采用四引物ARMS-PCR方法检测奶牛乳腺组织DGAT1基因单核苷酸多态性

Detection of Dairy Cow Mammary Tissue DGAT1 Gene Single Nucleotide Polymorphisms Using Tetra-primers ARMS-PCR Method

试验旨在快速有效地检测泌乳奶牛的二酰甘油转酰基酶1(diacylgycerol acyltransferase 1,DGAT1)乳脂量性状的优势等位基因K232A单核苷酸多态性,确定DGAT1基因型进而确定泌乳性状,为中国荷斯坦奶牛分子标记辅助选择提供技术支持。选取6头泌乳初期(3头为高乳产量牛,3头为低乳产量牛)中国北方荷斯坦奶牛为研究对象,提取乳腺组织基因组DNA,分别设计1对外引物和1对内引物,建立一种四引物ARMS-PCR体系快速检测奶牛乳腺组织DGAT1基因单核苷酸多态性。结果发现,外引物扩增片段长度为512 bp,为PCR反应的阳性对照,基因型为232K扩增片段长度为369 bp,基因型为232A扩增片段长度为181 bp。PCR结果显示,6头牛的乳腺组织样本均由外部引物扩增出长度为512 bp的片段,泌乳期高乳产量奶牛和泌乳期低乳产量奶牛乳腺组织的特异性扩增片段长度均为181 bp。表明本研究选取的6头奶牛样本DGAT1基因K232A多态性均为232A型。提示该PCR鉴定方法能够快速有效地鉴定奶牛DGAT1基因型,可用于中国荷斯坦奶牛分子标记辅助选择。

This study was aimed to detect the single nucleotide polymorphisms of diacylgycerol acyltransferase 1（ DGAT1） K232 A,and determine the DGAT1 genotype and milk traits of dairy cow,which would provide a new technique for marker-assisted selection in China Holstein dairy cows. In the present study,six Northern China Holstein dairy cows（ three were lactating cows with high quatity milk and three were lactating cows with low quatity milk） were used to detect mammary tissue DGAT1 gene K232 A polymorphisms. Genome DNA was extracted from each cow,a pair of external primers and a pair of internal primers were designed to amplify DGAT1 gene. The results showed that PCR-amplified fragments were512 bp（ external band）,369 bp（ 232 K allele） and 181 bp（ 232 A allele）,respectively. The exterenal band functions as the internal PCR-positive control. The tetra-primer ARMS-PCR amplifications yielded a512 bp fragment and a 181 bp fragment,indicating that the six dairy cows were all homozygous 232 A.The results indicated that the tetra-primers ARMS-PCR was a quick and convenient method to identify dairy cow DGAT1 gene K232 A polymorphisms,which was suitable for marker-assisted selection in China Holstein dairy cows.