中国荷斯坦奶牛TLR1和CBLB基因SNP位点筛选及分析

Screening and analysis of SNP loci of TLR1 and CBLB genes in Chinese Holstein cows

目的研究炎症反应相关基因Toll样受体1(Toll-like receptor 1,TLR1)和Cbl原癌基因B(Cbl proto-oncogene B,CBLB)基因的单核苷酸多态性(single nucleotide polymorphism,SNP)与中国荷斯坦奶牛炎性疾病的相关性。方法以中国荷斯坦奶牛为试验对象,采用DNA混池法对中国荷斯坦奶牛TLR1基因CDS区和CBLB基因18外显子进行PCR扩增,筛选出SNP位点。估算SNP位点的等位基因频率,运用在线软件预测TLR1和CBLB基因突变前后mRNA二级结构、蛋白质理化性质、疏水性和亲水性、N-糖基化位点、二级和三级结构等的变化。结果 TLR1基因CDS区共发现4个SNP位点,分别为C1424A、A1475C、G1496A和G1550A。其中C1424A和G1496A位点为错义突变,分别由原来的苯丙氨酸(Phe)和色氨酸(Trp)变为亮氨酸(Leu)和不翻译;A1475C和G1550A位点为同义翻译。CBLB基因18外显子发现1个SNP位点,为G222406T,为错义突变,由原天冬氨酸(Asp)变为酪氨酸(Tyr)。TLR1基因4个SNP位点突变前后等位基因频率均有明显差异,而CBLB基因SNP位点突变前后等位基因频率差异较小。TLR1和CBLB基因编码蛋白均属于不稳定蛋白,总体表现为疏水,TLR1编码蛋白质中有6个潜在的N-糖基化位点,分别为Asn76、Asn187、Asn293、Asn306、Asn368和Asn443,CBLB编码蛋白质中有4个潜在的N-糖基化位点,分别为Asn142、Asn602、Asn744和Asn797。TLR1和CBLB基因发生错义突变后,m RNA二级结构稳定性和蛋白质的二、三级结构也发生了改变。结论中国荷斯坦奶牛TLR1和CBLB基因5个SNP位点的发现可能影响奶牛炎症疾病的发生发展。

Objective To investigate the association between single nucleotide polymorphism (SNP) in the Toll-like receptor 1(TLR1)or Cbl proto-oncogene B(CBLB)gene and inflammatory diseases in Chinese Holstein cows.Methods The CDS region of TLR1 gene and the 18th exon of CBLB gene were amplified with DNA pool template by PCR,and the SNP loci in Chinese Holstein cows were screened.The allele frequencies of the SNP loci were estimated,and changes in m RNA secondary structure as well as physicochemical properties,hyrophobicity,hydrophilicity,N-glycosylation site,secondary and tertiary structures of protein before and after TLR1 and CBLB gene mutations were predicted by online software.Results Base substitutions at four SNP loci,i.e.C1424A,A1475C,G1496A and G1550A,were found in the CDS region of TLR1 gene,among which the C1424A and G1496A were missense mutations,resulting in the changes from phenylalanine(Phe)and tryptophan(Trp)to leucine(Leu)and untranslated,respectively.However,the A1475C and G1550A were synonymous translations.A SNP locus was found in exon 18 of CBLB gene,at which the base substitution(G222406T)was a missense mutation,resulting in the change from aspartic acid (Asp)to tyrosine (Tyr).The allele frequencies of four SNP loci before and after mutation showed significant difference,while that of SNP locus showed no significant difference.Both the proteins encoded by TLR1 and CBLB genes were unstable and were hydrophobic in general.Six potential N-glycosylation sites in the protein encoded by TLR1 gene,i.e.Asn76,Asn187,Asn293,Asn306,Asn368 and Asn443,as well as four N-glycosylation sites in that encoded by CBLB gene,i.e.Asn142,Asn602,Asn744 and Asn797,were observed.Bioinformatics analysis revealed that,after the missense mutations of TLR1 and CBLB genes,the stability of secondary structure of m RNA as well as the secondary and tertiary structures of protein showed also changes.Conclusion The occurrence of five SNP loci of TLR1 and CBLB genes in Chinese Holstein cows may affect the development of inflammatory diseases in dairy cows.