Screening And Expression Of The Candidate Genes Associated With ETEC F4 Receptor In Piglet

Piglets diarrhea is a widespread disease in swine industry and one of the important factors in the economic benefits of pig production. Enterotoxigenic Escherichia coli (ETEC) is the primary pathogenic bacterium leading to the diarrhea of piglet. The reason of ETEC to cause the piglet to diarrhea depend on their capability of Settlement and producing enterotoxins in the small intestine epithelial cells. F4 adhesion can combine to the piglet's small intestine epithelial cells depends on whether there is a small intestine receptor. No receptors in the pig are not the incidence of bacterial infection, for the performance of resistance, therefore, the resistance breed pigs is of great significance. So, the use of molecular biology tools to find the receptor and screening of genetic markers ETEC is an important way for breeding disease-resistant.In this paper, we use the technique of SSRs and DDRT-PCR to do experiment in Shaziling piglets and Yorkshire piglets. Make the difference research between breeds in the small intestine and spleen organizations, screening the candidate gene and genetic markers related of ETEC F4 adhesion, analysis the relational candidacy gene's expression by fluorescence quantitative PCR. The main conclusions of this study are as follows:1. The test detected the adhesion phenotype beween the intestinal epithelial cells and ETEC F4 three-antigen (ab, ac, ad) in 63 newborn piglets. The results showed that F4ab antigen was the highest rate of adhesion, Shaziling pig adhesion individual ratio was 0.727, Yorkshire pig adhesion individual ratio was 0.667. Thus, the F4ab antigen was the mainly reason cause newborn piglets suffering from dysentery. It was significantly in the two breeds of F4ad adhesion rate, the adhesion Shaziling pig individual ratio was 0.400, higher than the approximately Yorkshire was 0.242 (P <0.05).2. The 4 loci in the four locas all are highly polymorphic, heterozygosity (H) ranged from 0.6117 to 0.7500. multi-information content (PIC) reached to over 0.5749, the highest was 0.7247. The allele frequencies are significant differences in various miao-satellite seats in Chinese and foreign breeds. Generally speaking, the genetic diversity of local pigs are higher than foreign. In microsatellite loci on the SW458, AC genotype in two varieties of three serum adhesion in the form of no-adhesion, which is the choice of resistance pig provides the possibility, SW458 seats is expected as the resistance gene E.coliF4 Genetic markers.3. Through DDRT-PCR technology, a 24 primer combination of primers, we studied the different spleen structures and small intestine different adhesion phonetype between the shaziling pig and the Yorkshire pig. It detected a total of 57 different segments. Among 57 differential segments, 6 segments were not re-amplified and did not get further studied. Northern hybrid-point verification, 5 different regions showed false positive, the pilot found a total of 46 positive difference in fragments, the positive rate was 80.7% (46/57).4. This study Choose 22 different fragments to a sequencing, after BLAST homology Comparison, there were 5 sequence homology with known functional genes with high Similarity and the remaining 17 sequences, were found a lower homologous EST but did not know that the function, most of them were new EST sequence. 20.ESTs were deposited into GenBank and their accession No. were FE597044-FE597057 and FE861134-FE861141.5. In the small intestine, ATPase, Ca²⁺ transporting, plasma membrane 1 (ATP2B1) gene and Histamine receptor H1 (HRH1) gene were 93% (183/193, 420/450) homology identity. ATP2B1 is the enzyme ATP Ca2 + transit membrane 1, to control intestinal cells Na⁺ and Ca²⁺ balance, and its function is closely related to diarrhea. At the same time, ATP2B1 gene in the pig 13 q31-q32,was closely linked the F4ab receptor gene. HRH1 receptor mainly located in peripheral organizations, high expression in the inflammatory response associated with the organization and hematopoietic cells, HRH1 was so excited after Ca²⁺ cells increased, the release of vascular endothelial relaxation factor (EDRF) and PGI2, it can makes the expansion of small vessels and Permeability increase. This makes the gastrointestinal permeability increase, excessive secretion of gastric acid, resulting in diarrhea and other symptoms. With ATP2B1, HRH1 genetic identity and function, respectively them as F4ab, F4ac receptor gene is related to the candidate for the next phase of research.6.In the spleen, Pre-B-cell colony enhancing factor 1 (PBEF1) gene was 97% (351/360) homology identity. PBEF1 gene can transfer the sugar base / nicotinamide-ribose Meideng transfer enzyme, Involved the regulation and control process in cell signal transduction and cell proliferation, so it has a broad impact on the immune, metabolic and so on. May be it was the product of spleen immune reaction in response to piglets diarrhea. Comprehensive PBEF1 gene identity and function can be used as the candidate of the F4ad receptor gene related to conduct further research.7. According to the fluorescence quantitative PCR technology, we analyzed the three related gene's expression analysis, to verify the PBEF1 gene may be related to diarrhea in pigs as a candidate gene. ATP2B1 gene is expected as E.coli F4ab receptor-related candidate genes, gene and the HRH1 need further study.