中国荷斯坦牛TLR1基因SNPs快速筛查及蛋白功能预测

为探究中国荷斯坦牛Toll样受体1(TLR₁)基因与中国荷斯坦牛乳房炎抗性的关联性,以中国荷斯坦牛为试验对象,构建DNA混合池,采用PCR扩增和直接测序法对中国荷斯坦牛TLR₁基因进行单核苷酸多态性(SNP)检测,从而筛选出对中国荷斯坦牛免疫方面有显著影响的SNPs位点,同时应用在线软件对中国荷斯坦牛TLR₁基因编码的蛋白质进行功能预测。结果表明,中国荷斯坦牛TLR₁基因编码727个氨基酸,组成了一种不稳定的水溶性蛋白,蛋白质二、三级结构均以α-螺旋为主;PCR扩增后,在扩增片段处共发现8个SNPs位点,分别为A⁶¹T-TLR₁、C⁶³²A-TLR₁、C¹⁴⁰⁸T-TLR₁、C¹⁴⁵¹T-TLR₁、A¹⁴⁶¹G-TLR₁、A¹⁴⁷⁵C-TLR₁、G¹⁵⁵⁰A-TLR₁、G¹⁵⁹⁶A-TLR₁,其中A⁶¹T-TLR₁、C⁶³²A-TLR₁、C¹⁴⁰⁸T-TLR₁、A¹⁴⁶¹G-TLR₁、G¹⁵⁹⁶A-TLR₁属错义突变,分别由原来的赖氨酸(Lys)、苯丙氨酸(Phe)、丙氨酸(Ala)、异亮氨酸(Ile)、缬氨酸(Val)突变为甲硫氨酸(Met)、亮氨酸(Leu)、缬氨酸(Val)、缬氨酸(Val)、异亮氨酸(Ile),SNPs位点的等位基因频率在突变前后均存在差异。DNA池结合直接测序技术检测到TLR₁基因8个SNPs位点,可作为中国荷斯坦牛乳房炎的遗传标记进行深入研究;蛋白质功能预测结果表明,有多种与免疫相关的因子几率较高。本研究结果为中国荷斯坦牛在分子领域的抗病育种提供了理论依据。

Rapidly Screening of TLR1 Gene SNPs and Prediction of Protein Function in Chinese Holstein Cattle

In order to investigate the association between Toll like receptor 1 (TLR₁) gene and mastitis resistance in Chinese Holstein cattle, a DNA pooling was constructed in Chinese Holstein cattle. The single nucleotide polymorphism (SNP) of TLR₁ gene of Chinese Holstein cattle was detected by PCR amplification and direct sequencing, and the SNPs loci which had a significant effect on the immunity of Chinese Holstein cattle were screened. At the same time, the online software was used to predict the function of the protein encoded by the TLR₁ gene of Chinese Holstein cattle. The results showed that Chinese Holstein TLR₁ gene encodes 727 amino acids and constitutes an unstable water-soluble protein. After PCR amplification, a total of 8 SNPs loci were found in the amplified fragments, namely A⁶¹T-TLR₁, C⁶³²A-TLR₁,C¹⁴⁰⁸T-TLR₁, C¹⁴⁵¹T-TLR₁, A¹⁴⁶¹G-TLR₁, A¹⁴⁷⁵C-TLR₁, G¹⁵⁵⁰A-TLR₁ and G¹⁵⁹⁶A-TLR₁. Among them, C⁶³²A-TLR₁, C¹⁴⁵¹T-TLR₁, A¹⁴⁶¹G-TLR₁, A¹⁴⁷⁵C-TLR₁ and G¹⁵⁹⁶A-TLR₁ resulted in missense mutation. The allele frequencies of SNPs loci with five missense mutations were mutated from the original lysine(Lys), phenylalanine(Phe), alanine(Ala), isoleucine(Ile), andvaline(Val) to methionine(Met), leucine(Leu), valine(Val), valine(Val) and isoleucine(Ile), and the allele frequencies of the five missense mutation SNPs were slightly different before and after the mutation, and the rest are significantly different. The DNA pooling combined with direct sequencing technology to detect 8 SNPs of TLR₁ gene, which can be used as a genetic marker for Chinese Holstein cow mastitis. The results of protein function prediction show that there are many factors associated with immune related factors. The results of this study provide a theoretical basis for the resistance breeding of Chinese Holstein cattle in molecular field.