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

白菜型油菜自交不亲和性（SI）受一个S位点控制，具有多个等位基因。到目前为止，已经确定了两个多态性基因在S位点。我们确定S受体激酶（SRK）决定柱头的S单倍型特异性，S位点糖蛋白（SLG）基因增强SI反应。然而，在我们开始这个项目时，花粉S决定簇还没有被鉴定出来。在我们之前的研究中，我们分离了含有SLG ^9和SRK ^9的76-kb连续基因组片段，并确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，SAE 1和SP11。然而，我们没有确定哪些基因，SAE 1和SP 11，是真实的花粉S决定子从它们的序列信息。因此，我们开始利用转化技术和等位基因多态性来确定真实的花粉S决定子，在SAE 1的情况下，我们没有发现S等位基因之间的等位基因多态性。对照 关于我们 最后，我们从S^等位基因中分离到了类SP11的cDNA克隆<52>。此外，<12>通过与NAIST的矶贝教授小组的合作研究，还分离了来自S^8和S^等位基因的SP11样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）。它们位于每个S等位基因中SLG/SRK基因的侧翼区，表明这4个类SP11基因是等位的。它们的核苷酸序列在S等位基因之间高度转移。此外，由于该基因在绒毡层细胞中表达，我们可以解释芸苔属植物的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。为了确定该基因是真实的花粉S决定子，将SP11-9基因转化到S^<52>S^<60>杂合子中。在S^9柱头上，花粉被排斥，柱头侧没有观察到表型变化，表明SP11基因是真实的花粉S决定子。通过RT-PCR和CHEF分析相结合的方法，共分离到16个新的SP11基因，均位于S位点，大小在60 ~ 104 kb之间。表明这些半胱氨酸残基对三级构象的形成是重要的。在构建SLG、SRK和SP11的系统发育树时，此外，我们还分离了SP11基因，将其归类为II类S单倍型，并观察到与I类S单倍型相似的趋势。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