Isolation and functional analysis of the self-incompatibility gene in the grasses (Poaceae)

利用AFLP技术，从具有配子体自交不亲和性的野生大麦(2x)雌蕊和花药中筛选到S基因的特异转录本。利用AMF分析得到的多个克隆构建了S座位周围的详细遗传连锁图谱。将该连锁图谱与大麦染色体1的物理图谱进行比较，结果表明球茎拟青霉的S基因座位于一大片横跨着丝粒的染色体区域，重组受到严重抑制。在连锁分析中，我们获得了5个与S基因座没有重组的克隆。其中两个无性系表现出雌蕊或花药特异表达。序列分析表明，雌蕊特异克隆(HPS10)编码一个未知的小蛋白，可能是一种分泌型信号分子。该克隆具有高度的等位基因多态性，有望成为雌蕊S基因的候选基因。因此，我们一直在尝试用其RNAi构建转化球茎海棠愈伤组织，但到目前为止还没有获得转化子。对于花药特异克隆(122-7)，序列分析表明它将编码一个带有F-box基序的蛋白质。这一发现之所以值得注意，是因为最近在其他配子体SI种(茄科等)中发现了花粉S因子。是一种F-box蛋白。该克隆的等位基因多态性有待进一步研究。在最后的实验中，我们对球茎海棠雌蕊蛋白进行了2D-Gel分析，并通过LC-MS/MS鉴定了一个S基因型特有的蛋白，分离到一个编码该蛋白的cDNA克隆，发现该蛋白与S基因座紧密连锁。

The AFLP-based mRNA, fingerprinting (AME) analysis was conducted to find S genotype-specific transcripts from pistils and anthers at anthesis in Wild barley, Hordeum bulbosum (2x) that possesses the gametophytic self-incompatibility (SI). A detailed genetic linkage map around the S locus was constructed using several clones obtained by the AMF analysis. Comparison of the linkage map with a physical map of barley choromosome 1 indicated that the S locus of H. bulbosum was located in a large chromosomal region across the centromere, where recombination is severely suppressed. In the linkage analysis, we have obtained five clones that showed no recombination with the S locus. Two of those clones showed pistil- or anther-specific expression. From sequence analysis of, the full length cDNA, the pistil-specific clone (HPS10) was shown to encode a small unknown protein that seems to be a putative secreted signaling molecule. Together with a high degree of allelic polymorphism, this clone turned out to be a promising candidate for the pistil S gene. Thus, we have been trying, to transform H. bulbosum calli with its RNAi construct, but so far we have obtained no transformants. For the anther-specific clone (122-7), sequence analysis revealed that it would encode a protein carrying an F-box motif. This finding is notable because the pollen S factor recently identified in the other gametophytic SI species (Solanaceae, etc.) is found to be an F-box protein. Further investigation on allelic polymorphism of the clone is needed. In the final experiment, we conducted 2D-gel analysis of pistil proteins in H. bulbosum and identified an S genotype-specific protein by LC-MS/MS. A cDNA clone coding for the protein was isolated and revealed to be tightly linked to the S locus.