荷斯坦牛耐热性RAPD和SCAR标记的研究

Study on RAPD and SCAR Markers for Heat-tolerance in Holstein

以52头高产耐热组和41头高产不耐热组中国荷斯坦牛的DNA为材料,选用200条RAPD引物,利用RAPD-PCR技术,对荷斯坦牛的耐热性进行研究。结果发现,使用S441引物和S463引物在高产耐热组中分别发现了581 bp(RAPD-S441标记)和680 bp(RAPD-S463标记)2条特异性片段。通过测序和序列比较显示:581 bp的片段与KCNN2基因的同源性达90%;680 bp片段与NW_001024067.1|BtUn_WGA9442_2的同源性达99%。根据已知KCNN2基因的功能,初步判断KCNN2基因为影响耐热性能的候选基因。得到GenBank登录号EF123743和EF123744。利用NCBI的ORF finder在581 bp中没有发现ORF,与预测其是KCNN2基因内含子3的结论相符合。在680 bp中发现4个开放阅读框(ORF),分别为ORF1、ORF2、ORF3、ORF4。这4个阅读框分别编码48AA、34AA、45AA、34AA。根据RAPD-S441、RAPD-S463标记的测序结果,利用Premier 5.0软件设计对应的SCAR引物。成功将RAPD-S441标记转化为耐热SCAR标记。

The heat-tolerance of the Chinese Holstein （52 herds high yield and heat-tolerance, to- gether with 41 herds high yield and heat-sensitivity） was studied using RAPD-PCR （ 200 RAPD primers）. The results indicated that the 581 bp （primer $441） and 680 bp （primer S463） fragments were found in heat-tolerance group. The homology between 581 bp fragment and KCNN2 gene got to 89%, as well as the homology between 680 bp fragment and NW_001024067.1 ｜ BtUn _WGA9442_2 got to 99%. According to its function it seems that the KCNN2 gene was a candidate of heat-tolerance gene. The 581 and 680 bp fragments were submitted to GenBank and the accession numbers of EF123743 and EF123744 were got, respectively. The ORF was not found in 581 bp fragment, however, 40RFs （ORF1, coding 48AA; ORF2, coding 34AA; ORF3, coding 45AA; ORF4, coding 34 AA）in 680 bp gene fragment were found. Using the sequence of RAPD- S441 and RAPD-S463, together with the SCAR primer designed by Premier 5.0 software, the RAPD-S441 marker （581 bp） was converted to SCAR marker.