Isolation of pollen S-determinant gene in Brassica species by using transformation and allelic polymorphysim

甘蓝型油菜自交不亲和性由多个等位基因控制，称为S。到目前为止，在S基因座上已经发现了两个多态基因。我们确定，S受体蛋白(SRK)决定了柱头的S单倍型特异性，而S位点糖蛋白(SLG)基因增强了SI反应。然而，当我们开始这个项目时，还没有鉴定出花粉S的决定簇。在我们之前的研究中，我们分离了含有SLG^9和SRK^9的76kb的相邻基因组片段，并测定了片段中的核苷酸序列和表达基因。在该片段中，我们鉴定了两个花药特异基因SAE1和SP11。然而，我们并不能从序列信息中确定哪些基因是真正的花粉S决定簇。因此，我们开始利用转化技术和等位基因多态性来确定真正的花粉S决定因素，但在SAE1号中，我们没有发现S等位基因之间的等位基因多态性。在控制…中此外，我们还可以从S等位基因中分离到类似SP11的cDNA克隆。此外，通过与Isogai教授的团队的合作研究，还从S^8和S^lt；12&gt；等位基因中分离到了类似SP11的cDNA克隆。这四个克隆编码新的富含半胱氨酸的花粉外壳蛋白(PCP)。它们均位于S等位基因SLG/SRK基因两侧，表明这4个类SP11基因是等位基因。它们的核苷酸序列在S等位基因之间发生了高度差异。此外，由于该基因在绒毡层细胞中表达，我们可以解释油菜的SI是由孢子体控制的。因此，我们选择该sp11基因作为花粉S决定簇的候选基因。为了确定该sp11基因是真正的花粉S决定基因，将sp11-9基因转化到S^&lt；52&gt；S^&lt；60&gt；杂合子中。转化子的花粉在S 9号柱头上被拒收，柱头侧没有观察到表型变化，表明SP11基因是真正的花粉S决定基因。通过RT-PCR法和Chef分析相结合的方法，分离到16个新的Sp11基因，它们位于S基因座，大小在60~104kb之间。对SP11的氨基酸序列进行比对，发现6个半胱氨酸残基在所有SP11中都是完全保守的，表明这些半胱氨酸残基对三级构象的形成具有重要的作用。在构建SLG、SRK和SP11的系统发育树时，这3个基因的进化模式相似，表明这3个基因共同进化形成了一个新的等位基因。此外，我们分离到的SP11基因被归入II类单倍型。并观察到与上述第一类S单倍型相似的趋势。较少

Self-incompatibility (SI) in Brassica campestris is controlled by a single locus, termed S, with multiple alleles. To date, two polymorphyic genes have been identified at the S locus. We determined that the S receptor kinase (SRK) determines the S haplotype specificity of stigma, and the S locus glycoprotein (SLG) gene enhances the SI response. However, pollen S determinant was not identified, when we started this project. In our previous study, we isolated 76-kb contiguous genomic fragment containing SLG^9 and SRK^9, and determined the nucleotide sequence and expressed genes in the fragments. In the fragment, we identified two anther-specific genes, SAE1 and SP11. However, we did not determine which genes, SAE1 and SP11, were real pollen S determinant from their sequence information. Thus, we started to determine the real pollen S determinant by using transformation technique and allelic polymorphism.In the case of SAE1, we could not find allelic polymorphism among S alleles. In contr … More ast, we could isolate SP11-like cDNA clone from S^<52> allele. Furthermore, SP11-like cDNA clones from S^8 and S^<12> alleles were also isolated by co-operative research with Prof. Isogai's group, NAIST. These four clones encode the novel cysteine-rich pollen coat protein (PCP). They were located at the flanking region of SLG/SRK gene in each S allele, indicating that these four SP11-like genes were allelic. Their nucleotide sequences were highly diverted among S alleles. Furthermore, because this gene expressed in tapetum cells, we could explain that SI in Brassica is sporophytically controlled. Therefore, we selected this SP11 gene as a candidate gene for pollen S determinant. In order to determine that this SP11 gene is real pollen S determinant, SP11-9 gene were transformed into S^<52>S^<60> heterozygote. The pollen of transformant was rejected on S^9 stigma, and no phenotype change was observed in stigma side, indicating that the SP11 gene is real pollen S determinant. By using the combination of RT-PCR and CHEF analysis, 16 novel SP11 genes were isolated, and were located at the S locus, whose size was from 60 to 104 kb.When aligned the estimated amino acid sequences of SP11, six cysteine residues were completely conserved in all SP11, indicating that these cysteine residues are important for formation of tertiary conformation.When constructed the phylogenetic trees in SLG, SRK, and SP11, the pattern of the trees were similar among these three genes, indicating that these three genes co-evolved to make a new S allele.Furthermore, we isolated SP11 gene classified into class II S haplotype, and observed a similar trend to class I S haplotypes as described above. Less