| In the swine industry, E. coli F18is one of the most prevail and harmful enterotoxigenic Escherichia coli (ETEC), which always adhere to the small intestine epithelial cells by its pili. Moreover, the product of it, intestinotoxin, can cause piglets Diarrhea. Nowadays, despite the occurrence of the piglets Diarrhea have been reduced, to some extent, by using the means of enforcing management, improving hygienic conditions, using many antibiotics and subunit vaccine, but it did not fundamentally eliminate the incidence and prevalence of the weaning diarrhea and edema disease. In the long-term, breeding for disease resistance during the selection process is a kind of feasible and efficient method. As the research showed transporter associated antigen processing (TAP)(TAP1is one of the subunits of the TAP protein) played an important role in transporting the endogenous antigen peptide. In this study, TAP1gene polymorphisms, gene expression profiling and the methylation status of TAP1upstream sequences CpG island have been detected systematically, coincide combining with the level of some immune response indicators, analyzing and discussing the affection of the genetic variation of TAP1to immunity response and the resistance to E. coli F18. The achievement of this study will provide a basis for obtaining molecular marker and researches on regulation mechanism of TAP1gene. The main results showed as following:1. TAP1gene was analyzed by mismatch PCR-RFLP method in11pigs breeds, the results showed that one alleles and three kinds of genotypes AA, AB and BB were detected, in which Tibetan pig was only detected AA and BB genotype, and other pig breeds appeared in the genotype of AA, BB and AB. The frequency of allele B was0.44-0.95. Overall, allele B is advantage allele. The frequency of genotype BB in Large White, Landrace, Duroc and Sutai pigs is rather high, about0.906,0.826,0.736, and0.603, respectively. The results of sequence analysis showed that729bp of exon2of TAP1gene have a G/A mutation, and it did not induce the change of amino acid. The result of Chi Square test indicated that except for Tibetan and Sutai pigs, the other populations were all fit with Hardy-Weinberg equilibrium at this locus (P>0.05).2. The expression of TAP1gene in11tissues of different ages from newborn to weaning (8,18,30,35days), weaning piglets of different breeds (Sutai, Meishan, Large White), Sutai F18resistance and sensitivity weaning piglets was analyzed by real-time fluorescent quantitative PCR. The results revealed that TAP1gene in pig are widely expressed in11tissues. It was performed that the TAP1gene was relatively highly expressed in lung, duodenum, jejunum and immune tissue, such as spleen, thymus and lymph. The expression in lung and jejunum were the highest imply TAP1relate with the pathogenic bacteria in the lung and jejunum. In the different phase, there had no significant different among8,18,30age. But had a great enhance in35age, significant in Liver, thymus duodenum (P<0.05) and very significant in lung, Spleen, lymph, jejunum (P<0.01). The expression of TAP1had great increase in weaning age (35age), it initial indicate that the TAP1gene may be related to the F18E, coli resistance. The result about the TAP1expression of F18resistance and sensitivity pig showed that the expression of TAP1gene in sensitive individuals were higher than in resistant individuals generally, the ratio of sensitive individuals and resistant individuals in all tissues ranged from1.430to4.579, the ratio in Thymus, lymph nodes, jejunum, duodenum was4.579,2.822,2.512,1.892, respectively. This further indicated that TAP1gene maybe relate to the resistance of E. coli F18. In different breeds, normally the Sutai pig had a higher expression than other pigs, there were a significant different in the tissues of liver, lung, lymph nodes (P<0.05) and had great significant in the tissues of thymus, spleen, duodenum and jejunum (P<0.01). This also indicated that TAP1gene maybe relate to the resistance of E. coli F18. Last the analysis between different genotypes showed that TAP1express in all their tissues, and BB genotype have higher TAP1expression than AA and BB genotype, particularly have significant different in liver, lung, kidney, thymus, lymph nodes, duodenum and jejunum (P<0.05). Such a result implied this polymorphism had a potential to be a marker gene.3. The level of cytokine (IL-2, IL-6, IL-10, and IL-12) in different pigs (Meishan pigs, Sutai pigs and Large White) was measured with ELISA kit in this study. The results showed that the normal range was different among three pig populations, which may be associated with resistance to disease in various breeds. All the Immunity indicator among three pig populations were contrasted, which showed the level of IL-2in Large White and Meishan pigs were significantly higher than in Sutai pigs. But the situation was opposite in IL-6(P<0.05), the level of IL-10of Meishan pigs significantly higher than Sutai pigs (P<0.05). There was no significant difference among IL-12level of three pig populations. The result of immune response impact by TAP1gene exon2G729A polymorphic in piglets showed:In Large White, the BB genotype of IL-12level was significantly lower than the AA genotype (P<0.05), combined with function of IL-12and IL-12normal range of Large White, indicating that TAP1exon2polymorphic point BB genotypemay have a strong immune response.4. Three CpG islands were found in the TAP1promoter region by bioinformatics analysis. Combined with the information of promoter and transcription factor binding sites, the region from-594to-499maybe a regulation CpG island. Then methylated primer was designed on the basis of this site. The methylation statuses of CpG islands were analysed by BSP method. The results in different tissues showed that all tissues except jejunum were in demethylation statuses, and the methylation rate of the CpG islands was low, moreover, only the jejunum was in the status of methylation, which indicated the CpG islands has no effect on the TAP1expression in this tissues but jejunum. Methylation statuse was analyzed only in jejunum and duodenum of piglets including different ages (8,18,30and35days), different breeds and F18resistance and sensibility groups in consideration E. coli F18mainly existing in this two tissues. The results showed that the jejunum was in methylation and the duodenum was in demethylation statuses. However, not only the methylation degree declined by the age of pig grown, but also the methylation rate of F18resistance group was higher than the sensitive group. The methylation rate was accordance with the TAP1expression. The results suggested that the methylation of TAP1might activate its expression. The results build a technology platform for futher detection of methylation, and we need to augment the sample number to enhance our data’s scientificalness. |
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