miR-215和miR-192对断奶仔猪F18大肠杆菌抗性调控的分子机制

Little is known about the functional genes and mechanism regualtion of weaning piglets' resistance to E. coli F18 in Chinese native breeds, which has become a bottleneck problem to solve the anti- E. coli disease breeding of Chinese native breeds. Differential miRNAs have been selected by deep sequencing, Agilent double labeled cDNA microarray and proteomics analyses conducted on the paired full-sib weaning piglets that were characterized as resistant or sensitive to E. coli F18. Our previous research combined with the results of other researchers showed confidently that target miRNAs of miR-215 and miR-192 played important roles in regulating the weanling piglets' resistance to E. coli F18. In this study, function verification of differential miRNAs will be carried,and the target genes and sites of miRNAs will be predicted and varified from the transcription and translation level aiming at identifying the key target genes which were related to the piglets' resistance to E. coli F18 and then constructing regulation network. Integrating overexpression and RNA inerference of target genes in piglets' intestinal epithelial cells line, the type V secretion system and receptor binding experiments, and E. coli F18 infection experiment will be established for further functional analysis of these target genes. This research would be benefit for further understanding of the mechanism that miR-192/miR-215 and miRNA-mediated gene regulation plays in regulating the Meishan weanling piglets' resistance to E. coli F18 and laid a solid foundation for the breeding of disease resistance to E.coli in Chinese native pig breeds.

中国地方猪种断奶仔猪F18大肠杆菌抗性的功能基因和调控机制不甚明了，已成为解决中国地方猪种大肠杆菌病抗性育种的瓶颈问题。课题组前期高通量测序筛选F18大肠杆菌全同胞抗性和敏感型断奶仔猪小肠上皮差异miRNAs、表达谱芯片和蛋白质组学等试验结果和国内外相关研究，充分显示了miR-192和miR-215在断奶仔猪F18大肠杆菌抗性过程中发挥了重要调控作用，本研究继续对miR-192和miR-215进行功能验证，预测其靶基因和靶位点，并同时从转录和翻译水平对预测结果进行验证，确定起核心调控作用的关键靶基因，构建miRNA及靶基因调控网络；通过RNAi和过表达、黏附和黏附抑制、大肠杆菌攻毒等试验，在细胞和个体水平精确分析和验证关键靶基因的功能，以期阐明miR-192/miR-215及其关键靶基因在梅山猪断奶仔猪F18大肠杆菌抗性中的调控作用和分子机制，为中国地方猪种抗大肠杆菌病育种提供依据和指导。

产肠毒素性大肠杆菌F18（ETEC F18）是初生和断奶仔猪腹泻最常见和最重要的病原菌之一，目前中国地方断奶仔猪抗E. coli F18细菌性腹泻的分子机制尚未清楚，已成为解决中国地方猪种大肠杆菌病抗性育种的瓶颈问题。课题组前期从microRNA调控作用入手，通过F18大肠杆菌抗性型和敏感型全同胞个体小肠上皮差异miRNAs筛选、表达谱芯片和蛋白质组学等一系列分析，已经筛选出miR-192/215在断奶仔猪F18大肠杆菌抗性过程中发挥重要的调控作用。本研究继续对miR-192/215进行功能验证，利用靶基因预测、双荧光素酶报告、TALEN敲除以及转录组测序等一系列试验技术来筛选与鉴定其关键靶基因，并且从个体水平（组织表达谱、抗性与敏感型肠道组织差异表达、FISH定位等）到细胞水平（LPS诱导及菌体刺激、RNA干扰及过表达、细菌粘附试验等）、从mRNA（qPCR）到蛋白质水平（western blot）等方面系统验证关键靶基因表达水平与F18大肠杆菌抗性的关系；采用BSP+Miseq测序的方法，对关键靶基因启动子区CpG岛进行了超高精度的甲基化定量检测，同时分析了重要甲基化位点对基因mRNA表达量的影响和转录因子调控作用；此外，利用PCR-SSCP方法检测了miR-192/215前体序列突变，并分析其对仔猪E. coli F18抗性的影响。结果显示，miR-192敲除后猪小肠上皮细胞与大肠杆菌粘附能力明显上升，TLR5作为其关键靶基因；靶基因TLR5主要表达在细胞质和细胞膜上，并且表达水平下调与大肠杆菌抗性密切相关，TLR5启动子区中15个CpG位点发生不同程度的甲基化修饰，其中CpG6位点甲基化修饰与mRNA表达水平呈现显著负相关（P<0.05），并且CpG6位点位于核心启动子区Sp1转录因子结合位点上；miR-215前体序列43 bp处存在C/T突变，并且其对E. coli F18粘附能力具有显著影响。本研究鉴定出miR-192/215调控的关键靶基因—TLR5，并且发现TLR5基因启动子区CpG6位点甲基化修饰抑制了Sp1转录因子与启动子DNA的结合，来降低TLR5基因表达水平，进而提高断奶仔猪F18大肠杆菌抗性，系统阐明了miR-192/miR-215及其关键靶基因在断奶仔猪F18大肠杆菌抗性中的调控作用和分子机制，为中国地方猪种抗大肠杆菌病育种提供依据和指导