以417头中国荷斯坦奶牛为研究对象, 根据体细胞评分(Somatic cell score, SCS)的大小将该奶牛群体划分为感染牛群(100头)和健康牛群(317头)。通过PCR-RFLP和CRS-RFLP方法检测了肿瘤坏死因子a(Tumor necrosis factor-alpha, TNF-a)基因在荷斯坦奶牛群体中的多态性, 并分析这些多态位点和奶牛乳房炎的相关性。研究发现了3个单核苷酸多态位点(Single-nucleotide polymorphism, SNP): 第2外显子39 bp处G→A的突变; 第4外显子293 bp处 C→T的突变; 5′侧翼区(5′-flanking region, 5′UTR)C→G的突变。这3个突变位点分别是DraⅠ、AfaⅠ和DdeⅠ限制性内切酶的酶切多态位点, 其中DraⅠ为创造酶切位点。经过基因型分析与χ2检验表明: 3个酶切多态位点在荷斯坦奶牛群中均未达到Hardy-Weinberg平衡状态。运用SPSS 13.0软件, 采用最小二乘拟合线性模型分析3个酶切多态位点与SCS的关系, 结果表明: AA基因型个体在DraⅠ酶切位点中的SCS显著大于BB及AB基因型个体(P<0.05), BB基因型表现出乳房炎抗性。AfaⅠ酶切位点中BB基因型个体的SCS显著大于AA及AB基因型个体(P<0.05), AA基因型表现出乳房炎抗性。DdeⅠ酶切位点中, AB基因型个体的SCS显著低于AA基因型个体(P<0.05), AB基因型为优良基因型。因此BB、AA、AB基因型分别为DraⅠ、AfaⅠ、DdeⅠ酶切位点中的优良基因型, 可作为分子标记应用于奶牛乳房炎抗性筛选。
Four hundred and seven Chinese Holstein cows were chosen and divided into 2 groups (healthy group (317), sub-clinical and clinical group (100)) to investigate variation in bovine Tumor necrosis factor-alpha (TNF-a) gene and analyze their associations with mastitis. By PCR-RFLP and CRS-RFLP analyses, three polymorphic sites were detected, with one G→A mutation at 39 bp in exon2 and one C→T mutation at 293 bp in exon4, and C→G mutation in 5´UTR. The three polymorphic sites were able to be cut by DraⅠ, AfaⅠ, and DdeⅠ restriction enzymes respectively, and the DraⅠlocus was Create Restriction Enzyme Cutting Site. Chi-square analysis suggested that the three polymorphic sites did not reach Hardy-Weinberg equilibrium (P<0.05). Least square linear model (LSM) analysis indicated that the DraⅠ, AfaⅠ, and DdeⅠ loci of TNF-a gene were associated with somatic cell score (SCS) (P<0.05). Genotype AA in DraⅠlocus and BB in AfaⅠ locus showed significantly higher SCS (P<0.05) and genotype AB in DdeⅠlocus showed significantly lower SCS than AA and BB genotypes in the population tested (P<0.05). This suggested that genotypes BB in DraⅠloci, AA in AfaⅠloci, and AB in DdeⅠloci can be used as candidate markers for mastitis resistance selection in dairy cattle.
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