Molecular Basis of Self-Incompatibility in Petunia

大多数开花植物的两性花都是雄性和雌性的生殖器官靠得很近。 在进化过程中，人们采取了许多繁殖策略，以使开花植物能够防止近亲繁殖并促进异交。 一种策略是自交不亲和（SI），它允许开花植物的雌蕊拒绝自花花粉并接受非自花花粉。 有花植物的三个科，茄科、蔷薇科和玄参科，都使用基于RNA酶的SI机制。 在这里，雌蕊S-RNase基因和尚未鉴定的花粉S-基因位于一个遗传位点，S-位点，控制授粉的结果。 在任何给定的物种中，存在许多不同的S-单倍型（即，S-基因座的S-单倍型（S-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。 在雌蕊中的花粉管生长期间，如果由花粉管携带的S-单倍型与由雌蕊携带的两个S-单倍型中的一个匹配，则生长将被抑制，因此花粉管不能将精细胞运送到子房进行受精。 据认为，雌蕊中产生的S-RNase被花粉管吸收，并且S-RNase的RNase活性负责花粉管的生长抑制。 虽然所有的S-RNase都可以进入花粉管，但只有自身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。 这就提出了一个问题，S-单倍型特异性抑制如何才能实现。 为了解决这个问题，花粉S-基因必须得到鉴定。 PI的研究小组一直在使用矮牵牛（Petunia inflata），一种花园矮牵牛的野生亲戚，作为研究基于S-RNase的SI的模型，最近已经确定了一个非常可能的候选者，命名为PiSLF，用于花粉S基因。 它位于S-RNase基因附近，在花粉中表达，并显示等位基因序列差异，所有这些都是花粉S-基因的预期特性。 此外，在玄参科金鱼草的S-RNase基因附近发现了一个类似的基因。 PiSLF将是该项目的重点。 将使用体内方法来确认PiSLF是否确实是花粉S基因。 大多数F-box蛋白是SCF复合物的组成部分，参与泛素介导的蛋白质降解。 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-RNases的特异性降解。 该项目的另一个重点将是完成S基因座的连续染色体区域（重叠群）的克隆。 该重叠群将用于鉴定其他S基因座连锁基因，并且在发现PiSLF不是花粉S基因的情况下，用于鉴定其他候选基因。该项目的完成不仅将显著推进对基于RNase的自我/非自我识别机制的理解，而且有助于理解由F-box蛋白介导的选择性蛋白质降解。 后者最近已成为一个重要的调节机制，在不同的生物体的各种细胞和发育过程。 在实践方面，如果PiSLF被证实是花粉S-基因，则可以探索通过转移S-RNase基因和PiSLF基因以促进杂交种子生产来将SI性状恢复到自交亲和栽培物种的可能性。 如果成功，这将产生非常重要的农业影响。 在美国，大多数作物都是由杂交种子种植的，因为这种植物比由自花授粉获得的种子种植的植物具有更大的活力和更高的产量。 目前，杂交种子生产需要人工或机械地从作为母本的植物上去除花药以防止自花受精。 这是一个非常劳动密集、成本高昂且效率低下的过程。 因此，迫切需要更经济有利的杂交种子生产方法。

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.