Toll-like receptors (TLRs) play important roles in building innate immune and inducing adaptive immune responses. Associations of the TLR genes polymorphisms with disease susceptibility, which are the basis of molecular breeding for disease resistant animals, have been reported extensively. Retrotransposon insertion polymorphisms (RIPs), as a new type of molecular markers developed recently, have great potential in population genetics and quantitative trait locus mapping. In this study, bioinformatic prediction combined with PCR-based amplification was employed to screen for RIPs in porcine TLR genes. Their population distribution was examined, and for one RIP the impact on gene activity and phenotype was further evaluated. Five RIPs, located at the 3' flank of TLR3, 5' flank of TLR5, intron 1 of TLR6, intron 1 of TLR7, and 3' flank of TLR8 respectively, were identified. These RIPs were detected in different breeds with an uneven distribution among them. By using the dual luciferase activity assay a 192 bp endogenous retrovirus (ERV) in the intron 1 of TLR6 was shown to act as an enhancer increasing the activities of TLR6 putative promoter and two mini-promoters. Furthermore, real-time quantitative polymerase chain reaction (qPCR) analysis revealed significant association (p < 0.05) of the ERV insertion with increased mRNA expression of TLR6, the neighboring gene TLR1, and genes downstream in the TLR signaling pathway such as MyD88 (Myeloid differentiation factor 88), Rac1 (Rac family small GTPase 1), TIRAP (TIR domain containing adaptor protein), Tollip (Toll interacting protein) as well as the inflammatory factors IL6 (Interleukin 6), IL8 (Interleukin 8), and TNFα (Tumor necrosis factor alpha) in tissues of 30 day-old piglet. In addition, serum IL6 and TNFα concentrations were also significantly upregulated by the ERV insertion (p < 0.05). A total of five RIPs were identified in five different TLR loci. The 192 bp ERV insertion in the first intron of TLR6 was associated with higher expression of TLR6, TLR1, and several genes downstream in the signaling cascade. Thus, the ERV insertion may act as an enhancer affecting regulation of the TLR signaling pathways, and can be potentially applied in breeding of disease resistant animals.

中文翻译：

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在猪 TLR6 的第一个内含子中插入 192 bp 的 ERV 片段可能作为与 TLR6 和 TLR1 表达增加相关的增强子

Toll 样受体 (TLR) 在构建先天免疫和诱导适应性免疫反应中发挥重要作用。TLR 基因多态性与疾病易感性的关联已被广泛报道，这是抗病动物分子育种的基础。逆转录转座子插入多态性（RIPs）作为近年来发展起来的一种新型分子标记，在群体遗传学和数量性状基因座定位方面具有巨大的潜力。在这项研究中，生物信息学预测结合基于 PCR 的扩增被用于筛选猪 TLR 基因中的 RIP。检查了它们的种群分布，并进一步评估了一个 RIP 对基因活性和表型的影响。五个 RIP，位于 TLR3 的 3' 侧翼、TLR5 的 5' 侧翼、TLR6 的内含子 1、TLR7 的内含子 1 和 3' 分别鉴定了 TLR8 的侧翼。这些 RIP 在不同品种中检测到，它们之间分布不均。通过使用双荧光素酶活性测定，TLR6 内含子 1 中的 192 bp 内源性逆转录病毒 (ERV) 显示作为增强子，增加 TLR6 推定启动子和两个微型启动子的活性。此外，实时定量聚合酶链反应 (qPCR) 分析显示，ERV 插入与 TLR6、邻近基因 TLR1 和 TLR 信号通路下游基因（如 MyD88（Myeloid分化因子 88）、Rac1（Rac 家族小 GTPase 1）、TIRAP（包含接头蛋白的 TIR 结构域）、Tollip（Toll 相互作用蛋白）以及炎症因子 IL6（白介素 6）、IL8（白介素 8），和 30 日龄仔猪组织中的 TNFα（肿瘤坏死因子 α）。此外，ERV 插入也显着上调了血清 IL6 和 TNFα 浓度（p < 0.05）。在五个不同的 TLR 基因座中总共鉴定出五个 RIP。TLR6 第一个内含子中的 192 bp ERV 插入与 TLR6、TLR1 和信号级联下游几个基因的更高表达有关。因此，ERV 插入可以作为影响 TLR 信号通路调节的增强子，并有可能应用于抗病动物的育种。TLR6 第一个内含子中的 192 bp ERV 插入与 TLR6、TLR1 和信号级联下游几个基因的更高表达有关。因此，ERV 插入可以作为影响 TLR 信号通路调节的增强子，并有可能应用于抗病动物的育种。TLR6 第一个内含子中的 192 bp ERV 插入与 TLR6、TLR1 和信号级联下游几个基因的更高表达有关。因此，ERV 插入可以作为影响 TLR 信号通路调节的增强子，并有可能应用于抗病动物的育种。