7个砂梨品种S基因型的确定及1个新S-RNase基因的分离鉴定

摘要

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基因犁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494 bp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仅在化柱中特异表达,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294 bp.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.%Pyrus pyrifolia is a commercially important fruit tree which exhibits gametophytic self-incompatibility (GSI). It is necessary to identify S-genotypes of cultivars for determination of cross-compatible combination prior to performing pear plantation and breeding programs. In this study, seven cultivars of P. pyrifolia were used for S-genotype analysis by PCR-based molecular method with primers designed from conserved sequences of known pear S-RNases. Amplified products were analyzed by 1.8% agarose gel electrophoresis. In each of the four cultivars ' Chubixiang', ' Huangpishui', ' Zhenghedaxueli' and ' Hongtaiyang', two expected bands were generated. The amplified products in the other three cultivars did not show length polymorphism, and therefore were further separated by 6% polyacrylamide gel electrophoresis (PAGE). A total of 14 purified fragments from the seven cultivars were cloned and sequenced. Sequence analysis revealed that 10 alleles with typical structural features of pear S-RNase were identified, of which one from 'Zhenghedaxueli', with 494 bp, was determined as a new S-RNase allele that was tentatively denominated as S43-RNase (GenBank accession No. EF566873). RT-PCR revealed that the S43-RNase was expressed specifically in the styles, which is consistent with the expression pattern of S-RNases. Comparison of genomic and cDNA sequences revealed there was an intron of 294 bp in the S43-RNase gene. The deduced amino acid sequence shared 65 % to 92% similarity with other Maloideae S-RNases. This study will be helpful in pear production and breeding programs.