藏猪睾丸高原适应性的DNA甲基化机制

DNA methylation plays a very important role in gene transcription and regulation. However, it is not yet clear about DNA methylation mechanism of Tibetan pig testicles plateau adaptability. It had been detected that mRNA expression levels of HIF2α and Dnmt3a in testicular tissue of Tibetan pig was significantly lower than that of Large White pigs’ during our earlier work. Next experiments will be carried on as follows：(1)To explore the differences between genomic DNA methylation level of Tibetan pigs in the testicular tissue and that of Large White pigs by fluorescence method (methylation Quantitative kit). (2) It will be detected that differences of Dnmt1, Dnmt3a, Dnmt3b and HIF2α protein expression in the testicular tissues of Tibetan pigs and Large White pigs by using Western Blot.(3)The mRNA expressive discrepancy of Dnmt1 and Dnmt3 will be detected between testicular tissue of Tibetan pig and that of Large White pigs by real-time fluorescence quantitative PCR technique. (4) It will be filtrate that genes of Dnmt1, Dnmt3a and Dnmt3b for 5 ' flanking region and in the coding region of SNPs by PCR-RFLP. Based on the experimental data above,it will be clarified that major DNA methylation mechanisms of Tibetan pig testicles plateau adaptability by this project. It will provided the technology for improving the boar Tibetan pig fecundity and establishing molecular breeding.

DNA 甲基化对基因转录水平的表达具有重要的调控作用，然而关于藏猪睾丸高原适应性的DNA 甲基化机制尚未明了。本项目在我们工作前期检测到藏猪睾丸组织HIF2α及Dnmt3a的mRNA表达水平显著低于大白猪的基础上，拟采用(1)荧光法（甲基化定量试剂盒）检测藏猪与大白猪睾丸组织基因组DNA甲基化水平差异；(2)采用Western Blot检测Dnmt1、Dnmt3a、Dnmt3b及HIF2α基因在藏猪与大白猪睾丸组织中的蛋白表达差异；(3)采用实时荧光定量 PCR技术检测Dnmt1、Dnmt3b在藏猪与大白猪睾丸组织中的mRNA表达差异；(4)采用PCR-RFLP技术筛选藏猪Dnmt1 、Dnmt3a、Dnmt3b基因5’侧翼区和编码区的SNPs。本项目拟通过对以上试验结果的分析，阐述藏猪睾丸高原适应性的主要DNA 甲基化机制，为提高藏猪繁殖力及分子育种等提供支撑。

DNA甲基化对基因转录水平的表达具有重要的调控作用，藏猪是我国特有的能适应高原环境的一个重要品种，其雄性生殖系统已适应高海拔条件，然而关于藏猪睾丸高原适应性的DNA甲基化机制尚未明了。本项目采用荧光法检测藏猪与大白猪睾丸组织基因组DNA甲基化水平差异；采用Western Blot检测4个基因（Dnmt1、Dnmt3a、Dnmt3b及HIF2α）在藏猪与大白猪睾丸组织中的蛋白表达差异；采用实时荧光定量PCR技术检测这4个基因在藏猪与大白猪睾丸组织中的mRNA表达差异；采用PCR-测序法筛选藏猪Dnmt1、Dnmt3a、Dnmt3b基因5’侧翼区和编码区的SNPs。结果显示，体成熟后藏猪睾丸基因组DNA甲基化水平、这4个基因的蛋白及mRNA的表达量均显著低于大白猪；这些SNPs（Dnmt1的G-1087A、A-929T、G3596A、Dnmt3a的C-2012T、Dnmt3b的G1459A）可以作为藏猪分子育种的候选标志。以上结果表明，在高原环境中，这些SNPs可能影响藏猪睾丸中DNA甲基转移酶的表达水平，而藏猪睾丸中DNA甲基转移酶的表达水平低于大白猪会导致藏猪低水平的基因组DNA甲基化，这可能会促进一些高原环境适应性基因的表达，从而使藏猪有较高的精子活力；藏猪睾丸中HIF2α的表达水平低于大白猪，表明藏猪睾丸由于适应高原环境而对高原低氧的回应较小，这可能是藏猪睾丸高原适应性的主要DNA甲基化机制，这将为提高藏猪繁殖力及分子育种等提供支撑。