Molecular analysis of gene-products associated with self-incompatibility in Ipomoea trifida

本研究旨在揭示甘薯野生近缘三裂甘薯的柱头蛋白与自交不亲和相关。从三裂甘蓝柱头cDNA文库中分离到一个编码SLG/SRK类蛋白的基因克隆IRK1，该蛋白已被鉴定为甘蓝型油菜自交不亲和基因(S基因)产物。然而，对分离S等位基因的F1群体进行的限制性片段长度多态性分析表明，IRK1基因在遗传上与S座位没有连锁。S核糖核酸酶已被证明参与茄科植物的自交不亲和反应。从三裂盾壳菌柱头cDNA文库中分离到的核糖核酸酶基因克隆IRN3也没有与S基因座连锁。这些结果表明，通菜属植物具有不同于十字花科和茄科植物的自交不亲和机制。本研究对6个S等位基因纯合子的柱头提取液进行了双向电泳分析。通过比较纯合子之间的电泳谱，我们确定了S等位基因特有的蛋白质点。这些S等位基因特异蛋白的分子质量范围为60-80kD，具有酸性和中性等位基因PI。在杂合子植物中，这些蛋白与S等位基因存在共分离。通过获得N端氨基酸序列和从柱头cDNA文库中筛选出完整的cDNA克隆，对这些蛋白的结构和功能进行了进一步的研究。

The present study was performed to reveal stigma proteins associated with self-incompatibility in a wild relative of the sweet potato, Ipomoea trifida. A cDNA clone, IRK1 isolated from stigama cDNA library of I.trifida was found to encode SLG/SRK-like proteins that have been identified as self-incompatibility gene (S-gene) products in Brassica. RFLP analysis of F1 population segregating for S-alleles, however, showed that the IRK1 gene is genetically unlinked to the S-locus. S-RNases have been shown to be involved in self-incompatibility reaction of solanaceous plants. An RNase cDNA clone, IRN3 isolated from stigma cDNA library of I.trifida, also showed no linkage to the S-locus. These results suggest that Ipomoea plants possessaself-incompatibility mechanism different from those of Brassica and solanaceous plants. In the present study, stigma extracts obtained from six S-allele homozygotes were analyzed by two-dimensional electrophoresis. From comparison of electrophoretic patterns among the homozygotes, we have identified the protein spots specific for S-alleles. These S-allele specific proteins have a range of molecular mass of 60-80 kD with acidic to neutral PI.Co-segregation of these proteins with S-genotypes was confirmed in heterozygous plants for S-alleles. Further studies on the structure and function of these protein are now conducted through getting the data on the N-terminal amino acid sequence and screening of full cDNA clone from the stigma cDNA library.