藏猪HIFs级联途径基因范围高原适应功能基因鉴定及其调控机制

The Tibet pig is a distinctive indigenous breed native to Tibet Plateau. The breed that has genetic mechanism of adaptation to high-altitude hypoxia is a good model for research of adaptation mechanism on high-altitude. In this project, base on the theory of hypoxic adaptation and previous evidences on genome-wide scanning of human populations in high-altitude, we enrich all genes sequence involved in HIFs-pathway cascade with the methods of custom sequence-capture arrays, and survey the DNA variation to analysed the genetic structure and origin and evolution of Tibet pig populations,and to indify the selective genes between Tibet and lowland pig breeds with high-through sequencing. Furthermore, we confirm polymorphism of the selective sites and gene to analyze the relationship between genotype and physiological phenotypes of highland adaptation and potential gene control function with the methods of PCR-RFLP and Sequenom Massarray. Using real-time PCR and western blotting we measure the expressions of related genes in key organs tissues of Tibet pigs to indentify the genes function and involved pathways and its controlled physiological process. Then, the electrophoretic mobility shift assay (EMSA) and luciferase reporter gene were used to verify the function of genes and regulating network for adaptation to high-altitude, and over-expression and RNAi of target genes in cell model in vitro were used to verify the functional gene for hypoxic adaptation, and to reveal the molecular mechanism of high-altitude hypoxic adaptation in Tibet pig.

藏猪是我国青藏高原特有的原始小型地方猪种，对高海拔低氧环境具有独特的适应性，是研究动物高原适应机制的良好模式动物。本项目根据低氧适应理论和高原人群全基因组扫描结果，采用靶向基因捕获芯片富集藏猪和对照猪HIFs信号级联途径所有基因，高通量测序分析藏猪群体遗传结构和起源进化，鉴定藏猪群体中高海拔环境正选择位点和基因。采用PCR-RFLP和飞行质谱技术进一步验证不同海拔分布的猪群体中高原适应正选择位点，分析基因型与高原适应性生理表型的相关性及其可能的基因调控作用。采用定量PCR、Western blotting测定相关基因在藏猪关键器官组织中的定量表达，分析功能基因作用的pathway调控途径。进而采用凝胶阻滞和荧光素酶报告基因等技术分析高原适应相关DNA突变位点对基因调控的作用，并利用超标达和RNAi技术在体外细胞模型中验证低氧适应的功能基因，揭示其对藏猪高海拔低氧适应的分子机制。

藏猪是我国青藏高原特有的原始小型地方猪种，对高海拔低氧环境具有独特的适应性，是研究动物高原适应机制的良好模式动物。本项目根据低氧适应理论和高原人群全基因组扫描结果，采用高通量测序技术对藏猪和对照猪HIFs信号级联途径所有基因分析藏猪群体遗传结构和起源进化，鉴定了猪群体中高海拔环境正选择位点和基因。采用转录组和蛋白质组鉴定了藏猪心脏组织高原适应相关基因，并从DNA甲基化水平分析了藏猪表观调控基因。采用定量PCR、Western blotting测定了VEGF、BNP和ANP等基因在藏猪关键器官组织中的定量表达。进而采用凝胶阻滞和荧光素酶报告基因等技术分析高原适应相关DNA突变位点对基因调控的作用，并利用超标达和RNAi技术在体外细胞模型中验证低氧适应的功能基因，揭示其对藏猪高海拔低氧适应的分子机制。