TLR5基因启动子区甲基化修饰与苏太断奶仔猪E.coli F18抗性的关系

摘要

旨在分析探讨TLR5基因启动子区甲基化修饰对断奶仔猪F18大肠杆菌抗性的调控作用.本研究首先利用qPCR和Western blot检测分析了TLR5基因在F18大肠杆菌抗性型和敏感型苏太断奶仔猪小肠组织(十二指肠和空肠)中的差异表达,然后利用生物信息学分析和双荧光素酶报告系统检测确定TLR5基因核心启动子区、CpG岛及其作用元件,进而检测并分析TLR5基因启动子区甲基化修饰与TLR5基因在F18大肠杆菌抗型和敏感型断奶仔猪小肠组织中表达水平的相关性.结果表明,TLR5基因在敏感型断奶仔猪十二指肠和空肠组织中的mRNA表达水平分别显著(P＜0.05)和极显著(P＜0.01)高于在抗性型个体中的表达,且十二指肠和空肠组织中敏感组蛋白表达水平均显著高于抗性组(P＜0.05).TLR5基因启动子区包含2个CpG岛和16个作用元件,启动子区第2个CpG岛第6个CG位点甲基化水平对TLR5基因的表达具有一定的调控作用,该位点位于转录因子Sp1结合的核心启动子区域.本研究结果表明,猪TLR5基因的表达水平和F18大肠杆菌的抗性有关,其低表达可能有利于F18大肠杆菌抗性;TLR5基因启动子区第2个CpG岛第6个CG位点甲基化能够显著抑制TLR5基因的表达,并最终影响断奶仔猪对大肠杆菌的抗性.%The aim of this study was to investigate the regulation function of methylation in promoter region of TLR5 gene on the resistance to E.coli F18 of weaned piglets.The differential expressions of TLR5 gene and protein in small intestine (duodenum and jejunum) between E.coli F18-resistant and E.coli F18-sensitive Sutai weaned piglets were detected by qPCR and Western blot,respectively.The core promoter region,CpG islands and their acting elements of TLR5 were determined by bioinformatics analysis and dual luciferase reporter system,and the correlation between the methylation degree in promoter region and the expression of TLR5 gene in E.coli F18-resistant and E.coli F18-sensitive Sutai weaned piglets was detected and analyzed.The results showed that expression levels of TLR5 gene in duodenum and jejunum of sensitive individuals were significantly and very significantly higher than that in resistant individuals (P＜0.05 and P＜0.01),respectively.And the protein expressions of TLR5 in duodenum and jejunum of sensitive individuals were significantly higher than that of resistant individuals (P＜0.05).The core promoter region of TLR5 gene included 2 CpG islands and 16 acting elements,the methylation level of the mC 6 site in the second CpG island of the promoter region had a certain regulation effect on the expression of TLR5 gene.The site located in the core promoter bound by transcrip tion factor Spl.The above results indicated that TLR5 gene played an important regulating role in the process of E.coli invasion,whose low expression was conducive to the piglets' resistance to E.coli.The methylation of the mC-6 CG site in the second CpG island of the TLR5 gene promoter could inhibit the binding of the transcription factor Spl,which inhibited the expression of the TLR5 gene and ultimately affected the resistance of weaned piglets to E.coli.