利用高覆盖度甲基化测序技术系统解析猪时序特异、品种特异、组织特异的甲基化基因

DNA methylation plays important roles in cell fertilization, division, differentiation, senescence and apoptosis. After fertilization, the development and growth of fertilized eggs experienced the process of demethylation and re-methylation. Cells with same genomic information will differentiate into different tissues and organs by methylation or acetylation during growth and differentiation periods. DNA methylation also contains genetical factors, the heritability of DNA methylation is about 20% in human, and some of these methylated genes will be stably inherited to its progeny. Nevertheless, the methylated states affected by the environment and corresponding phenotypic changes will also transmitted to their offspring. Furthermore, methylation states can be used to predict the morbidity rates and ages. At present, the studies of methylation in pigs are merely focused on identification of tissue specific and environmental specific methylated genes and regions..In this study, we synthetically investigate age specific, breed specific and tissue specific methylated genes and loci and to estimate pig methylation heritability by two breeds, three generations and three age points design. A total of 26 samples is used for high coverage whole-genome methylation sequence. We want to systematically uncovered the differential methylated genes among age, breeds and tissue; We also want to dynamically explore the methylation pattern in the whole growth stages of pigs, to search the methylated genes associated with aging and meat traits, the molecular mechanism of trait associated methylated gene would further confirmed by functional experiment. These results can contribute to the understanding of the change of methylation during ageing and used for prediction for diseases and senility in pig.

DNA甲基化在生命发生、生长、分化和衰亡过程都发挥重要作用。生命之初，受精卵的生长发育就经历了去甲基化和重新甲基化的过程。随着生命的成长，相同基因组信息的细胞通过甲基化和乙酰化分化形成不同的组织和器官。同时，DNA甲基化也受遗传因素的影响；人的甲基化位点的遗传力约为20%，其中部分甲基化基因能稳定遗传给后代，受环境导致甲基化的基因和表型变化也会遗传给后代。再次，不同甲基化水平能预测被检样本患病概率和年龄。目前猪中甲基化的研究仅聚焦于组织特异和环境特异甲基化区域挖掘。.本项目设计两个品种、两种组织、三个世代幼、中、老三个年龄段26个样本用于甲基化重测序，拟系统地解析猪年龄特异、品种特异、组织特异的甲基化基因和位点并动态展示其生活周期中甲基化图谱变化、估算其甲基化遗传力，搜寻与衰老、肉质表型相关的甲基化基因并进行功能验证分析，研究结果将有助于综合理解猪中甲基化水平的变化及其对年龄和疾病的影响。

DNA甲基化在生命发生、生长、分化和衰亡过程都发挥重要作用。生命之初，受精卵的生长发育就经历了去甲基化和重新甲基化的过程。随着生命的成长，相同基因组信息的细胞通过甲基化和乙酰化分化形成不同的组织和器官。.本项目采集了大白和巴马香中外两个品种的肌肉和睾丸两种组织，对1岁、4岁和9岁三个世代幼、中、老三个年龄段的26个样本进行全基因组亚硫酸盐测序，利用BisSNP软件将测序数据比对到猪11.1参加基因组并鉴定甲基化位点和区域。分别在组织间、年龄段、品种间和性别间分别鉴别到5.3M、0.2M、1.2M和0.16M的差异甲基化位点。组织间鉴别到的5.3M差异甲基化位点可以聚类成1.2万DMRs，分布在基因组的不同部分，其中有5K个 DMR位于启动子区域。.结合转录组数据进行关联分析，发现位于启动子区的25%差异甲基化区域中的甲基化水平与基因表达量呈显著的正相关或负相关。其中，TNNT3启动子区域的甲基化水平与表达呈显著的负相关，切片结果显示该基因甲基化水平的变化解释了快肌纤维比例随年龄变化趋势的一致性。SPACA1和MEI1基因的启动子存在与表达成显著负相关的DMR，该区域的甲基化水平随年龄升高，同时切片结果显示生精能力和精细胞比例减小，证明甲基化导致了SPACA1和MEI1基因这两个基因表达降低。在肌肉组织中发现HOXC基因簇的的基因内存在大量的CpG，接近40%的差异CpG位于CpG岛内，功能分析发现肌肉组织HOXC家族的去甲基化有利于肌肉的生长，但不利于肌内脂肪的沉积。