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为鉴定中国梨S-RNase基因及其S基因型,根据日本梨S-RNase基因保守区设计引物,对13个中国梨品种进行基因组PCR特异扩增,并对PCR产物克隆、测序、序列分析.结果鉴定出13个S等位基因,其中11个S基因与GenBank中已知的S1,S7,S12,S15,S16,S18,S19,S22,S27,S29,S34-RNases相同,另外2个则为新的S-RNase基因,命名为S37-RNase和S38-RNase,GenBank接受号分别为DQ839238、DQ839239.13个S等位基因中,在推导氨基酸水平上,S18与S27相似性最低,为58%,S7与S27,S12与S19,S15与S37及S15与S38间相似性最高,为94%.经分析,S19为中国梨中出现频率最高的等位基因,对其DNA序列进行限制性酶切位点分析,建立了一种快速、经济鉴定S19的方法,该方法无需测序而仅使用特异的限制性内切酶AflⅡ对PCR产物进行酶切消化.根据分子鉴定结果,13个中国梨品种S基因型被确定为:冰糖(S16S19),六棱(S16S19),锦香(S34S37),鹅酥(S15S38),蜜梨(S19S29),甜橙子(S7S12),大青皮(S19S34),秋白(S19S34),紫酥(S19S34),花长把(S19S22),灌阳雪梨(S18S27),早蜜(S19S29),青面(S1S18).
To identify the Chinese pear S-RNase gene and its S genotype, thirteen Chinese pear cultivars were amplified by PCR and cloned, sequenced and sequenced. The primers were designed according to the conserved regions of Japanese pear S-RNase genes. As a result, 13 S alleles were identified, of which 11 were identical to the known S1, S7, S12, S15, S16, S18, S19, S22, S27, S29 and S34-RNases in GenBank and the other 2 The new S-RNase genes were named as S37-RNase and S38-RNase. GenBank accession numbers were DQ839238 and DQ839239, respectively. Among the 13 S alleles of DQ839239, the deduced amino acid level had the lowest similarity between S18 and S27 %, S7 and S27, S12 and S19, S15 and S37 and S15 and S38 the highest similarity of 94% .After analysis, S19 is the highest occurrence in Chinese pear alleles, the DNA sequence of the restriction enzyme A site-specific analysis was carried out to establish a rapid and economical method for the identification of S19, which did not require sequencing but used restriction endonuclease AflII to digest the PCR product.According to molecular identification results, 13 Chinese pear cultivars The genotypes of S were identified as: S15S38, S16S19, S16S19, S34S37, S15S38, S19S29 and S7S 12, S19S34, S19S34, S19S34, S19S22, S18S27, S19S29, S1S18.