整合基因组和转录组测序解析藏绒山羊高海拔适应的分子机制

The high-altitude environment (altitude >2500 m) extremely challenges mammals and humans physiologically，and thus unveiling the molecular mechanism of high-altitude adaptation for animals inhabiting in such area has long been highlighted in biology research. Currently, a hand of genes potentially associated with high-altitude adaptation have been scanned by employing genome or transcriptome sequencing solely, which lay the foundation for the future research, but could not integrated animal’s high-altitude adaptation systematically from genome structure to gene expression. Here, using Tibetan cashmere goat (Ritu county, altitude ~4500 m) and Inner Mongolia cashmere goat (Alashan Left Banner, altitude ~1200 m) as materials, first of all, we will identify DNA variation and relating candidate genes (including mRNA and lncRNA genes) between two breeds by using genome re-sequencing, and then integrate these results with RNA-seq data from heart, lung and kidney, including sequence mutation, gene alternative splicing and gene enrichment. Further, we selectively verify 10 to 15 critical DNA mutations and their effect on gene expression in two goat groups. Finally, we will get the core genomic variation of genes relating to high-altitude adaptation of Tibetan goat, and construct the corresponding gene regulation networks. This study will reveal the mechanism underpinning high-altitude adaptation of animals systematically, from DNA structure mutations to RNA mutation and gene expression difference. And it will be valuable for similar researches in the future.

高海拔环境（海拔>2500m）给哺乳动物和人带来极端的生理挑战。长期以来，解析高海拔地区动物环境适应的分子机制是生物学研究的热点之一。目前，单一地从基因组或转录组分析获得的候选基因为揭示动物的高原适应性奠定了基础，但不能系统性地从基因组结构到基因表达一体化角度对动物的高海拔适应性作出解析。本项目拟以藏绒山羊（日土县，海拔4500m）和内蒙古绒山羊（阿拉善盟，海拔1200m）为材料，首先进行重测序筛选基因DNA变异及候选基因（包括mRNA和lncRNA基因），然后整合RNA-seq获得的心、肺和肾组织中这些候选基因的表达变化（包括序列突变、可变剪切和基因表达），筛选10-15个关键位点并进行大群验证，最终获得藏绒山羊适应高海拔环境的核心基因变异和相应的基因调控网络。本研究从DNA变异-RNA变异-基因表达差异这三个层面系统准确揭示动物高海拔适应的分子机制，对类似研究具有重要参考价值。

为解析藏绒山羊高海拔适应的分子机制，以藏绒山羊（日土县，海拔4500m）和内蒙古绒山羊（阿拉善盟，海拔1200m）为材料，首先进行重测序筛选候选基因及基因的DNA变异及候选基因，在线粒体ATP6和Cytb中分别鉴定出33和67个单核苷酸多态性位点（SNPs），其中错义突变均为6个。采用PCR克隆的方法得到山羊EDNRA Knockout Lentivirus （EDNRA）基因的CDS区序列，采用实时荧光定量PCR(qPCR)的方法检测了EDNRA基因在山羊心脏、肝、脾、肺、肾、大脑和脂肪组织中mRNA的表达量。基于全基因组FST分析方法 ，一些与家山羊和野山羊之间高度分化的 CNV 重叠的基因主要富集了几种免疫相关途径，而与高海拔地区山羊和低海拔地区山羊之间高度分化的 CNV 重叠的基因主要与维生素和脂质代谢有关。6 号染色体上FGF5下游约 14 kb 处的 507 bp 缺失显示 高海拔地区山羊和低海拔地区山羊之间存在高度差异 ( FST = 0.973)。对西藏自治区仲巴县的正常耳和小耳藏山羊群体及前期测定的5个山羊群体的基因组序列，采用比较基因组学鉴定影响山羊耳朵大小的遗传位点。2号染色体56.475～56.575 Mb基因组区域在两种耳型藏山羊之间分化程度最高（FST=0.36）。在7号染色体45.05～59.76 Mb区域内，受选择基因数量多达52个。相关研究可为动物和人类高原健康医学研究提供一定的见解，为高海拔地区分子选育西藏绒山羊新品种/品系提供新的理论参考。