| Porcine post-weaning diarrhea (PWD) and porcine edema disease (ED) are two of the worst diseases resulting in tremendous damage and loss to the pig industry. Enterotoxigenic Escherichia coli (ETEC) is the main pathogen causing PWD, while Verotoxigenic Escherichia coli (VTEC) is related to ED. It has been reported that the (1,2) fucosyltransferase1gene (FUT1) is linked to the receptor of E.coli F18and can be considered as the candidate gene for controlling the adhesion of E.coli F18. An M307G/A point mutation occurs in FUT1, and the G allele is phenotypically dominant over the A allele. Pigs of genotype AA are resistant to E.coli F18, and pigs of genotypes GG and AG are sensitive to E.coli F18. Based on this principle, the researchers initiated E.coli F18-resistant breeding in foreign pigs. However, many Chinese scientists discovered an extremely skewed distribution in Chinese domestic pig breeds. Except for GG genotype, all Chinese pig breeds don’t carry the AA and AG genotype. This clearly suggests that the molecular mechanism between foreign and Chinese native pig breeds is different and the FUT1genetic marker, although suitable for foreign pig breeds, does not work well for Chinese domestic breeds.In previous studies, we identified a few FUT1AG animals in a Sutai pig population. After several years of continuous selection and breeding, two pig resource populations were established with one carrying the E.coli F18-resistant AA genotype and the other harboring E.coli F18-sensitive AG or GG genotypes. Simultaneously, we also constructed a type V secretion system to express E.coli F18adhesin. The display of functional adhesin through the type V secretion system was combined with receptor binding experiments to further analyze and verify the resistance/sensitivity to the E.coli F18strain among these pig resource populations. This study utilized the established E.coli F18-resistant (genotype AA) and-sensitive (genotypes AG and GG) pig resource populations to analyze and screen for candidate pig genes and differential proteins conferring E.coli F18resistance using gene expression profiling microarrays proteomics and bio informatics techniques.The identification of relevant genes and differential proteins may shed light on the genetic basis of E.coli F18-resistance in Chinese domestic pig breeds, and it could solve the crucial scientific issues in the breeding of E.coli F18-resistant lines for domestic Chinese pig breeds.The main results were as follows:1. The differential genes were identified by microarray screening between E.coli F18-resistant and-sensitive groups.(1) Using a two-fold change minimum threshold, we found13differentially expressed genes, with6up-regulated and7down-regulated in the pairs of GG and AA animals. And there were6genes had a fold-change>2in the pairs of AG and AA group with4up-regulated and2down-regulated. The FUT1gene was found to be differentially expressed between E.coli F18-sensitive (AG) and-resistant (AA) animals.(2) Our Gene Ontology (GO) analysis revealed that the differential genes with known functions are involved in a plethora of processes including immune responses, extracellular modification (such as glycosylation), cell adherence, signal transduction, transcriptional regulation, and metabolism. Subsequently, according to the KEGG database and the referances, we focused on the following two kinds of pathways:immune-related pathways paticipating in resistance to E.coli F18and the Glycosphingolipid biosynthesis-globo series related to the formation of E.coli F18receptor.(3) The expression validation results of7genes (SLA-1, SLA-3, ST3GAL1, A, FUT1, TAP1and SLA-DQA gene) between resistant and sensitive pairs to E.coli F18duodenal tissues matched the results of the microarrays, which showed that the microarray results was reliable.2. The relationship between the expressions of differential genes SLA-1and SLA-3and E.coli F18infection in post-weaning pigs was detected here.(1) The expression profiles showed that the expression levels of SLA-1and SLA-3measured by3different inner genes were found to be almost the same. SLA-1and SLA-3transcripts were expressed in all tissues. SLA-1was expressed at relatively high levels in lung, immune tissues such as spleen, thymus, lymph node and tissues of the digestive system (stomach, duodenum and jejunum). However, SLA-3was expressed at relatively low levels in piglet heart, liver, spleen, kidney, stomach, muscle, thymus, duodenum and jejunum compared to its expression in lung and lymphoid tissues.(2) The difference of SLA-1and SLA-3expression between E. coli F18-resistant and-sensitive groups was not significant, both genes expressed comparatively higher in the resistant group in the tissues including spleen, lung, stomach, thymus, lymph node, jejunum and duodenum.(3) The linear correlation result showed that a weak positive correlation was observed in the tissues of spleen, lung, stomach, thymus, lymph node duodenum and jejunum analyzed between the expression of SLA-1and SLA-3and disease resistance. Furthermore, on the whole, SLA-3was expressed at higher levels than SLA-1in the resistant group. However, in the sensitive group, a weak negative correlation was observed in lymph node, a relatively strong positive correlation in the stomach, thymus, duodenum and jejunum, and a weak positive correlation in the spleen and lung.