猪m6A甲基化酶WTAP基因与F18大肠杆菌感染的关系

doi:10.11843/j.issn.0366-6964.2021.06.025

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (6): 1709-1716.doi: 10.11843/j.issn.0366-6964.2021.06.025

猪m⁶A甲基化酶WTAP基因与F18大肠杆菌感染的关系

肖叶懿¹, 郜重丞¹, 包文斌^1,2, 吴正常^1,2*, 吴圣龙^1,2*

1. 扬州大学动物科学与技术学院, 扬州 225009;
2. 教育部农业与农产品安全国际合作联合实验室, 扬州 225009

收稿日期:2020-10-20 出版日期:2021-06-23 发布日期:2021-06-22
通讯作者: 吴圣龙,主要从事猪抗病育种和健康养殖研究,E-mail:pigbreeding@163.com;吴正常,主要从事猪抗病育种和健康养殖研究,E-mail:wuzhengchang@126.com
作者简介:肖叶懿(1997-),男,江苏扬州人,硕士,主要从事猪抗病育种研究,E-mail:yeyixiao@126.com
基金资助:
国家自然科学基金（31972535；31772560）；江苏省自然科学基金（BK20180899）；江苏省农业科技自主创新资金项目（CX（20）3011）；扬州大学科技创新培育基金项目（2019CXJ166）；扬州大学大学生科技创新基金（X20200646）

Relationship between Porcine m⁶A Methylase WTAP Gene and F18 Escherichia coli Infection

XIAO Yeyi¹, GAO Zhongcheng¹, BAO Wenbin^1,2, WU Zhengchang^1,2*, WU Shenglong^1,2*

1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China;
2. Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou 225009, China

Received:2020-10-20 Online:2021-06-23 Published:2021-06-22

摘要/Abstract

摘要： 本研究旨在探讨猪m⁶A甲基化酶WTAP表达水平与大肠杆菌（E. coli）感染抗性的关系。选取35日龄苏太断奶仔猪（Sus scrofa）大肠杆菌抗性型和敏感型个体各4头，采集十二指肠和空肠组织，利用RT-qPCR检测WTAP在E. coli抗性型和敏感型个体十二指肠、空肠的表达差异，并分别利用产肠毒素大肠杆菌（F18ab、F18ac）刺激和内毒素（LPS）诱导猪小肠上皮细胞（IPEC-J2），检测WTAP基因的表达变化。同时构建WTAP基因干扰载体并转染IPEC-J2细胞，通过菌毛定量、菌落计数以及间接免疫荧光试验检测该基因沉默对大肠杆菌黏附能力的影响。结果显示：在十二指肠和空肠组织中，WTAP基因在E. coli抗性型个体中的表达量显著高于敏感型个体（P<0.01）；并且在F18ab和F18ac刺激后表达量显著下降，与LPS诱导6 h后结果相一致（P<0.01）。沉默WTAP基因后，大肠杆菌黏附能力极显著上升（P<0.01）。本研究在细胞和个体水平上验证发现，m⁶A甲基转移酶WTAP的高表达可能有助于仔猪抗大肠杆菌感染，为进一步揭示仔猪抗大肠杆菌感染的RNA甲基化调控机制奠定基础。

关键词: 猪, N6-甲基腺嘌呤(m⁶A), RNA甲基化酶, 肾母细胞瘤1-相关蛋白, 大肠杆菌

Abstract: This study aimed to investigate the relationship between the expression level of porcine m⁶A methylases WTAP and resistance to E. coli infection. In this study, the small intestine tissues of 35-day-old Sutai piglets (Sus scrofa) were collected from 4 individuals of E. coli resistant-and sensitive-type. RT-qPCR was used to detect the expression level of WTAP in the duodenum, jejunum of E. coli resistant-and sensitive-individuals. The mRNA expression level of the WTAP gene in intestinal epithelial cells (IPEC-J2) was detected respectively after Escherichia coli F18ab and F18ac stimulation and lipopolysaccharide (LPS) induction. At the same time, the interference vectors of the WTAP gene were constructed and transfected into IPEC-J2 cells, and the effect of gene silencing on the adhesion ability of E. coli was detected by pili protein detection, colony count and indirect immunofluorescence assay. Results showed that the expression level of the WTAP gene in duodenum and jejunum of resistant individuals were significantly higher than that of sensitive individuals (P<0.01). After F18ab and F18ac stimulation, the expression level of the WTAP gene was significantly decreased, which was consistent with the results after 6 h LPS induction (P<0.01). Meanwhile silencing the WTAP gene could significantly increase the adhesion ability of E. coli (P<0.01). This study verified that the high expression level of m⁶A methyltransferase WTAP may improve the ability of piglets to resist E. coli infection and laid a foundation for further RNA methylation investigation of the regulatory mechanism of E. coli infection in piglets.

Key words: pig, N⁶-methyladenosine (m⁶A), RNA methylation, WTAP, E. coli

中图分类号:

S813

肖叶懿, 郜重丞, 包文斌, 吴正常, 吴圣龙. 猪m⁶A甲基化酶WTAP基因与F18大肠杆菌感染的关系[J]. 畜牧兽医学报, 2021, 52(6): 1709-1716.

Relationship between Porcine m, GAO Zhongcheng, BAO Wenbin, WU Zhengchang, WU Shenglong. Relationship between Porcine m⁶A Methylase WTAP Gene and F18 Escherichia coli Infection[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1709-1716.

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猪m⁶A甲基化酶WTAP基因与F18大肠杆菌感染的关系

Relationship between Porcine m⁶A Methylase WTAP Gene and F18 Escherichia coli Infection

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