Molecular basis of mechanisms of dominance relationships among S haplotypes in self-incompatible Brassicaceae.

自交不亲和（SI）系统通过排斥来自具有相同S单倍型的植物的花粉来防止自交受精并促进异交。芸苔属SI系统在孢子体上由单个位点的多个等位基因控制，称为S位点。由于受孢子体的控制，S单倍型在柱头和花粉中都存在显性关系。我们对其中四种II类S单倍型进行了S杂合体与其各自的亲本S纯合体之间的测交，并观察到花粉侧存在线性显性关系。为了确定这种显性关系是如何控制的，RNA凝胶印迹分析进行了6个S杂合子和它们各自的父母S纯合子通过使用相应的SP11 cDNA克隆作为探针。在所有6个S杂合子中，来自显性S单倍型的SP11主要表达，来自隐性S单倍型的SP11被抑制。因此，花粉侧SI表型的线性显性关系受SP11基因表达的调控。我们测定了含有相应SP11的3个II类S单倍型基因组克隆的核苷酸序列。这些包含相应SRK的5 '侧翼区的部分。SP11和SRK在各S单倍型中的距离约为10 kbp。这一结果表明，SP11和SRK的相对位置是保守的。因此，SP11和SRK之间的区域被命名为“S-基因间区”。然后，我们克隆了II类SRK，并与其他II类SRK进行了比较。II类SRK内的预测氨基酸序列相似性为约90%至95%，尽管S结构域的高变区被转移。这些结果表明，不仅相似的S基因，但其SP11和SRK的物理位置是保守的II类S单倍型。

Self-incompatibility (SI) systems prevent self-fertilization and promote out-crossing by rejecting pollen from plants with the same S haplotype. The Brassica SI system is controlled sporophytically by multiple alleles at the single locus, termed S locus. Because of sporophytic control of Brassica SI, dominance relationships among S haplotypes are observed in both stigma and pollen. We performed test-crosses between S heterozygotes and their respective parental S homozygotes for four of these class-II S haplotypes, and observed a linear dominance relationship on the pollen side. In order to determine how this dominance relationship is controlled, RNA gel blot analyzes were performed for six S heterozygotes and their respective parental S homozygotes by using the corresponding SP11 cDNA clone as a probe. In all six S heterozygotes, SP11 derived from a dominant S haplotype was predominantly expressed, and SP11 derived from a recessive S haplotype was repressed. Therefore, the linear dominance relationship of the SI phenotype on the pollen side is regulated by the expression of SP11 gene.We determined the nucleotide sequences of the genomic clone of three class-II S haplotypes that contained corresponding SP11. These contain the part of the 5'-flanking region of the respective SRK. The distance of SP11 and SRK is about 10kbp in each S haplotypes. This result indicates that the relative position of SP11 and SRK is conserved. Therefore, the region between SP11 and SRK was named the "S-intergenic region". Then, we cloned class-II SRK, and compared these with other class-II SRK. The predicted amino acid sequence similarity within class-II SRK is about 90 to 95%, though the hypervariable region of the S domain is diverted. These results indicate not only similarity of S genes but that the physical position of their SP11 and SRK is conserved in the class-II S haplotypes.