Molecular Basis of Self-Incompatibility in Petunia

[235176 .高]大多数开花植物都是两性花，雄性和雌性生殖器官的位置很近。开花植物在进化过程中采用了许多生殖策略，以防止近亲繁殖和促进异交。一种策略是自交不亲和（self-incompatibility， SI），它允许开花植物雌蕊拒绝自交花粉而接受非自交花粉。三科开花植物，茄科，蔷薇科和玄参科，都使用基于rase的SI机制。在这里，雌蕊S-RNase基因和位于遗传位点S-locus的尚未确定的花粉s -基因控制授粉的结果。在任何给定的物种中，s -位点有许多不同的s -单倍型（即变体），每个s -单倍型携带S-RNase基因和花粉s -基因的特定等位基因。在雌蕊花粉管生长过程中，如果花粉管携带的s -单倍型与雌蕊携带的两个s -单倍型中的一个相匹配，花粉管的生长就会受到抑制，从而无法将精细胞送到卵巢受精。认为雌蕊产生的s -RNase被花粉管吸收，s -RNase的活性是抑制花粉管生长的原因。虽然所有的S-RNase都能进入花粉管，但只有自S-RNase（与花粉管具有相同s单倍型的S-RNase）能够在花粉管内发挥作用。这就提出了如何实现s -单倍型特异性抑制的问题。为了解决这个问题，必须鉴定花粉s基因。PI的研究小组一直以矮牵牛花（Petunia inflata）作为模型来研究基于S-RNase的SI，最近发现了一个很可能的花粉s基因候选株PiSLF。矮牵牛花是花园矮牵牛花的野生亲戚。它位于S-RNase基因附近，在花粉中表达，并表现出等位基因序列的差异，这些都是花粉s基因的特性。此外，在玄参科植物Antirrhinum hispanicum的S-RNase基因附近发现了一个相似的基因。PiSLF将是这个项目的主要焦点。体内方法将用于确认PiSLF是否确实是花粉s基因。大多数F-box蛋白是参与泛素介导的蛋白质降解的SCF复合物的组分。因此，将使用几种体外方法来检查PiSLF是否介导花粉管中非自身s - rnase的特异性降解。该项目的另一个重点将是完成s位点的连续染色体区域（contig）的克隆。该序列将用于鉴定其他s位点连锁基因，如果发现PiSLF不是花粉s基因，则用于鉴定其他候选基因。该项目的完成不仅将大大促进对基于rnase的自我/非自我识别机制的理解，而且有助于理解F-box蛋白介导的选择性蛋白质降解。后者最近已成为多种生物中各种细胞和发育过程的重要调节机制。在实践方面，如果PiSLF被证实是花粉s -基因，可以探索通过转移S-RNase基因和PiSLF基因来恢复自交亲和栽培物种的SI性状的可能性，以促进杂交种子的生产。如果成功，这将产生非常重要的农艺影响。在美国，大多数农作物都是用杂交种子种植的，因为这种植物比用自花授粉的种子种植的植物更有活力，产量更高。目前，杂交种子生产需要人工或机械去除作为母本的植物的花药，以防止自花受精。这是一个劳动密集、成本高昂且效率低下的过程。因此，迫切需要经济上更有利的杂交制种方法。

0235176Kao Most flowering plant species produce bisexual flowers with both male and female reproductive organs located in close proximity. A number of reproductive strategies have been adopted during the course of evolution to allow flowering plants to prevent inbreeding and promote out-crosses. One strategy is self-incompatibility (SI), which allows pistils of flowering plants to reject self-pollen and accept non-self pollen. Three families of flowering plants, Solanaceae, Rosaceae and Scrophulariaceae, all use an RNase-based SI mechanism. Here, the pistil S-RNase gene and the yet unidentified pollen S-gene located at a genetic locus, the S-locus, control the outcome of pollination. In any given species, there are many different S-haplotypes (i.e., variants) of the S-locus, with each S-haplotype carrying a specific allele of the S-RNase gene and the pollen S-gene. During pollen tube growth in a pistil, if the S-haplotype carried by the pollen tube matches one of the two S-haplotypes carried by the pistil, the growth will be inhibited and thus the pollen tube cannot deliver the sperm cell to the ovary for fertilization. It is thought that S-RNases produced in the pistil are taken up by pollen tubes, and that the RNase activity of S-RNases is responsible for growth inhibition of pollen tubes. Although all S-RNases can enter a pollen tube, only the self S-RNase (the S-RNase of the same S-haplotype as the pollen tube) is able to function inside the pollen tube. This raises a question as to how S-haplotype specific inhibition can be achieved. To address this question, the pollen S-gene must be identified. The PI's group has been using Petunia inflata, a wild relative of garden petunia, as a model to study S-RNase based SI, and has recently identified a very likely candidate, named PiSLF, for the pollen S-gene. It is located near the S-RNase gene, expressed in pollen, and shows allelic sequence differences, all of which are properties expected of the pollen S-gene. Moreover, a similar gene has been found to be located near the S-RNase gene of Antirrhinum hispanicum, a species in the Scrophulariaceae. PiSLF will be a major focus of this project. In vivo approaches will be used to confirm whether PiSLF is indeed the pollen S-gene. Most F-box proteins are components of SCF complexes that are involved in ubiquitin-mediated protein degradation. Thus, several in vitro approaches will be used to examine whether PiSLF mediates specific degradation of non-self S-RNases in the pollen tube. Another focus of the project will be on the completion of the cloning of a contiguous chromosome region (contig) of the S-locus. This contig will be used for identifying other S-locus linked genes and, in the event that PiSLF is found not to be the pollen S-gene, for identifying additional candidates. Accomplishment of this project will not only significantly advance understanding of the RNase-based self/non-self recognition mechanism, but also contribute to the understanding of selective protein degradation mediated by F-box proteins. The latter has recently emerged as an important regulatory mechanism for a variety of cellular and developmental processes in diverse organisms. On the practical side, if PiSLF is confirmed to be the pollen S-gene, one can explore the possibility of restoring the SI trait to self-compatible cultivated species by transferring the S-RNase gene and the PiSLF gene to facilitate hybrid seed production. If successful, this will have a very important agronomic impact. In the United States, the majority of crops are grown from hybrid seed, because such plants have greater vigor and produce higher yield than plants grown from seed obtained from self-pollination. Currently, hybrid seed production requires manual or mechanical removal of anthers from the plants serving as female parent to prevent self-fertilization. This is a very labor-intensive, costly, and inefficient process. Thus, there is a pressing need for more economically advantageous methods for hybrid seed production.