Bao Wenbin

Isolation of new alleles of the swine TLR4 gene and analysis of its genetic variation

Using the PCR-SSCP method, the genetic variation in exon 1 of the TLR4 gene was detected among 893 animals, including Asian wild boars, 3 imported commercial and 10 Chinese indigenous swine breeds. This was conducted to analyze the polymorphisms of exon 1 of TLR4 gene in native and foreign pig breeds and aimed at providing a theoretical foundation for further research on the role that TLR4 gene played in immune and defense system. New alleles were isolated for exon 1 of the swine TLR4 gene for the first time, There were 6 genotypes and 3 alleles, in which the Duroc appeared AA, BB, CC, AB, AC and BC genotypes; Sutai pig, which has Duroc pig origin, were detected to be BB, CC, and BC genotypes; Yorkshire and Landrace were detected to be CC and BC genotypes. Wild boar and all 10 Chinese native pig breeds appeared highly conserved in exon 1 of TLR4 gene, with only CC genotype. Among the 3 homozygous genotypes, the CC genotype matches the sequence in GenBank, while a G93C synonymous mutation and a G194A nonsense mutation were found in the BB and AA genotypes, respectively. The correlation between these two mutation points of TLR4 gene with resistance to stress and disease is worthy of further study.Using the PCR-SSCP method, the genetic variation in exon 1 of the TLR4 gene was detected among 893 animals, including Asian wild boars, 3 imported commercial and 10 Chinese indigenous swine breeds. This was conducted to analyze the polymorphisms of exon 1 of TLR4 gene in native and foreign pig breeds and aimed at providing a theoretical foundation for further research on the role that TLR4 gene played in immune and defense system. New alleles were isolated for exon 1 of the swine TLR4 gene for the first time, There were 6 genotypes and 3 alleles, in which the Duroc appeared AA, BB, CC, AB, AC and BC genotypes; Sutai pig, which has Duroc pig origin, were detected to be BB, CC, and BC genotypes; Yorkshire and Landrace were detected to be CC and BC genotypes. Wild boar and all 10 Chinese native pig breeds appeared highly conserved in exon 1 of TLR4 gene, with only CC genotype. Among the 3 homozygous genotypes, the CC genotype matches the sequence in GenBank, while a G93C synonymous mutation and a G194A nonsense mutation were found in the BB and AA genotypes, respectively. The correlation between these two mutation points of TLR4 gene with resistance to stress and disease is worthy of further study.

TLR 5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

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。