DNA池快速筛查中国荷斯坦牛TLR1~TLR6基因SNPs及等位基因频率估算

Rapidly Screening SNPs of Chinese Holstein Cattle TLR^TLR Gene and Estimating Allele Frequency by DNA Pooling

Toll样受体(Toll-like receptor,TLRs)是固有免疫系统的重要组成成分,在免疫防御、炎症反应等方面发挥重要的作用,并与乳腺炎抗性显著相关。为筛选出对荷斯坦牛乳腺炎有显著影响的单核苷酸多态性(single nucleotide polymorphisms,SNPs)位好点,本研究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荷斯坦牛TLR1~TLR6基因进行SNPs检测。结果表明,在扩增片段处共发现22个SNPs,其中C^1451T-TLR1、A^1475C-TLR1、G^1550A-TLR1、T^189G-TLR2、C^1632T-TLR7、T^1515C-TLR6、G^1578A-TLR6,T^1620C-TLR6、A^1644C-TLT6和G^1719A-TLR6为同义突变;A61T-TLR1、C632A-TLR1、C^1408T-TLR1、A^1461G-TLR1、G^1596A-TLR1、G^455A-TLR2、G^602A-TLR2、A^631G-TLR2、G^640A-TLR6、C^1577T-TLR6、G^1630A-TLR6为错义突变,导致11个氨基酸发生改变;SNPs突变前后等位基因频率均有明显差异。DNA池结合直接测序技术可快速筛选SNPs位点,检测到TLR1~TLR6基因22个SNPs,可作为荷斯坦奶牛乳腺炎的遗传标记进行深入研究。

Toll-like receptors(TLRs)are important components of the innate immune system.They play an important role in immune defense and inflammatory response,and were significantly correlated with mastitis resistance.In order to screen the single nucleotide polymorphisms site that has a significant impact on Holstein cattle mastitis,in this study,Holstein cattle were chose for testing subjects,DNA pooling was constructed,PCR amplification and direct sequencing were used to detect the TLR1~TLR6 gene SNPs in Holstein cattle.The result showed that 22 SNPs were found in the amplified fragment,among them C^1451T-TLR1,A^1475C-TLR1,G^1550A-TLR1,T^189G-TLR2,C^1632T-TLR2,T^1515C-TLR6,G^1578A-TLR6,T^1620C-TLR6,A^1644C-TLR6 and G^1719A-TLR6 were synonymous mutations;A^61T-TLR1,C^632A-TLR1,C^1408T-TLR1,A^1461G-TLR1,G^1596A-TLR1,G^455A-TLR2,G^602A-TLR2,A^631G-TLR2,G^640A-TLR6,C^1577T-TLR6 and G^1630A-TLR6 were missense mutation,11 amino acid were changed;It was shown that the allele frequency were significant differences before and after SNPs mutation.DNA pooling combined with direct sequencing technology can quickly screen out SNPs site and detect 22 SNPs that TLR1 to TLR6 gene,which can be used as a genetic marker for Holstein cattle.