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砂梨是重要的经济树种,表现出典型的配子体自交不亲和性,在生产和育种上需鉴定品种的S基因型以确定品种间的亲和性。选取7个砂梨品种为试验材料,使用梨S-RNase(S基因)通用引物进行基因组PCR扩增,产物通过1.8%的琼脂糖凝胶电泳分析。结果表明:‘楚比香’等4个品种中产生了预期的2条电泳条带,而其他3个品种都只产生1条带产物,通过6%的聚丙烯酰胺电泳对该3个品种的PCR产物进一步分析,结果产物被成功分离。将7个品种中分离到的14个条带分别回收、克隆、测序及序列分析,从中鉴别出10个具有梨S-RNase基因序列特征的S基因,其中‘政和大雪梨’中494bp的基因片段被鉴定为新的S基因,暂命名为S43-RNase(GenBank接受号EF566873)。RT-PCR试验证明S43-RNase仅在花柱中特异表达,符合S-RNase的表达特征。通过比对S43-RNase的基因组序列和cDNA序列,确定其内含子大小为294bp。在推导氨基酸水平上,S43-RNase与苹果亚科其他S-RNase表现出65%～92%的相似性。
Sand pear is an important economic tree species, showing the typical self-incompatibility of gametophyte, in the production and breeding needs to identify the S genotypes of varieties to determine the compatibility between varieties. Seven sand-pear cultivars were selected as experimental materials, genomic PCR amplification was performed using common primers for pear S-RNase (S gene), and the products were analyzed by 1.8% agarose gel electrophoresis. The results showed that the expected two electrophoresis bands were produced in four varieties such as ’Chu-Xiang’, while the other three cultivars produced only one band. The products were analyzed by 6% polyacrylamide electrophoresis The PCR product was further analyzed and the product was successfully isolated. The 14 bands isolated from 7 cultivars were recovered, cloned, sequenced and sequenced respectively. Ten S genes were identified with the sequence of pear S-RNase genes, of which 494bp Was identified as a new S gene, tentatively named S43-RNase (GenBank accession number EF566873). The results of RT-PCR showed that S43-RNase was only expressed in style, which accorded with the expression characteristics of S-RNase. By comparing the genomic sequence and cDNA sequence of S43-RNase, the intron size was determined to be 294 bp. At the deduced amino acid level, S43-RNase exhibited 65% to 92% similarity to other S-RNases in the apple subfamily.