Sequence Analysis and Expression Level of FUT3 Gene in Pig (Sus scrofa) and Its Relationship with Escherichia coli F18 Resistance
YANG Li1, ZHOU Ya-Jing1, BAO Wen-Bin1,2, WU Zheng-Chang1,2*, WU Sheng-Long1,2*
1. Key Laboratory for Animal Genetics,Breeding,Reproduction and Molecular Design of Jiangsu Province,Animal Science and Technology College,Yangzhou University,Yangzhou 225009,China;
2. Joint International Research Laboratory of Agriculture and Agri-Product Safety,the Ministry of Education of China,Yangzhou University,Yangzhou,225009,China
Abstract：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 1 068 bp in length,encoding 361 amino acids,which was a lipophilic hydrophilic and unstable non-secretory protein. FUT3 protein sequence included 2 glycosylation sites,29 phosphorylation sites and 2 conserved domains (Glyco_tran_10_N and Glyco_transf_10). 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 understood from the bioinformatics level. Besides,the important regulatory role of porcine FUT3 expression level on E. coli F18 resistance was preliminarily verified at the individual and cellular levels,providing a certain theoretical basis for in-depth exploration of porcine FUT3 gene function and expression regulation mechanism in the future.
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