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

油菜自交不亲和（SI）是由一个称为S的单位点和多个等位基因控制的。迄今为止，在S位点已鉴定出两个多态基因。我们发现S受体激酶（SRK）决定柱头S单倍型特异性，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，花粉S的决定因素还没有被确定。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性基因，SAE1和SP11。然而，我们并没有从SAE1和SP11的序列信息中确定哪个基因是真正的花粉S决定因子。因此，我们开始利用转化技术和等位基因多态性来确定花粉S的真实决定因素。在SAE1的情况下，我们没有发现S等位基因之间的多态性。相比之下，我们更快速地从S^<52>等位基因中分离出SP11-like cDNA克隆。此外，通过与Isogai教授小组的合作研究，还从S^8和S^<12>等位基因中分离出sp11样cDNA克隆。这4个克隆编码了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SLG/SRK基因的侧翼区域，说明这4个sp11样基因是等位基因。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的SI受孢子生理控制。因此，我们选择SP11基因作为花粉S决定因子的候选基因。为了确定该SP11基因是真正的花粉S决定因子，将SP11-9基因转化为S^<52>S^<60>杂合子。转化花粉在S^9柱头上被拒绝，柱头侧未见表型变化，表明SP11基因是花粉S的真正决定因素。利用RT-PCR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位点，大小在60 ~ 104 kb之间。当对SP11的估计氨基酸序列进行比对时，6个半胱氨酸残基在所有SP11中都是完全保守的，这表明这些半胱氨酸残基对三级构象的形成很重要。在SLG、SRK和SP11中构建系统发育树时，这三个基因的树模式相似，表明这三个基因共同进化产生了一个新的S等位基因。此外，我们分离到的SP11基因被分类为II类S单倍型，并观察到与上述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