lncRNAs对哺乳仔猪C型产气荚膜梭菌腹泻抗性的调控机制研究

Piglet diarrhea is one of the primary factors responsible for the benefits of swine industry. Improving the diarrhea-resistance of piglets from genetic predispositions, will contribute to reduce the episodes of diarrhea. As the key regulators, the mechanism of lncRNAs regulating gastrointestinal tract disease by Helicobacter pylori and Salmonella has been studied. However, which lncRNAs take part in piglets diarrhea caused by Clostridium perfringens type C, and how to realize its regulation function are remain unexplored. This project will launch studies by utilization of C. perfringens type C attacking 7-day piglets, the small intestinal epithelial and spleen tissues in the tolerance, susceptible and control group piglets will be selected for RNA-seq analyses. (1) Obtaining significantly differential expressed profiles of mRNA and lncRNA. (2) By constructing the lncRNA-mRNA co-expression network, to predict the target genes for lncRNA, and to understand the relationship between lncRNA and target genes, as well as the signal pathway network which related to piglet diarrhea. (3) lncRNAs in the key signal pathways which have important regulatory function will be further functionally analyzed at the cellular level, to shed light on the regulation mechanism of lncRNA resisting to diarrhea caused by C. perfringens type C in suckling piglets, and to obtain regulation lncRNA. These results will provide references for diarrhea-resistant studies in pigs.

仔猪腹泻是影响养猪效益的重要因素之一。从遗传基础上提高仔猪对腹泻的抗性将有助于减少腹泻发生。lncRNAs作为关键调控因子，其调控幽门螺旋菌、沙门氏菌等导致胃肠道疾病的机制已有研究，但目前就哪些lncRNAs参与了仔猪C型产气荚膜梭菌腹泻抗性的调控及如何调控尚未见报道。项目用C型产气荚膜梭菌对7日龄仔猪进行攻毒，对耐受组、易感组及对照组仔猪小肠上皮和脾脏组织进行转录组测序，(1)获取差异表达显著的mRNA和lncRNA；(2)构建lncRNA-mRNA网络，预测lncRNA的靶基因，并对差异表达基因及共表达基因进行GO和KEGG分析，了解lncRNA与靶基因之间的关系及影响仔猪腹泻的信号通路网络；(3)选取关键信号通路上重要的调控lncRNA，在细胞水平验证其功能，揭示lncRNA对仔猪C型产气荚膜梭菌腹泻抗性的调控机制，找出具有重要调控功能的lncRNA，为猪腹泻抗性相关研究提供参考。

C型产气荚膜梭菌是引起仔猪腹泻病的主要致病菌之一，从遗传基础上提高仔猪对腹泻的抗性将有助于减少腹泻疾病的发生。lncRNAs作为关键调控因子，其调控大肠杆菌、沙门氏菌等导致胃肠道疾病的机制已有研究，但目前就哪些lncRNAs参与仔猪C型产气荚膜梭菌腹泻抗性的调控及如何调控未见报道。本项目采用C型产气荚膜梭菌对7日龄仔猪进行攻毒，通过仔猪腹泻指标评定和组织形态学表型分析，筛选耐受型和易感型仔猪作为试验对象，通过仔猪小肠和脾脏组织转录组学测序及分析，鉴定与仔猪C型 产气荚膜梭菌腹泻抗性相关的lncRNA、mRNA和miRNA，探究了lncRNA、miRNA与靶基因之间的关系及影响仔猪腹泻的信号通路网络；成功构建C型产气荚膜梭菌重组CPB2毒素蛋白，建立CPB2毒素处理猪IPEC-J2细胞的体外细胞模型，确定体外接毒的最适宜条件为浓度20μg/mL CPB2毒素处理24h；通过基因功能研究验证了miR-21-5p通过靶向PDCD4缓解C型产气荚膜梭菌CPB2毒素诱导IPEC-J2细胞的凋亡和炎症损伤；构建了由3个miRNA，17个lncRNA和9个mRNA构成的共表达ceRNA网络（如lnc001186-ssclet-7i-TARBP2等）；通过过表达、干扰实验，在细胞水平验证了lnc001186 能够减轻 CPB2 毒素引起的 IPEC-J2细胞损伤及炎症反应，证明了其在仔猪抗C型产气荚膜梭菌腹泻的潜在功能。揭示了lncRNA对仔猪腹泻抗性的调控机制，课题研究结果可为仔猪腹泻的分子抗病育种提供参考依据。