F18大肠杆菌抗性型与敏感型苏太断奶仔猪十二指肠组织比较转录组分析

doi:10.11843/j.issn.0366-6964.2018.10.007

摘要/Abstract

摘要：

旨在揭示培育品种-苏太猪（杜洛克×梅山猪）F18大肠杆菌抗性的调控通路和重要候选基因，同时进一步探究中外猪品种F18大肠杆菌抗性调控遗传基础的差异。本研究以课题组前期获得的苏太猪和梅山猪F18大肠杆菌抗性型与敏感型断奶仔猪全同胞个体为研究对象，通过转录组测序筛选苏太猪F18大肠杆菌抗性相关的调控通路以及重要候选基因，并在细胞水平，利用qPCR和Western blot分析F18大肠杆菌刺激小肠上皮细胞IPEC-J2后重要候选基因（蛋白）的表达变化，同时利用qPCR检测重要候选基因在苏太和梅山断奶仔猪F18大肠杆菌抗性型与敏感型个体十二指肠组织中的差异表达情况。结果显示：1）在苏太断奶仔猪F18大肠杆菌抗性型与敏感型个体中筛选出238个差异表达基因，主要参与Toll样受体信号通路（toll-like receptor signaling pathway）和糖脂类通路（glycosphingolipid biosynthesis-lacto and neolacto series），其中TLR5、IL-1β、FUT2为重要候选基因；2）不同血清型F18大肠杆菌（F18ac、F18ab）菌体分别刺激小肠上皮细胞IPEC-J2后，FUT2、IL-1β、TLR5基因mRNA表达水平显著上调（P<0.05），并且其蛋白表达水平也表现为明显的上调，由此表明，TLR5、IL-1β和FUT2在断奶仔猪F18大肠杆菌感染过程中发挥重要的调控作用；3）组织差异表达分析显示，TLR5、IL-1β和FUT2在苏太猪抗性型与敏感型个体十二指肠组织中表达差异均达到显著水平（P<0.05），而梅山猪中TLR5、IL-1β表达差异达到极显著水平（P<0.01），FUT2表达水平差异不显著（P>0.05）。结合课题组前期关于梅山猪及外来引进品种F18大肠杆菌抗性相关分子机制研究及报道，本研究进一步证明中外猪品种F18大肠杆菌抗性调控的遗传基础确实存在差异，Toll样受体信号通路及CD14、TLR5等基因可能在中国地方品种-梅山猪抵抗F18大肠杆菌感染过程中发挥着免疫调控作用，而鞘糖脂合成通路及FUT2等基因可能在外来猪品种F18大肠杆菌受体形成过程中起关键作用。

Abstract:

To reveal the regulatory pathways and important candidate genes related to E. coli F18 resistance in Sutai pig (Duroc×Meishan pigs), and further explore the differences of hereditary basis in regulating E. coli F18 resistance between Chinese domestic and foreign pig breeds, Sutai and Meishan populations previously established with resistance and sensitivity to E. coli F18 were selected as experiment objects. Using transcriptome sequencing, we screened out the regulatory pathways and candidate genes related to E. coli F18 resistance in Sutai pig, then analyzed the expressions of candidate genes (proteins) in different serotype of E. coli F18 (F18ac and F18ab)-stimulated pig intestinal epithelial cell (IPEC-J2) by qPCR and Western blot. Additionally, the differential expressions of candidate genes in duodenal tissues between E. coli F18-resistant and E. coli F18-sensitive weaned piglets were detected by qPCR. The results showed that:1) there were 238 differentially expressed genes in duodenum between Sutai E. coli F18-resistant and E. coli F18-sensitive weaned piglets, which were mainly involved in toll-like receptor signaling pathway and glycosphingolipid biosynthesis-lacto and neolacto series pathway, including TLR5, IL-1β and FUT2 genes. 2) The mRNA expression levels of IL-1β, TLR5, FUT2 were all significantly up-regulated after F18ac-, F18ab-stimulated IPEC-J2, respectively (P<0.05), and the corresponding proteins expression were also obviously increased, which indicated that IL-1β, TLR5 and FUT2 might play important regulatory roles in the process of Sutai piglets responsing to E. coli F18 infection. 3) Tissue expressions analysis showed that the expression levels of TLR5, IL-1β, FUT2 genes in duodenum tissues between Sutai E. coli F18-resistant and E. coli F18-sensitive individuals were all significantly different (P<0.05). The expressions of TLR5, IL-1β genes in duodenum tissues between Meishan E. coli F18-resistant and E. coli F18-sensitive individuals showed extremely significant difference (P<0.01), but FUT2 expression was not significantly different (P>0.05). Combined with previous studies of molecular mechanism of resistance to E. coli F18 in Meishan and foreign pig breeds, this study revealed the differences of hereditary basis in regulating E. coli F18 resistance between Chinese domestic and foreign pig breeds. Toll-like receptor signaling pathway including CD14 and TLR5 probably played immune roles in regulating E. coli F18 resistance in Chinese domestic pig breeds such as Meishan pigs, while the glycosphingolipid biosynthesis-lacto and neolacto series pathway including FUT2 was associated with the formation of E. coli F18 receptor in foreign pigs.

中图分类号:

S828.2

吴正常, 冯海悦, 黄焱杰, 吴丽思, 吴圣龙, 包文斌. F18大肠杆菌抗性型与敏感型苏太断奶仔猪十二指肠组织比较转录组分析[J]. 畜牧兽医学报, 2018, 49(10): 2112-2123.

WU Zheng-chang, FENG Hai-yue, HUANG Yan-jie, WU Li-si, WU Sheng-long, BAO Wen-bin. Comparative Transcriptome Analysis of Duodenal Tissues between Sutai E. coli F18-resistant and E. coli F18-sensitive Weaned Piglets[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(10): 2112-2123.

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