哈萨克羊脾脏转录组分析及免疫相关基因鉴定

The Kazakh sheep is an ancient breed with disease resistance characteristics in Xinjiang, which was bred by long-term natural and artificial selection. Our previous study found that the immune indices, general resistance to disease and lamb survival of Kazakh sheep and hybrid progeny were significantly higher than those of Suffolk sheep. So we speculated that the specific immune-related genes were existed in Kazakh sheep. However, molecular genetic studies of general resistance to disease are less in sheep. Newborn lambs of Kazakh and Suffolk will be used as experimental materials. The de novo assembly of transcriptomes from spleens will be carried out between Kazakh and Suffolk sheep by RNA-Seq (RNA sequencing) technology and bioinformation. By contrasting the differentially expressed mRNA, some real target gene of immune response in Kazakh sheep will be determined. The full-length cDNA of candidate genes will be cloned by rapid amplification of cDNA ends (RACE). The immune-related genes of Kazakh sheep will be verified by Real-time PCR for the measurement of gene expression profiles, gene overexpression and RNA interference (RNAi).The study is in order to reveal the molecular mechanism of disease resistance in Kazakh sheep，and lays the basis for molecular regulation of immune and carrying out molecular breeding of disease resistance in sheep.

哈萨克羊是新疆古老的绵羊品种，是经过长期自然选择和人工选育形成的地方良种，具有抗病力强的种质特征。我们前期研究结果表明：哈萨克羊及其与萨福克羊杂交后代免疫指标、抗病力及羔羊存活率显著高于萨福克羊，推测哈萨克羊存在品种特异的免疫相关基因。但针对绵羊一般抗病力的分子遗传机理研究很少。本项目以哈萨克和萨福克初生羔羊为实验材料，利用RNA-Seq（RNA sequencing）技术分析绵羊品种间脾脏转录组，通过生物信息学比较分析，发现与哈萨克羊免疫反应相关的、其转录水平发生显著变化的基因，利用cDNA末端快速扩增技术（RACE）克隆候选基因的全长cDNA，通过实时荧光定量PCR（Real-time PCR）技术分析基因表达谱、基因过表达和抑制表达（RNAi）进行候选基因的功能验证，获得哈萨克羊免疫相关基因，从而明确哈萨克羊抗病力强的分子机理，为免疫的分子调控以及绵羊抗病分子育种提供理论基础。

抗病育种已成为当前疾病控制的有效方法，哈萨克羊是新疆古老的绵羊品种，具有抗病力强的种质特征。本项目通过RNA-Seq技术寻找绵羊品种间脾脏免疫相关的差异表达基因，应用生物信息学、基因克隆、qRT-PCR、RNAi、基因过表达、细胞生物学和遗传学等方法和技术对候选基因的功能进行了初步研究。首先应用ELISA方法测定并分析新疆部分绵羊的10种免疫分子，建立基于免疫分子的一般抗病力遗传选择方法，证明绵羊免疫分子的分泌具有年节律和昼夜节律特征，本地哈萨克羊的免疫力或抗病性比引进的萨福克羊更强，可通过免疫力间接选择抗病力，实现绵羊抗病育种。进而利用RNA-Seq技术分析哈萨克羊和萨福克羊品种间脾脏组织转录组和数字基因表达谱（DGE），获得1158个差异表达基因，经qRT-PCR技术验证、生物信息学和候选基因分析，对14个基因进行克隆和测序，2个基因序列登录Genbank数据库，发现15个重要的SNPs位点。通过qRT-PCR、RNAi和基因过表达等方法研究IDO1基因、LTB基因和FCER1G基因功能，证明这3个基因是哈萨克羊免疫相关的重要基因。遗传关联分析证明IDO1基因突变的1072G＞A AA基因型和1125A＞G GG基因型个体的IFN-α、IL-2和IL-12水平极显著高于其他基因型个体，证明在生产中选择肉用体型好、生长发育快、周岁体重大、1072G＞A位点AA基因型或1125A＞G位点GG基因型的羔羊进行组群，是进行肉羊抗病育种的有效方法。针对抗病育种建立了绵羊IDO1基因一般抗病力PCR-RFLP等基因突变检测方法。研究成果为进一步研究免疫的分子调控以及实现绵羊抗病分子育种提供了基础。项目资助发表5篇文章，1篇SCI文章投稿中，获1项发明专利授权，培养硕士研究生1名，本科生1名。