奶牛CCL11基因启动子、外显子的扩增及多态性分析

Amplification of promoter and exon of dairy cow CCL11 gene and analysis of exon polymorphism

为了探讨CCL11基因启动子和外显子的多态性,试验采集88头中国荷斯坦奶牛的血液样品,通过PCR技术扩增了中国荷斯坦奶牛CCL11基因外显子和启动子序列,同时用限制性内切酶酶切的方法检测了中国荷斯坦奶牛CCL11基因外显子的多态性,酶切产物经琼脂糖凝胶电泳判断突变位点的基因型,通过ESEfinder 3.0在线软件预测差异位点基因序列可能存在的ESE基序,利用Expasy-Translate tool在线翻译软件将基因测序结果翻译成氨基酸序列。结果表明:试验成功扩增出中国荷斯坦奶牛CCL11基因启动子序列,第1段长度为794 bp,第2段长度为1060 bp,经测序未检测出多态位点,3个外显子序列长度分别为524,539,1130 bp,其中第2,3外显子经测序未检测出多态位点。在第1外显子80 bp处存在一个T>G的突变,且突变导致氨基酸序列由缬氨酸变为甘氨酸,为有义突变,突变与ESE基序之间存在着较强的相关性。CCL11基因有TT(野生型)、TG(杂合型)、GG(突变型)3种基因型,基因型频率分别为17.05%、75.00%和7.95%。说明奶牛CCL11基因的突变可能与奶牛乳腺炎的发生相关。

In order to explore the polymorphism of CCL11 gene promoter and exon, in this experiment blood samples of 88 Chinese Holstein dairy cows were selected, and the CCL11 gene exon and promoter sequences of Chinese Holstein cows were amplified by PCR technology, and the polymorphism of the exon of CCL11 gene of Chinese Holstein dairy cows was detected by restriction enzyme digestion at the same time. The agarose gel electrophoresis was used to determine the genotype of the mutation site of the digested products. ESEfinder 3.0 online software was used to predict the difference sites of CCL11 gene in Chinese Holstein dairy cows, and the Expasy-Translate tool online translation software was used to translated the results of gene sequencing into amino acid sequences of the CCL11 gene sequence of Chinese Holstein dairy cows. The results showed that the experiment successfully amplified the CCL11 gene promoter sequence of Chinese Holstein dairy cows, with the first segment was 794 bp, and the second segment was 1 060 bp. The lengths of the three exons were 524 bp, 539 bp and 1 130 bp, respectively. No polymorphic sites were detected in the second and third exons after sequencing. There was a T>G mutation at 80 bp in the first exon, and the mutation caused the amino acid sequence to change from valine to glycine, which meant it was a sense mutation. There was a strong correlation between mutations and ESE. The CCL11 gene had three genotypes, TT(wild type), TG(heterozygous type), and GG(mutant type), and the genotype frequencies were 17.05%, 75.00% and 7.95%, respectively. The results suggested that the mutation of CCL11 gene in dairy cows may be related to the occurrence of dairy cow mastitis.