circRNA_16225和circRNA_04962对断奶仔猪大肠杆菌抗性的调控机制分析

The functional genes and mechanism regulation of weaning piglet’ resistance to E. coli in Chinese native breeds remain unclear, which has become a bottleneck problem to solve the anti-E. coli disease breeding of Chinese native breeds. Our research group previously performed the comprehensive analysis of circRNAs, transcriptome, miRNAs sequencing of intestinal tissues between Meishan resistant and sensitive to E. coli piglets. On this basis, we preliminary constructed the ceRNA regulatory relationships among circRNA_16225—miR-143-3p—TLR5 and circRNA_04962—miR-195—FUT2, which further indicates circRNA (circRNA_16225, circRNA_04962) and target genes (FUT2, TLR5) play an important regulating role in the resistance to E. coli of weaning piglets. In this study, we systematically verified the function of two circRNAs by Northern blot, RNA-fluorescence in situhybridization, overexpression and siRNA interference. Then, the interaction among circRNA-miRNA-mRNA was verified by luciferase reporter system, RNA pull down, RNA immune coprecipitation (RIP). Finally, we further analyzed the relationship between two circRNAs and target genes and the resistance to E. coli infection by adhesion test of small intestinal epithelial cell in vitro. This research aims at clarifying the mechanism of two circRNAs regulating the resistance to E. coli, and is expected to obtain function genes related to E. coli infection, which provide guidance for solving key scientific problems about the breeding of disease resistance to E. coli in Chinese native pig breeds.

中国地方猪品种仔猪大肠杆菌抗性的调控机制和功能基因尚不清楚，已成为抗性育种中的瓶颈问题。课题组已对大肠杆菌抗性和敏感型梅山断奶仔猪circRNA、转录组和miRNA的测序结果进行综合分析，构建了ceRNA调控网络并初步确定circRNA_16225—miR-143-3p—TLR5和circ RNA_04962—miR-195—FUT2在大肠杆菌抗性过程中发挥了重要调控作用。本研究利用Northern blot、FISH、过表达和siRNA敲降等试验对这2个circRNA进行系统功能验证，并利用双荧光素酶报告系统、pull down和RIP技术验证circRNA-miRNA-mRNA的作用关系，最后通过细胞黏附等试验进一步分析circRNA及其靶基因与大肠杆菌抗性的关系，以期阐明circRNA对大肠杆菌抗性的调控机制并获得重要的抗性功能基因，解决地方猪品种抗大肠杆菌病育种的关键科学问题。

大肠杆菌F18（E. coli F18）是引起断奶仔猪细菌性腹泻的主要病原，地方品种猪对其抗性的调控机制有待系统阐明。前期基于多组学测序筛选出关键环状RNA-circRNA_04962，本研究在此基础上首先对circRNA_04962（circ_FUT2）进行了成环鉴定，并在细胞水平验证发现circ_FUT2表达下调与大肠杆菌F18抗性的有关；其次，利用双荧光素酶活性、RIP、western blot等验证circ_FUT2/miR-212/FUT3三者靶向结合关系，最终确定靶基因FUT3；最后，在细胞水平和敲除小鼠模型上分别系统验证FUT3表达对大肠杆菌F18细菌性腹泻的影响，同时利用蛋白质组、CoIP-MS和Pull down-MS分析靶基因FUT3通过影响鞘糖脂生物合成通路（受体形成）和Toll样信号通路（免疫应答）来影响大肠杆菌F18易感性。本研究深入揭示了环状RNA-circ_FUT2通过吸附miR-212介导ceRNA机制对大肠杆菌F18抗性具有调控作用，并且发现通过激活鞘糖脂生物合成通路及Toll样受体信号通路来最终影响机体对大肠杆菌F18易感性，同时本研究从细胞水平和个体水平上系统验证与确定了1个与大肠杆菌F18抗性密切相关的候选功能基因-FUT3，今后需要筛选与鉴定FUT3基因的重要功能变异位点，并在群体水平上进行多个世代的大规模上遗传效应验证，从而筛选出断奶仔猪抵抗F18大肠杆菌腹泻病的有效分子标记。本研究最终确定了断奶仔猪F18大肠杆菌病抗性候选非编码RNA分子和功能基因，为今后制订抗大肠杆菌病遗传选育策略奠定基础和提供理论依据。