Screening And Regulatory Mechanism Analysis Of Genes And Long Noncoding RNA(lncRNA)Related To E.Coli F18 Resistance In Weaned Piglets

Porcine post-weaning diarrhea?PWD?is an important infectious disease which resulted in the death for piglets and caused tremendous economic loss to pig industry.Escherichia coli strain F18 is the main pathogenic bacterium responsible for post-weaning diarrhea?PWD?in piglets.To further reveal the hereditary basis and regulatory mechanism of resistance to E.coli F18 in Chinese domestic weaned piglets breeds,this study used Meishan piglets as model animals to test their susceptibility to E.coli F18 by challenging with the pathogens through feeding different serotype of F18ab and F18ac strains,which was tested and verified by a series of experiments,such as E.coli F18 bacteria detection,bacteria counting and adhesion test of the pathogens to the epithelial cells of small intestine in vitro.Ultimately,we strictly obtained Meishan E.coli F18-resistant and-sensitive piglets.Using transcriptome and long non-coding RNA sequencing,this study systematacially screened out the regulatory pathway,functional genes and lncRNA related to E.coli F18 resistance.In addition,the functional mechanism of important pathway,key genes and lncRNA was analyzed in terms of mRNA,protein and cell,respectively.This study aimed at revealing the molecular mechanism of function genes and lncRNA regulating the resistance to E.coli F18 in Meishan piglets and providing certain theoretical reference for solving the key scientific problem about the breeding of resistance to E.coli F18 in Chinese domestic weaned piglets.Besides,this study also selected Sutai pig?a new hybrid between the Duroc and Meishan breeds?as experiment object based on previously established populations that are resistant and sensitive to E.coli F18.Using high-throughput sequencing,we analyzed the regulatory pathway and candidate genes related to E.coli F18 resistance in Sutai pig.Combining with the literature mining results of E.coli F18 resistance genes in foreign pig breeds,we speculated that the specific pathway and function genes were probably suitable for E.coli F18 resistance in foreign pig breeds,which further analyzed and verified the hereditary basis differences in regulating E.coli F18 resistance between Chinese domestic and foreign pig breeds.The main findings are as follows:1.Transcriptome analysis of duodenum between Meishan E.coli F18-resistant and-sensitive weaned piglets?1?There were 198 differential expression genes?DGEs?between Meishan E.coli F18-resistant and-sensitive weaned piglets,of which 125 genes were up-regulated in the resistant group.Most of the DGEs were involved in the "immune System" and "infectious Diseases" pathway,of which Toll-like receptor signaling pathway including key CD 14 gene was screened out.?2?qPCR and western blot showed most of the toll-like receptor signaling pathway genes?CD14,TLR4,IL 1?,ERK,IFN-?,JNK,p38,NFkB and TNF-??were significantly up-regulated in LPS-induced small intestinal epithelial cell?IPEC-J2?,which indicated that the toll-like receptor signaling pathway really plays an important role in regulating the process of E.coli F18 resistance.?3?Immunohistochemistry?IHC?results showed that CD 14 was widely distributed in intestinal tissue and the expression level in resistant group was significantly higher than sensitive group.After CD14 gene knockdown,the adhesion ability of F18ab fimbriae to IPEC-J2 showed extremely significantly increased?P<0.01?,while the adhesion ability of F18ac fimbriae to IPEC-J2 increased without significant level?P>0.05?;the expression of IL-1,IFN-?.TLR4 and TNF-a was down-regulated significantly?P<0.05?,while the expression of MyD88 gene was down-regulated without significant level?P>0.05?.Above results indicated that the increased expression of CD14 enhances E.coli F18 resistance;the expression level of IL-6 and IL-12 were significantly decreased in IPEC-J2 cell?P<0.05?,while the expression level of IL-8,MIP-1? and MIP-1? were decreased without significant level?P>0.05?.2.Transcriptome analysis of duodenum between Sutai E.coli F18-resistant and-sensitive weaned piglets?1?There were 238 differentially expressed genes?DGEs?between Sutai E.coli F18-resistant and-sensitive weaned piglets,of which 112 genes were up-regulated in the resistant group.DGEs is main involved in immune pathways such as antigen processing and presentation?including TAP2?,toll like receptor signaling pathway?including TLR5,IL1??,and glycosphingolipid biosynthesis-lacto and neolacto Series?including FUT2?.?2?qPCR and Western blot showed the expression levels of FUT2,TAP2,IL1? and TLR5 were all significantly up-regulated in LPS-induced,F18ac,F18ab and K88ac-stimulated IPEC-J2,respectively.Tissue