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钵细胞评分(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;X~2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BsiY Ⅰ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BstX Ⅰ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BsiY Ⅰ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Bcl Ⅰ酶切产生3种基因型II、IJ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 re-ceptor 6(TLR6)gene and mastiffs 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 ana-lyzed 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 fre-quency 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 signifi-cantly lower than that for AB (P<0.01) or AA (P<0.01), least squares mean of SCS for AB was significantly lower than that for AA (P<0.01). For G1793A polymorphic site, two geno-types (CC and CD) were found by digesting the PCR products with restriction endonuclease BsiY Ⅰ, 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 (P>0.05). For C1859A polymorphic site, two genotypes (EE and EF) were found by digesting the PCR products with re-striction endonuclease BstX Ⅰ, 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 (P>0.05). For G1934A poly-morphic site, two genotypes (GG and GH) were found by digesting the PCR products with re-striction endonuclease BsiY Ⅰ, 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 (P>0.05). For A1980G poly-morphic site, three genotypes (II, IJ and JJ) were found by digesting the PCR products with re-striction endonuclease Bcl Ⅰ, 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 (P<0.01) or II (P<0.01), least squares mean of SCS for IJ was significantly lower than that for II (P<0.01). 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.
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