Studies On GPI1Screening And Its Function On Piglet Diarrhea

33Piglets were relative cultivars and from Shaziling breed conservation farm and30Yorkshire Piglets which from Hunan Agriculture University were used in the experiment, all of them are only a week old. After slaughtering, we collected the tissue samples of liver and small intestine as soon as possible so that we can extract DNA and RNA. To identify F4receptor adhesion phenotype, we should collect the jejunum organization, which was used to manufacture Brush Border Membrane.In this experiment, we used aCGH to screen the CNVs related with F4receptor. Especially for ETEC F4ad receptor, we obtained some related sequence of this gene and predicted protein physicochemical properties, structure and function of the gene by bioinformatics software and tools. Furthermore, we analyze the GPI1gene quantitatively so that we had figured out the expression regular of this gene. To find some effective genetic markers and make it used in breeding practice, we take association analysis between two gene locus of SNP and adhesion characteristics. The article has got the results as follows:1. The position of the related CNVs of F4ab receptor, ac receptor and ad receptor found by screening the related CNVs of F4receptor in the whole gene of pig. At the same time, we have clarified the origin of CNVs produce. In these, F4ab receptor and F4ac respectively contain11and48CNVs, but F4ad receptor contaid67CNVs;2. By further analyzing the CNVs that we have got, we have found the related gene of CNVs. We also discovered that the related gene of glucose metabolism, SLC gene-family, ZNF gene-family, KIF gene-family. Toll like receptor5gene and U6gene appear frequently. All of these genes are closely related to animal disease and immunity, especially for GPI1. It's very likely that GPI1is the candidate gene of ETEC F4ad receptor.3. By cloning and sequencing for GPI1, we've got1515bp sequence of this gene. Meanwhile, we took BLAST analysis between this sequence and other sequences in GenBank. As the consequence of the analysis, GPI1of pig and human are high homology, and the consistency is more than90%.4. The GPI1gene sequence was submitted to the GenBank, and the submit accession number is JX125039. 5. By the prediction of amino acid of GPI1gene, GPI1consists of232amino acids, its relative molecular mass is24964.5, its theoretical isoelectric point is9.44. In the amino acid composition of GPI1, the leucine content of19.8%which is higher than the other amino acid content of8.2%-19.4%. its instability parameter is39.82, belongs to the stable protein. The half life of the protein period is30hours. The fat coefficient of the protein is122.80and the average hydrophobic degree is0.512. This amino acid sequence doesn't contain signal peptide area. We took the transmembrane domain analysis with TMHMM program, and the result show us that this protein is hydrophobic protein, it coincide with that GPI1as the basic characteristic of the F4ad receptor. There are3transmembrane domain among the232amino acids, and it coincide with the characteristic that GPI1is the important intracellular signal transduction. SOPMA program predict the secondary structure as follows:a-helical is50.86%; β-turn is4.74%; extend chain is10.78%; random coil is33.62%.6. GPI1in the small intestine without adhesion is higher than GPI1in the small intestine with adhesion. And in the same types of adhesion, expression level of GPI1of piglets from Shaziling is more than Yorkshire's. The expression levels of GPI1between two kinds of adhesive type and two two varieties are significant differences(P<0.05). In different species, the expression level of GPI1in the small intestine without adhesion is higher than others, too. And there is significant difference between adhesion and inadhension(P<0.05).The expression level of different organization in two speices has a significant difference(P<0.05).The expression level of GPI1in liver is higher than in small intestine.7. We predicted SNP and endonuclease by using bioinformatics. At the same time, we took verification with PCR-RFLP so that we have found that there is a C→T mutation at25bp of exon3and there is a G→C mutation at211bp of exon5. When taking association with SNP of two exons, we've found that the SNP of two exons is related to the adhesion characteristics, and it can be treated as diarrhea resistance of potential genetic marker.