荷斯坦母牛TLR6基因多态性及其与体细胞评分关系的研究

Polymorphism of TLR6 Gene and Its Relationship with Somatic Cell Score in Holstein Cows

本研究旨在阐明Toll样受体6(TLR6)基因的多态性与荷斯坦母牛乳房炎抗性之间的关系。采用PCR-SSCP和PCR-RFLP技术检测208头荷斯坦母牛TLR6基因多态性,用最小二乘法分析该基因多态性与荷斯坦母牛体细胞评分(somatic cell score,SCS)的关系。结果发现,TLR6基因mRNA序列中存在T853A、G855A、G1793A、C1859A、G1934A和A1980G 6个多态位点,其中T853A突变使编码氨基酸由缬氨酸变为谷氨酸,G855A突变使天冬氨酸变为天冬酰胺,A1980G突变使异亮氨酸变为缬氨酸。T853A和G855A突变位点经SSCP检测发现3种基因型AA、AB和BB,其频率分别为0.308、0.490和0.202;χ2检验表明荷斯坦母牛在该位点处于Hardy-Weinberg平衡;BB基因型SCS最小二乘均值极显著低于AB和AA基因型所对应的值(P<0.01);AB基因型SCS最小二乘均值极显著低于AA基因型所对应的值(P<0.01)。G1793A突变位点经BsiYⅠ酶切产生2种基因型CC和CD,其频率分别为0.332和0.668;荷斯坦母牛在该位点偏离了Hardy-Weinberg平衡;CC和CD基因型SCS最小二乘均值差异不显著(P>0.05)。C1859A突变位点经BstXⅠ酶切产生2种基因型EE和EF,其频率分别为0.178和0.822;荷斯坦母牛在该位点偏离了Hardy-Weinberg平衡;EE和EF基因型SCS最小二乘均值差异不显著(P>0.05)。G1934A突变位点经BsiYⅠ酶切产生2种基因型GG和GH,其频率分别为0.356和0.644;荷斯坦母牛在该位点偏离了Hardy-Weinberg平衡;GG和GH基因型SCS最小二乘均值差异不显著(P>0.05)。A1980G突变位点经BclⅠ酶切产生3种基因型III、J和JJ,其频率分别为0.260、0.567和0.173,荷斯坦母牛在该位点处于Hardy-Weinberg平衡;JJ基因型SCS最小二乘均值极显著低于IJ和II基因型所对应的值(P<0.01);IJ基因型SCS最小二乘均值极显著低于II基因型所对应的值(P<0.01)。对于奶牛乳房炎抗性,BB和JJ是有利基因型,AA和II是不利基因型。本研究结果初步表明TLR6基因T853A和G855A突变位点的B等位基因以及A1980G突变位点的J等位基因是提高奶牛乳房炎抗性的2个潜在的DNA标记。

The objective of the present study was to elucidate the relationship between Toll-like receptor 6（TLR6）gene and mastitis resistance of Holstein cows.Polymorphism of all exons of TLR6 gene was detected in 208 Holstein cows by PCR-SSCP and PCR-RFLP.The relationship between polymorphisms of TLR6 gene and somatic cell score（SCS） in Holstein cows was analyzed by least squares linear model.Six polymorphic sites T853A,G855A,G1793A,C1859A,G1934A and A1980G were found;and T853A mutation resulted in an amino acid change of valine→glutamic,G855A mutation caused an amino acid change of aspartic acid→asparagine,A1980G mutation caused an amino acid change of isoleucine→valine.For T853A and G855A polymorphic sites,three genotypes（AA,AB and BB） were found in 208 Holstein cows by SSCP,and the frequency of AA,AB and BB genotypes was 0.308,0.490 and 0.202,respectively.Holstein cows were in Hardy-Weinberg equilibrium at the sites.Least squares mean of SCS for BB was significantly lower than that for AB（P0.01） or AA（P0.01）,least squares mean of SCS for AB was significantly lower than that for AA（P0.01）.For G1793A polymorphic site,two genotypes（CC and CD） were found by digesting the PCR products with restriction endonuclease BsiYⅠ,Chi-square test showed that Holstein cows were not in Hardy-Weinberg equilibrium.Least squares mean of SCS for CC was not significantly lower than that for CD（P0.05）.For C1859A polymorphic site,two genotypes（EE and EF） were found by digesting the PCR products with restriction endonuclease BstXⅠ,and the frequency of EE and EF genotypes was 0.178 and 0.822,respectively.Holstein cows were not in Hardy-Weinberg equilibrium at the site.Least squares mean of SCS for EE was not significantly lower than that for EF（P0.05）.For G1934A polymorphic site,two genotypes（GG and GH） were found by digesting the PCR products with restriction endonuclease BsiYⅠ,and the frequency of GG and GH genotypes was 0.356 and 0.644,respectively.Holstein cows were not in Hardy-Weinberg equilibrium at the site.Least squares mean of SCS for GG was not significantly lower than that for GH（P0.05）.For A1980G polymorphic site,three genotypes（II,IJ and JJ） were found by digesting the PCR products with restriction endonuclease BclⅠ,and the frequency of II,IJ and JJ genotypes was 0.260,0.567 and 0.173,respectively.Holstein cows were in Hardy-Weinberg equilibrium.Least squares mean of SCS for JJ was significantly lower than that for IJ（P0.01） or II（P0.01）,least squares mean of SCS for IJ was significantly lower than that for II（P0.01）.BB and JJ were the most favorable genotypes,AA and II were the most unfavorable genotypes for mastitis resistance.The results preliminarily indicated that both B and J alleles of TLR6 gene are two potential DNA markers for improving mastitis resistance in dairy cattle.