expression profile showed that FUT2 gene expressed in the liver,spleen,lung,kidney,stomach,thymus,lymph node,duodenum and jejunum tissues,especially the duodenal expression was relatively high.Further qPCR and western blot showed that the expression level of FUT2 gene in duodenum and jejunum tissues of sensitive individuals was extremely significantly higher than that of resistant group?P<0.01?.After FUT2 gene knockdown,the adhesion ability of E.coli F18ab and F18ac to IPEC-J2 cells was significantly declined?P<0.05?.Above results indicated that the decreased expression of FUT2 enhances E.coli F18 resistance.?3?Methylation analysis of FUT2 promoter showed that there were different levels of methylation in 22 CpG sites,meanwhile the methylation level of mC-6 and mC-22 had significantly negative correlation with the mRNA expression?P<0.05?,and mC-22 is located in the Spl transcription factor binding sites.EMSA test showed that nuclear extracts from duodenal tissues could interact with unmethylated FUT2 wild-type probes.When we added Spl antibody,a strong supershifted band was observed with the unmethylated wild-type probe only,which indicated that FUT2 methylation of mC-22 site residue could inhibit the ability of Sp1 binding to the FUT2 promoter and reduce the mRNA expression to enhance E.coli F18 resistance.3.Long noncoding RNA analysis of duodenum between Sutai or Meishan E.coli F18-resistant and-sensitive weaned piglets?1?Based on IncRNA sequencing and bioinformatics analysis,a total of 2056 candidate lncRNA molecules were obtained in Meishan piglets and Sutai piglets.According to the principle of p-value<0.05,there were 24 differentially expressed IncRNA between Meishan F18-sensitive and resistant individuals,of which 21 lncRNA were up-regulated in the resistant group.Besides,23 differentially expressed IncRNA were screened out between Sutai F18-sensitive and resistant individuals,of which 7 lncRNA were up-regulated in the resistant group.Target prediction showed,59 target genes existed in the upstream and downstream lncRNA were predicted based on cis mechanism in Meishan piglets,while 67 target genes in Sutai piglets.517 target genes had significant correlation with IncRNA based on trans mechanism in Meishan piglets,while 97 target genes in Sutai piglets.Go function and KEGG pathway enrichment showed,target genes were involved in NF-kappa B signaling pathway,Toll-like receptor signaling pathway,Salmonella infection,Glycosaminoglycan biosynthesis-KS,etc.?2?3 common IncRNA existed in both Meishan and Sutai piglets related to E.coli F18 infection,that is TCONS₀0183659,TCONS₀0352975,TCONS₀0053650.Combining with target prediction and function analysis,we screened out an important IncRNA—TCONS₀0183659 related to E.coli F18 resistance,and a possible target gene FUT3 was found inside the 100 kb range of TCONS₀0183659 sequence.TCONS₀0183659 belonging to long intergenic noncoding RNA,was located in pig chromosome 2,with the length of 5831 bp and two exon?5746 bp and 85 bp?.?3?qPCR test results showed that the expression level of TCONS₀0183659 was extremely significantly higher in the duodenum of resistance group than that of the sensitive group in Meishan and Sutai piglets?P<0.01?.FISH analysis showed that TCONS₀0183659 was both distributed in the nucleus and cytoplasm.Porcine intestinal epithelial cell line with TCONS₀0183659 silencing was successfully established and the interference efficiency reached 69.58%.RNA pull down and western blot verification showed that the interaction was existed between TCONS₀0183659 and Histone H4.Proteomic analysis and western blot showed that the expressions of Mxl,Mx2,IFIT2 protein were up-regulated after TCONS₀0183659 knockdown.Above results indicated that TCONS₀0183659 could regulate Mxl,Mx2,IFIT2 protein expression by interactions with Histone H4.Combining with previous studies at home and abroad,this study revealed the hereditary basis differences in regulating E.coli F18 resistance between Chinese domestic and foreign pig breeds.CD14 in the Toll-like receptor signaling pathway played an immune role in regulating the resistance to E.coli F18 in Meishan weaned piglets,while FUT2 in the glycosphingolipid biosynthesis-lacto and neolacto Series pathway was associated with the formation of E.coli F18 receptor in Sutai weaned piglets.In addition,this study systematacially revealed the regulatory mechanism of long noncoding RNA in the process of E.coli F18 resistance in weaned piglets and screened out a key IncRNA:TCONS₀0183659,meanwhile it speculated that TCONS₀0183659 could regulate Mxl,Mx2,IFIT2 expression by interactions with Histone H4.In future,we further analyzed whether the function of TCONS₀0183659 was related to Histone H4 epigenetic modification.