Institute of Cattle Science, Nanjing Agricultural University, Nanjing, 210095, China.
Mol Biol Rep. 2011 Jan;38(1):83-8. doi: 10.1007/s11033-010-0080-8. Epub 2010 Mar 26.
PCR low ionic strength single-strand conformation polymorphism (PCR-LIS-SSCP) and DNA sequencing methods were used to analyze the polymorphisms within the coding region of bovine ATP1A1 gene. A novel C/A mutation was identified at the nucleotide position 2789 of the ATP1A1 mRNA, but it was silent with respect to the amino acid sequence of the protein. The LIS-SSCP banding pattern could be divided into three kinds of genotypes, named CC, CA, and AA. Also, the association of the novel SNP of ATP1A1 gene with heat tolerance traits was studied, we found that the individuals with genotype CC showed significantly higher heat resistance than those of genotype CA (P<0.05). Further, the mRNA levels of ATP1A1 gene in lymphocytes of peripheral blood in dairy cows among various temperature groups and genotypes were analyzed by using real-time RT-PCR. Results showed that the expression of ATP1A1 mRNA was highest in heat-stressed cows with CC genotype among the three genotypes (P<0.01), and the ATP1A1 mRNA level at temperature 32.5°C was higher than that at optimal temperature 12.5°C in dairy cows (P<0.01). Simultaneity, the plasma potassium (K+, mmol/l) and sodium (Na+, mmol/l) declined when the temperature dropped (P<0.05). Our findings implied that the novel SNP here could be a potential genetic marker for anti-heat stress trait in dairy cow breeding.
采用 PCR 低离子强度单链构象多态性（PCR-LIS-SSCP）和 DNA 测序方法分析了牛 ATP1A1 基因编码区的多态性。在 ATP1A1 mRNA 的核苷酸位置 2789 处发现了一个新的 C/A 突变，但它与蛋白质的氨基酸序列是沉默的。LIS-SSCP 带型可分为三种基因型，分别命名为 CC、CA 和 AA。此外，还研究了 ATP1A1 基因的新 SNP 与耐热性性状的关联，发现基因型为 CC 的个体比基因型为 CA 的个体具有更高的耐热性（P<0.05）。进一步，通过实时 RT-PCR 分析了不同温度组和基因型奶牛外周血淋巴细胞中 ATP1A1 基因的 mRNA 水平。结果表明，在三种基因型中，CC 基因型奶牛在热应激下的 ATP1A1 mRNA 表达最高（P<0.01），并且在奶牛 32.5°C 时的 ATP1A1 mRNA 水平高于最佳温度 12.5°C（P<0.01）。同时，当温度下降时，血浆钾（K+，mmol/l）和钠（Na+，mmol/l）下降（P<0.05）。我们的研究结果表明，该新 SNP 可能是奶牛抗热应激性状遗传标记的潜在候选基因。
