藏绵羊低氧适应的血液生理表征及其相关分子基础研究

Tibetan sheep is a rare model animal with high ability to adapt hypoxia. At present, the study on hypoxia adaptation for highlander focus on the transport capacity of oxygen and genes regulated by HIF. Comparative study will be performed between Tibetan sheep and the local breeds distributed across Tea-horse Ancient Road. Firstly, examine blood physiological and velocity of blood stream in samples with a large size; secondly, screen TagSNPs of genes selected by hypoxia and involved in the pathway regulated by HIF, and perform genotyping of TagSNPs by MassARRAY with a large sample, and analyze genetic effect of SNP together with physiological index; Thirdly, reveal network on hypoxia adaptation by mRNA-seq, and specially, clarify the regulation mechanism of HIF and find new pathways through verifying mRNA and protein expression profile under hypoxia of key genes. This study aims to explore the physiological representation on hypoxia adaptation, selective expression under hypoxia and genes on hypoxic direct selection in Tibetan sheep. The results in this study could provide physiological and molecular data for hypoxia studies and hypoxia sickness, and possibly contribute to find molecular markers for breeding hybrid of Tibetan sheep and lowland sheep.

藏绵羊是高原典型家养动物。目前高原民族和动物低氧适应研究热点集中于血液生理和低氧诱导因子（HIF）调控通路。本研究拟选择藏绵羊和茶马古道连续海拔分布的本地绵羊品种作对照研究，大样本测定血液和血流变相关生理指标，揭示藏绵羊低氧适应的血液生理表征；同时筛选HIF通路受到低氧正选择的基因和标签SNP(TagSNP)，通过MassARRAY进行大样本TagSNP分型，结合生理指标，剖分低氧正选择位点遗传效应；最后通过mRNA-seq揭示藏绵羊低氧适应性调控网络，通过qPCR和western blot验证关键基因表达量，重点阐明HIF通路在低氧适应方面发挥的作用及机制。结合上述生理、遗传和调控通路分析，找到遗传性变化的调控基因和功能位点。结果可以为高原低氧适应、低氧相关疾病等基础领域积累生理和分子资料；为快速、准确选育既耐低氧又高产的藏系杂优羊寻找分子标记。

项目采集了1208头1500-2500-3500-4500米连续海拔绵羊血液样品，现场测定了血常规和血液流变学指标，明确了藏绵羊低氧适应的血液生理表征。藏绵羊具有高的红细胞数和血红蛋白浓度，有利于氧运输，同时通过降低红细胞平均体积缓解因红细胞数增加导致的血液粘稠度增加对机体的不利影响，保证血液的正常流动。项目通过500-1500-2500-3500-4500米5海拔群体100个个体全基因组扫描，采用环境适应性混合效应模型（MEMEA），并结合Fst和XPEHH等群体遗传学指标，筛选到海拔趋势位点6650个和低氧正选择基因12个，并解析了候选基因EPAS1的血液生理效应。项目通过本地和易地饲养的藏绵羊和石屏青绵羊（各5只）骨髓转录组测定。高原适应基因应该同时受遗传和环境的影响，筛选到藏绵羊低氧适应性表达基因197个。仅环境和仅遗传影响基因不是高原适应基因，共280个。互作基因179个，占高原适应基因比例为90.86%，仅互作基因占高原适应基因33.50%。结果首次揭示了遗传环境互作在藏绵羊低氧适应的关键作用。藏绵羊是典型高原土著畜种，其低氧适应的生理机制、功能基因发掘、低氧正选择功能位点筛选等研究，可为高原民族和动物低氧适应、高原疾病等研究提供基础资料。筛选的低氧选择位点可为快速、准确选育高产耐低氧藏系绵羊品种提供低氧分子标记，对藏绵羊特色基因保存和利用具有重要意义。连续海拔大样本生理和群体基因组分析及高原、低地易地实验等思路可为高原低氧适应研究提供有益借鉴。