猪APN基因启动子区变异对流行性腹泻病毒抗性的调控作用及机制分析

Porcine epidemic diarrhea virus (PEDV) is the main pathogen for viral diarrhea in pigs. The pig aminopeptidase N (APN) gene encodes the functional receptor for PEDV entry to host cells, and PEDV infection is expression level-dependent of the APN gene. Expression level of the APN gene is critical for PEDV infection, while the regulatory mechanisms for its expression and the function of the APN gene in regulation of the resistance to PEDV infection remain largely unknown. This study aims to identify regulatory SNPs in the promoter region and analyze the relationship between promoter CpG methylation and mRNA expression of the APN gene. The regulatory roles and underlying mechanisms of promoter functional SNPs and key regulatory elements in APN gene expression will be systematically investigated with the assays including CHIP, EMSA and RNA interference and overexpression. The functional roles of the APN gene in the responses of porcine small intestinal epithelial cell line (IPEC-J2) to PEDV infection and in the modulation of cytokine production was further explored by the assays such as RNA interference and overexpression. This study will elucidate the regulatory roles and underlying mechanisms of promoter SNPs and regulatory elements in the regulation of APN gene expression and resistance to PEDV infection, laying the foundations for further functional analysis of the APN gene and identification of genetic markers for porcine epidemic diarrhea resistance.

猪流行性腹泻病毒（PEDV）是猪病毒性腹泻的主要病原，猪氨肽酶N（APN）基因编码PEDV感染宿主细胞的功能受体，PEDV感染具有APN基因表达水平依赖性。APN表达水平是影响PEDV能否侵入宿主细胞并增殖的关键所在，然而其表达的分子调控机制及其对PEDV抗性的调控作用尚不清楚。本项目首先鉴定启动子区具有调控效应的SNPs，同步分析并验证启动子区甲基化位点对mRNA表达的作用关系；利用CHIP、EMSA和干扰/过表达转录因子等试验，系统分析APN基因启动子区功能变异位点和重要调控元件对表达的调控作用和机制；在细胞水平上，利用干扰/过表达等手段进一步验证PEDV侵染猪小肠上皮细胞过程中APN基因的作用及其对重要细胞因子的影响。本项目将揭示启动子区关键变异位点和重要调控元件对APN基因表达、PEDV抗性的调控作用及调控机制，为APN基因的功能研究及猪流行性腹泻抗性遗传标记的筛选提供基础和依据。

猪流行性腹泻病毒（PEDV）是猪病毒性腹泻的主要病原，是制约规模化养猪业发展的重大疾病问题之一。筛选鉴定PEDV抗性功能基因及分子标记，实施猪流行性腹泻抗病育种是从根本上防控猪流行性腹泻的长久有效策略。PEDV感染具有APN基因表达水平依赖性，然而APN基因表达调控的分子调控机制尚不清楚。通过病毒体外感染及qPCR检测试验，本研究发现PEDV感染可诱导宿主细胞APN基因的表达；进一步通过CRISPR/Cas9敲除技术构建APN基因敲除的IPEC-J2细胞，病毒感染试验显示，APN基因敲除后细胞中PEDV病毒量显著下降，表明APN基因表达对PEDV的感染具有调控作用。利用BSP-PCR技术检测APN基因启动子DNA甲基化水平，结果显示，病毒感染后DNA甲基化水平无明显变化；遗传变异分析发现，APN基因启动子区存在SNP（rs326030589），荧光素酶及EMAS试验结果显示该SNP显著影响APN基因转录活性。组织及细胞水平转录组分析显示，KLF4、ISG15、OASL、IRF8等基因可能参与调控PEDV与宿主细胞的互作。本项目揭示了APN基因表达与PEDV感染的作用关系，鉴定获得影响APN基因表达的SNP（rs326030589），筛选出可能参与调控PEDV与宿主细胞互作的等重要候选基因。研究结果拓展了人们对 PEDV致病机制的认识，并为进一步开展PEDV抗性基因鉴定以及猪流行性腹泻抗性遗传标记的筛选与应用提供理论依据。