猪FUT3基因序列分析及其表达水平与F18大肠杆菌抗性的关系  

Sequence Analysis and Expression Level of FUT3 Gene in Pig(Sus scrofa)and Its Relationship with Escherichia coli F18 Resistance

作　　者：杨荔 周雅静 包文斌[1,2] 吴正常 吴圣龙[1,2] YANG Li;ZHOU Ya-Jing;BAO Wen-Bin;WU Zheng-Chang;WU Sheng-Long(Key Laboratory for Animal Genetics,Breeding,Reproduction and Molecular Design of Jiangsu Province,Animal Science and Technology College,Yangzhou University,Yangzhou 225009,China;Joint International Research Laboratory of Agriculture and Agri-Product Safety,the Ministry of Education of China,Yangzhou University,Yangzhou,225009,China)

机构地区：[1]扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009 [2]扬州大学教育部农业与农产品安全国际合作联合实验室,扬州225009

出　　处：《农业生物技术学报》2020年第11期2002-2010,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31772560);江苏省自然科学基金(BK20180899);扬州市自然科学基金(YZ2018102);江苏高校优势学科建设工程资助项目(PAPD)。

摘　　要：断奶仔猪腹泻(post-weaning diarrhea,PWD)对国内外规模猪场造成了严重的经济损失,其中F18大肠杆菌(Escherichia coli F18,E.coli F18)是引起仔猪(Sus scrofa)细菌性腹泻的主要病原,本课题组前期通过测序和验证分析确定了一个E.coli F18受体相关分子—α-1,3岩藻糖转移酶基因(α-1,3-fucosyltransferase,FUT3)。为了探究猪FUT3基因的基本结构特征和功能,本研究以梅山猪为实验材料,利用PCR扩增克隆FUT3基因编码区,并结合生物信息学方法预测其蛋白质结构与功能,q RT-PCR检测其组织表达谱,免疫组化方法分析其组织定位与表达分布情况,同时检测E.coli F18菌体感染猪小肠上皮细胞(intestinal porcine epithelial cell line J2,IPEC-J2)前后FUT3 mRNA和蛋白质表达变化。结果显示,梅山猪FUT3基因编码区全长1068 bp,编码361个氨基酸,为脂溶亲水性、不稳定的非分泌蛋白,其蛋白质序列中包括2个糖基化位点、29个磷酸化位点和2个不同的保守结构域(Glycotran10N和Glycotransf10);组织表达谱显示,FUT3基因在各个组织中均有一定表达,尤其在肠道组织(十二指肠,空肠和回肠)中表达水平较高;免疫组化显示,FUT3在E.coli F18敏感型仔猪十二指肠中表达水平显著高于抗性型(P<0.05),并且FUT3主要分布在小肠上皮细胞黏膜表面;不同E.coli F18菌体刺激IPEC-J2细胞后,FUT3 mRNA和其蛋白质表达水平均表现出极显著的上调(P<0.01);FUT3基因在E.coli F18敏感型断奶仔猪十二指肠中的表达水平极显著高于抗性组(P<0.01)。本研究成功克隆获得了梅山猪FUT3基因编码序列全长,从生物信息学层面了解其蛋白结构和功能,并在个体和细胞水平上初步证实了猪FUT3表达水平对E.coli F18抗性具有重要的调控作用,为今后深入探究猪FUT3基因功能和表达调控机制提供了一定的基础资料。Post-weaning diarrhea(PWD)causes serious economic losses to large-scale pig(Sus scrofa)farms at home and abroad,of which Escherichia coli F18(E.coli F18)is the main pathogen causing bacterial diarrhea in piglets.E.coli F18 receptor-related molecule,α-1,3-fucosyltransferase gene(FUT3)was identified by relevant studies and sequencing.In order to investigate the basic structural characteristics and functions of FUT3 gene in pigs(Sus scrofa),the coding region of FUT3 gene was amplified and cloned by PCR.Then,this study predicted its protein structure and function regions by bioinformatics methods,detected the tissue expression profile by qRT-PCR,analyzed its localization and expression distribution by immunohistochemical methods,and detected the mRNA and protein expression levels of FUT3 in E.coli F18-stimulated intestinal porcine epithelial cell line J2(IPEC-J2).The results showed that the coding region of FUT3 gene in Meishan pigs was 1068 bp in length,encoding 361 amino acids,which was a lipophilic hydrophilic and unstable nonsecretory protein.FUT3 protein sequence included 2 glycosylation sites,29 phosphorylation sites and 2 conserved domains(Glycotran10N and Glycotransf10).Tissue expression profile showed that FUT3 gene was expressed in various tissues of pigs,especially intestinal tissues(duodenum,jejunum and ileum).Immunohistochemistry analysis showed that the expression level of FUT3 in sensitive duodenum was significantly higher than that in resistant duodenum,and FUT3 was mainly distributed on the mucosal surface of small intestinal epithelial cells.After E.coli F18 bacteria stimulation in IPEC-J2 cells,the expression levels of FUT3 mRNA and protein were extremely significantly up-regulated(P<0.01).The expression levels of FUT3 gene in duodenal tissues of weaned piglets in F18-sensitive group were extremely significantly higher than those in resistant group(P<0.01).In this study,the full-length coding sequence of Meishan pig FUT3 gene was successfully cloned,and its protein structure and function were unde

关 键 词：猪 α-(1 2)岩藻糖转移酶基因3(FUT3) 表达调控 F18大肠杆菌(E.coli F18) 

分 类 号：S828.8[农业科学—畜牧学]