个目标中，将使用四种方法来确定花粉S基因的潜在候选基因：S蛋白亲和层析、酵母双杂交蛋白-蛋白相互作用试验、肽展示文库筛选和mRNA差异展示。鉴定出的蛋白、多肽和PCR片段的cdna将被分离并检测其对应的基因是否与S等位基因共分离，以及等位基因之间是否存在序列差异，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满足上述两个标准的花粉基因中是否有花粉S基因。第三个目的是使用两种方法来确定花粉的等位基因特异性抑制是否在于选择性地将自身S蛋白摄取到花粉管中。第一种方法是研究在转基因植物S3花粉细胞质中产生缺乏RNase活性的S3蛋白突变体是否对S3花粉的自交不亲和行为产生显性的负面影响。第二种方法是通过免疫定位分别用S2和S3花粉授粉的S3S3雌蕊切端生长出的花粉管显微切片，来检测S3蛋白是否被S2（非自体）和/或S3（自体）花粉管吸收。使用rnase作为识别分子是本文研究的自交不亲和性类型所特有的，因此本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rnase生物学功能的理解范围。在实践方面，一旦花粉S基因被鉴定出来，就可以探索在自交不亲和的栽培物种中恢复自交不亲和性状的可能性，以促进杂交种子的生产。如果成功，这将对农业产生非常重要的影响。

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.