基于聚肌胞刺激的仔猪免疫应答表达谱分析及抗性基因的鉴定

Disease is the major challenge in the pig production. Improving the immune and disease resistance of pig population by breeding is an effective way to resolve the disease problem. Indigenous pig breeds of China have stronger disease resistance than imported pig breeds. However, little is known about the genetic basis and immune mechanisms of the different disease resistance between indigenous breeds and imported ones. In the project, piglets of Dapulian pigs, one Chinese indigenous breed with higher disease resistance, and Landrace with lower disease resistance, will be selected as experiment animals. Peripheral blood mononuclear cell (PBMC) will be separated and stimulated by polyinosinic:polycytidylic acid (Poly I:C). Then the mRNA of the PBMCs before and after stimulation will be sequenced using RNA sequencing (RNA-Seq) based on the next-generation sequencing for the first time in the world. The gene expression profiles will be analyzed for two goals. One is to select the differentially expressed genes before and after Poly I:C stimulation and to construct gene regulatory network of immune response to Poly I:C stimulation. The other is to select differentially expressed genes between the two breeds studied, and to identify the unique disease-resistance genes of Dapulian pigs. Implement of the project will not only be beneficial to explore the regulatory mechanism of immune response to RNA viruses, but provide important information for mining the disease-resistance genes of our indigenous breeds and disease-resistant breeding using marker assisted selection.

疾病是困扰当前养猪业发展的主要难题，通过育种措施提高猪群自身免疫力和.抗病力，成为解决疾病问题的有效途径。我国地方猪种在抗病和免疫力方面明显优于国外品种，但我们对其抗病力差异的遗传基础和免疫机理了解甚少。本项目选取抗病力强的大蒲莲和抗病力弱的长白仔猪为研究对象，分离外周血单核细胞（PBMC），利用聚肌胞(Poly I:C)免疫刺激，然后采用基于高通量测序平台的RNA 测序技术对免疫刺激前后PBMC 的mRNA 进行测序。通过对刺激前后PBMC 表达谱分析，一方面筛选Poly I:C 免疫刺激下仔猪的免疫应答基因并构建调控网络，另一方面筛选大蒲莲和长白免疫应答差异表达基因，鉴定大蒲莲猪特殊的抗性基因。本项目首次利用RNA 测序技术进行猪免疫应答研究，项目的实施不仅有利于探索仔猪对RNA 病毒感染的免疫应答调控机理，而且可为我国地方猪种高抗病力优异基因资源的挖掘及标记辅助抗病育种提供科学依据。

疾病是困扰当前养猪业发展的主要难题，通过育种措施提高猪群自身免疫力和抗病力，成为解决疾病问题的有效途径。我国地方猪种在抗病和免疫力方面明显优于国外品种，但我们对其抗病力差异的遗传基础和免疫机理了解甚少。针对以上问题，本项目开展了以下几方面的研究：. 1、聚肌胞浓度和免疫刺激时间对猪外周血单核细胞基因表达的影响：系统研究不同的Poly I:C 免疫剂量和免疫刺激时间对猪全血和外周血单核细胞（PBMC）各细胞因子和受体基因表达的影响，确定PolyI:C 进行免疫试验的最佳浓度和体外免疫刺激培养时间，为利用Poly I:C进行RNA 病毒感染的免疫应答调控机理研究提供试验依据。. 2、poly I:C免疫刺激下PBMC和外周血样本中内参基因筛选：利用荧光定量PCR法测定6个常用的候选内参基因（ACTB、GAPDH、B2M、TBP、RPL4和PPIA）在poly I:C 免疫刺激下的PBMC和外周血样本中表达量，利用geNorm软件进行稳定性评价分析，筛选仔猪外周血RNA基因定量分析中稳定表达的内参基因。. 3、基于聚肌胞刺激的仔猪免疫应答表达谱分析：选取大蒲莲和长白仔猪各3头为研究对象，分离其PBMC，利用Poly I:C进行免疫刺激，然后采用基于高通量测序平台的RNA 测序技术（mRNA-seq）对免疫刺激前后PBMC 的mRNA 进行测序。通过对刺激前后PBMC 表达谱进行系统分析，筛选Poly I:C免疫刺激下仔猪的免疫应答基因及大蒲莲（290个）和长白（85个）两品种间的差异表达基因，并对这些基因进行gene ontology和基因集富集分析。绝大多数显著富集的GO 和基因集是免疫相关的，这些条目为我们进一步挖掘机体对poly I:C免疫刺激应答基因和导致品种间遗传差异基因奠定了良好的基础。. 4、对6个候选基因进行大样本的初步验证：采集了124头大蒲莲仔猪和187头长白仔猪的血液样品，测定了血常规和T淋巴细胞亚群指标。对6个候选基因（CD4、LAG3、LTBR、AKT3、CD27、TNFRSF1A）进行SNP基因型和免疫指标的关联分析以及mRNA表达分析。. 本项目的实施不仅有利于探索仔猪对RNA 病毒感染的免疫应答调控机理，而且可为我国地方猪种高抗病力优异基因资源的挖掘及标记辅助抗病育种提供科学依据。