lncRNA—XLOC_014316和XLOC_020181对断奶仔猪大肠杆菌抗性的调控机制分析

Little is known about the functional genes and mechanism regulation of weaning piglet’ 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 lncRNAs, transcriptome, miRNAs throughput sequencing have been selected on the paired full-sib weaning Meishan piglets that were characterized as resistant or sensitive to E. coli F18 by previous detection and selection. Comprehensive analysis of sequencing results shows that two lncRNAs named XLOC_014316 and XLOC_020181 play an important regulating role in the resistance to E. coli F18 of weaning piglets. Further systematical verification and validation was performed. We constructed the IPEC-J2 cell line with knocking out lncRNAs, which was preliminary analyzed the target genes via transcriptome analysis. Combining with previous predicted target genes based on cis and trans theory and systematical verification, we screened out potential target genes. We finally confirmed the key target genes of E. coli F18 resistance and constructed the regulatory network using RNA pull down and RIP experiments. Meanwhile we accurately analyzed and verified the function of key target genes at the level of cell and individual. This research would be benefit for further understanding of the mechanism that two lncRNAs of XLOC_014316 and XLOC_020181 and key target genes regulation play in regulating the Meishan weaning 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.

中国地方猪品种断奶仔猪大肠杆菌抗性的功能基因和调控机制不甚明了，已成为大肠杆菌病抗性育种中的瓶颈问题。课题组对前期梅山猪抗性型和敏感型断奶仔猪小肠组织lncRNA、转录组和miRNA测序的结果进行综合分析，充分显示lncRNA—XLOC_014316和 XLOC_020181在断奶仔猪大肠杆菌抗性过程中发挥了重要调控作用，本研究继续对这2个lncRNA进行系统功能验证，构建敲除lncRNA的IPEC-J2小肠上皮细胞系，通过转录组测序分析其作用的靶基因，整合lncRNA的cis和trans机制分析及一系列实验验证结果，筛选潜在的关键靶基因，结合RNA pull down和RIP等试验，最终确定起核心调控的关键靶基因并构建调控网络，并同时在细胞和个体水平精确分析和验证关键靶基因的功能，以期阐明这2个lncRNA及其关键靶基因的调控作用和分子机制，为地方猪品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（ETEC）是初生和断奶仔猪腹泻最常见和最重要的病原菌，其中E. coli F18是养猪业中发生最普遍、危害最大的大肠杆菌病原之一，目前常规的预防性措施并没有从根本上消除断奶仔猪腹泻的发生与流行，因此从遗传角度提高仔猪对E. coli F18的抵抗力以及深入研究断奶仔猪对E. coli F18抗病力的分子机制具有非常重要的理论和实践意义。然而，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，课题组前期已经在梅山猪和苏太猪F18大肠杆菌抗性型和敏感型个体lncRNA测序筛选出的重要的lncRNA—XLOC_014316和XLOC_020181，本研究进一步通过生物信息学分析和表达验证锁定了1个反义长链非编码RNA—FUT3as/XLOC_014316，并且从FUT3as功能和分子调控机制入手，首先利用FISH定位、qPCR、Northern blot、siRNA干扰以及大肠杆菌F18体外黏附等一系列试验分析lncRNA-FUT3as功能，结果发现FUT3as表达水平下调有利于提高仔猪对大肠杆菌F18感染的抗性。其次，通过RNA-pull down结合RIP-PCR、western blot验证表明，FUT3as与组蛋白H4存在相互作用。干扰FUT3as前后iTRAQ蛋白质组学筛选出1个重要的信号通路—鞘糖脂生物合成（glycosphingolipid biosynthesis）以及FUT2、B3GALNT1、ST3GAL3基因，并利用qPCR和western blot验证FUT3as对鞘糖脂生物合成信号通路具有调控作用，其通路基因表达水平与大肠杆菌F18感染有关。此外，利用ChIP-qPCR检测发现H4组蛋白可以与FUT3基因启动子区结合，并且H4K16ac乙酰化修饰对基因表达起主要作用。靶基因FUT3主要在肠道组织黏膜表面分布，敏感型个体表达水平显著高于抗性型，本研究建立了shRNA干扰、过表达以及CRISPR-Cas9稳定的猪小肠上皮细胞系，并结合大肠杆菌体外黏附能力检测系统验证表明，靶基因FUT3表达水平下调与大肠杆菌F18抗性有关。通过CoIP结合质谱以及western blot验证表明，FUT3基因在鞘糖脂生物合成信号通路中发生重要调控作用。因此，本研究利用比较lncRNA测序筛选出1个关键反义长链非编码RNA。