猪TLR4基因在F18大肠杆菌抗性型和敏感型资源群体间的差异表达分析

Differentiation of Porcine TLR4 Gene mRNA Expression between Resistant and Sensitive Resource Populations to ETEC F18

本研究旨在检测TLR4基因mRNA在猪各个组织的分布以及在F18大肠杆菌抗性型和敏感型群体间差异表达水平,为探讨该基因在免疫识别和F18大肠杆菌抗性中发挥的作用提供理论依据。本试验运用SYBR GreenⅠ实时荧光定量PCR技术进行定量测定。首先,各取8头35日龄F18大肠杆菌抗性型和敏感型苏太仔猪组织样,包括心脏、肝脏、脾脏等11个组织,提取总RNA,以GAPDH基因为内参基因,进行实时荧光定量PCR。对TLR4基因mR-NA进行均一化处理,利用荧光阈值(Ct值)计算TLR4基因mRNA在各个组织以及F18大肠杆菌抗性型和敏感型群体间表达量。结果显示,TLR4 mRNA在猪各种组织中广泛表达,在肺、淋巴结、肾脏和脾脏等免疫器官中表达量较高,F18大肠杆菌敏感型的TLR4基因表达量普遍高于抗性型个体的表达量,在各个组织中(除胃外)TLR4表达量差异倍数从1.083到2.980不等;在肺、淋巴结、肾脏和胸腺组织中,F18大肠杆菌敏感型的TLR4基因表达量显著高于抗性型个体的表达量(P<0.05)。本研究结果表明,TLR4基因通过天然免疫识别机制对猪F18大肠杆菌病等革兰氏阴性疾病有一定的影响,TLR4基因表达量的下调与F18大肠杆菌的抗性具有一定程度的关系。

This study was conducted to detect the expression of TLR4 gene in various tissues of pig,and then to analyze the expression difference between resistant and sensitive resource populations to ETEC F18,which aimed to discuss the role that TLR4 gene played in immunity and resistance to ETEC F18.Porcine TLR4 mRNA was amplified by SYBR-GreenⅠ semi-quantitative RT-PCR,total RNA was extracted from 11 organizations of sixteen 35-day-old piglets（eight ETEC F18 resistant piglets and eight F18 sensitive piglets） including heart,liver,spleen,and so on.Using GAPDH as an inner control gene,the relative TLR4 gene mRNA expression level in various tissues and then in resistant and sensitive resource populations to ETEC F18 was calculated according to the fluorescence threshold（Ct value）.The results showed that TLR4 gene was expressed in all tissues and higher expression levels were detected in immune organs like lung,lymph node,kidney,spleen and thymus gland.The mRNA expression of TLR4 gene in sensitive individuals were higher than that in resistant individuals generally,the ratio of sensitive individuals and resistant individuals in all tissues apart from stomach ranged from 1.083 to 2.980.In tissues of lung,lymph node,kidney and spleen,the mRNA expression of TLR4 gene in sensitive individuals were significant higher than that in resistant individuals（P0.05）.The results speculated that TLR4 gene function may be related to innate immune recognition,particularly have a certain impact on porcine Gram-negative bacteria such as ETEC F18 etc.The resistance to ETEC F18 in pig is related to down regulation of mRNA expression of TLR4 gene to some degree.