(4) Gene ontology and pathway analysis was used to determine the potential SLA-1and SLA-3interactions. Both SLA-1and SLA-3can be included in37potential gene ontology biological processes, mainly relating to antigen processing and presentation, and regulation of immune responses. Results showed potential participation in nine pathways, five of which were related to immune function. Such information regarding these genes represents the basis for further study.3. The expression analysis of key genes belong to Glycosphingolipid biosynthesis-globo series in E.coli F18-resitant and-sensitive post-weaning pigs has been detected here, especially in the tissues of intestinal tract.(1) The expression profiles showed that the expression levels of FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1and NAGA were found to be almost the same in11tissues. On the whole, all the7genes expressed at relatively high level in liver, lung, kidney and stomach, followed by duodenum and jejunum. The expression was lower in immune tissues such as spleen, thymus, lymph node, and they expressed lowest in heart and muscle.(2) The expression result of those7pathway genes between E. coli F18-resistant and-sensitive groups in the tissues of intestinal tract was compared here. It indicated that in jejunum FUT1and NAGA genes expressed comparatively higher in the resistant group. Genes FUT2, ST3GAL1, HEXA, HEXB and B3GALNT1all expressed higher in E. coli F18-sensitive groups, in which ST3GAL1gene had a fold-change>2. While in the tissue of duodenum, except for the FUT1gene, other6genes expressed higher in the E. coli F18-sensitive group also with FUT2〠HEXA and NAGA genes had a fold-change>2.(3) The linear correlation among7genes in jejunum and duodenum tissues in E. coli F18-resistant group has been analysed in this research. The result showed that in jejunum in E. coli F18-resistant group there was a weak negative correlation observed in the genes of FUT1and HEXA, HEXB, NAGA, genes between FUT2and ST3GAL1, and genes of ST3GAL1and HEXA, HEXB, B3GALNT1. The correlation among other genes all showed a weak positive relation. In addition, in the tissue of duodenum in E. coli F18-resistant group a weak negative correlation was detected in the genes of FUT1and HEXA, ST3GAL1, NAGA, genes between FUT2and ST3GAL1, and genes of ST3GAL1and HEXA, HEXB, B3GALNT1. Also, the correlation among other genes all showed a weak positive relation.(4) The linear correlation among7genes in jejunum and duodenum tissues in E. coli F18-sensitive group has been studied in this research. The result showed that in jejunum in E. coli F18-sensitive group there was a weak negative correlation observed in the genes of NAGA and FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1, as well as genes of FUT1and FUT2, HEXB, B3GALNT1. The linear correlation between genes FUT2and B3GALNT1was significant (P<0.05). The correlation among other genes all showed a weak positive relation. Besides, in the tissue of duodenum in E. coli F18-resistant group a weak negative correlation was only detected in the genes of NAGA and FUT1, FUT2. And there was a strong positive relation in the genes of FUT1and FUT2, HEXB, genes among FUT2and HEXB, B3GALNT1, and genes between HEXB and B3GALNT1(P>0.05).4. The differential proteins were identified by differential proteomics technology between E.coli F18-resistant and-sensitive groups.(1) A total of20differential protein spots showed a significant change in expression among which10spots were highly expressed in the E. coli F18-resistant group and10different spots were highly expressed in the E. coli F18-susceptible group(2) ESI-MS/MS analysis was carried out for20differentially expressed proteins identified by comparison of the two groups and determinations of peptide fingerprint spectra were completed for16protein spots. According to the parameters of spectra, target proteins were identified by searching homologous proteins and peptides in the NCBInr database.A total of12proteins were identified among the16differential protein spots. Among these, two groups of spots (2287,2589,2484and2074,1967,1912) represented actin alpha2(ACTA2) and albumin (ALB) respectively. Furthermore,10significant differential proteins represented four different proteins in the E. coli F18-resistant group:upregulated transferrin (TF), similar to collapsin response mediator protein-2A (LOC100151886), ribosomal protein SA (RPSA), similar to AGAP005293-PA (LOC100153507) and six proteins in the E. coli F18-suscepitable group:upregulated vinculin (VCL), aconitase2(ACO2), actin, alpha cardiac muscle1(ACTCl), actin beta (ACTB), heat shock protein27kDa (HSP27) and smooth muscle protein22-alpha (SM22A).(3) GO analysis was used to identify genes with known functions including muscle contraction, cell surface protein localization, response to extracellular stimulus and activation of MAPKK. Pathways of differential protein-corresponding genes identified in the KEGG database included regulation of the actin cytoskeleton, adherens junction, leukocyte transendothelial migration and focal adhesion.(4) Network diagrams of interactions between differential proteins were constructed based on the KEGG database information by combining proteins in all significant pathways in order to identify the correlation of target proteins. Due to the small number of differential proteins identified in this study and limitations in the information in the database, the network diagram included only five differential proteins (others were connecting proteins):AC02, LOC100151886, ACTC1, VCL and HSP27. These five proteins were also the downstream of the network and of particular importance in this study. Combined with the function analysis of differential proteins, ACTB, VCL, Transferrin and HSP are the important protains related to E.coli F18infection. |
