应用多组学技术进一步解析藏猪高原适应性的分子机制

Many studies were focused on the molecular mechanism of high altitude adaptation, which would help us prevent and treat the hypoxia related diseases for people who travel to or live on the plateau region, deal with the emergencies happened on plateau during the activities and operations there, and provide scientific supports to prevention and treatment of human hypoxia-related cardiovascular, lung and brain diseases. Tibetan pigs have colonized and adapted to the harsh environment of high plateau for a long time, and many scientists have pay much attention to elucidate the molecular genetic basis on high altitude adaptation in Tibetan pigs. Previously we have scanned the whole-genome SNP genotypes on Tibetan pigs and other Chinese indigenous pigs by Illumina porcine 60K SNP chips, and recently we have resequenced 131 Chinese local pigs, including 46 Tibetan pigs, with high quality, we achieved lots of variants. With these variant data, we have made studies on high altitude adaptation in Tibetan pigs with population genetic and evolutionary analysis. We have identified several important candidate genes (EPAS1, NOS1, RGCC, and so on), which might play key roles on high altitude adaptation in Tibetan pigs. In this project, we plan to collect blood samples and Hematopoietic cells from red bone marrow in Tibetan pigs. We will perform integrative systems biological analysis on these samples, including genomics, transcriptomics, protomics and metabolomics. And we will survey plasma components, blood gas levels and metabolic intermediates in the blood samples, test the relationship among these biochemistry traits, multi-omics data and key selective genes. Meanwhile, we will validate the biological function of key candidate causative genes in high altitude adaptation. The predicted results might comprehensively and deeply elucidate the molecular genetic basis and biological function of high altitude adaptation in Tibetan pigs.

高原适应性机理研究有利于人们预防和治疗高原上发生的低氧相关疾病，应对高原活动中的突发事故，同时可为人类因缺氧而引发的相关心血管、肺和脑部疾病的防治提供重要科学依据。藏猪长期适应高原低氧环境，其高原适应的分子机理备受科研工作者的关注。申请者前期通过高质量全基因组重测序及高密度SNP芯片对藏猪的高原适应性进行了全基因组水平的群体遗传和选择进化分析，发现了一些重要基因（如：EPAS1、NOS1和RGCC等）与其高原适应性紧密相关。本项目在此基础上，拟通过进一步采集藏猪和低海拔地方猪的血液及红骨髓组织，进行转录组、蛋白质组和代谢组等组学分析，并测定血常规、血气及血液生化指标等表型特征，结合前期基因组学分析结果，从多层面多水平系统地探究藏猪高原适应性分子机理，并对其高原低氧适应的关键因果基因及相关基因网络进行生物学功能验证，以此更为全面深入地阐释藏猪适应高原环境的分子机制。

藏猪长期生活在高海拔低氧寒冷的环境中，是我国六大地理类群地方猪种非常独特的一种，其高原适应的分子机理备受科研工作者的关注。本项目试图系统地探究藏猪高原适应性分子机理，并对其高原低氧适应的关键因果基因及相关基因网络进行生物学功能验证。.挖掘了46头藏猪的131头中国地方猪种的SNP变异位点，对高原藏猪的种群结构、历史和演化进行了研究。确定了我国藏猪种群主要分为两大类群：西藏、四川和云南藏猪起源于西南，与内江猪、保山猪关系近；甘肃藏猪起源于华北，与八眉猪、河套猪关系近。两种起源的藏猪在历史上存在交流。应用位点特定枝长法（LSBL）进行了藏猪的选择性清除分析，验证了之前报道的藏猪高原适应性位点(如：EPAS1等)，鉴别到一些新基因位点（如：BPGM,GRIK2和GRM8等）。.利用自主研发全基因组高密度SNP芯片判定了709头中西方猪的基因型，验证了上述的藏猪种群历史演化结果。同时，揭示了164个甘肃藏猪受选择的SNP位点，对应到67个基因，包括与嗅觉相关的OR13CB、OR8U9和OR5R，与无氧酵解相关的LDHB，与血液或心脏功能相关的EPAS1、PED4D、CACNA2D3和KCNIP3等。利用305头中西方猪种的重测序数据，挖掘得到112890个可靠的结构变异，进行了藏猪的结构变异选择分析。发现候选基因MYPBC1（56bp缺失）和ADAMTS12（277bp缺失）与藏猪的高原适应性有关。利用405头中西方猪种的重测序数据，进行了短串联重复（STR）基因型分型。研究发现，35.8%的多态STRs与海拔高度显著相关,其中EPAS1、LARS2和PLAAT5基因存在显著关联。.重点开展了EPAS1和BMGP基因的功能研究。构建了EPAS1的单倍型，进行了血红蛋白含量的关联分析。发现EPAS1的HapI主要存在于较高海拔的甘肃藏猪群，保山猪、八眉猪等分布于较高海拔的群体携带少量该单倍型，其他猪种则不携带。采集了海拔3200米以上的甘肃陇南藏猪54头的血样，关联分析表明携带HapI单倍型个体的血红蛋白含量显著高于不携带该单倍型个体，说明EPAS1单倍型HapI有利于藏猪在高原缺氧环境下血液的携氧运输。BMGP基因与血氧结合能力的关联分析表明，BMGP基因的突变与血氧结合能力无关。

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