Studie On Functional Molecular Marker Of CXCL10 Gene Associated With Mastitis Susceptibility/Resistance In Dairy Cattle

Mastitis is a common symptom of controlling a variety of factors genetic and environmental subject, which has complexity, diversity and refractory characteristics, and there are a variety of immune-related genes involved in the process of its occurrence. CXCL10 belongs to the CXC chemokine superfamily non-ELR type, involved in various autoimmune diseases. CXCL10 expresses in neutrophils, dendritic cells, monocytes, epithelial cells, endothelial cells, fibroblasts and other cells. CXCL10 mainly mediates Thl-type inflammatory response,which chemokines leukocyte chemotaxis to sites of inflammation aggregation. CXCL10 enhances Thl response process and destructes of the Th2 response process. With molecular biology, gene in the promoter, miRNA and SNP level could be screened for mastitis resistance/susceptibility functional SNPs, and explore the molecular regulation mechanism of these functional SNPs based. The main results are as follows: 1. Expression of CXCL10 geneUsing the method of plate dilution count, we cultivated standard bacterial strains(Escherichia coli and Staphylococcus aureus), and respectively calculated per unit volume in the number of viable cells(CFU). The E coli and S aureus were added at the multiplicity of infection of 5? 15? 25? 35? 45?55 or 65, infecting cells 1h. By extraction of RNA, fluorescence quantitative PCR, we found that both high density or low density bacteria infection, CXCL10 mRNA expression were increased, and its expression increased with the bacterial concentration increased. Bovine neutrophils were isolated from freshly drawn venous blood samples of healthy(n=5) and infected(n=5) mammary cows, and then extracted RNA, fluorescence quantitative PCR. We found that The infected mammary gland tissues showed a significantly higher CXCL10 mRNA expression compared with those healthy mammary cows(P <0.05). It is showed that CXCL10 is involved in the process of the occurrence of mastitis, and plays an important role in the immune process. 2. Activity analysis of core promoter and identification of functional SNPs in CXCL10 geneFirst, by using online software Genomatix, we predicted gene promoter region of CXCL10(g.-602~g. +102). Different lengths of the promoter fragment(pGL3-1183, pGL3-958, pGL3 pGL3-669 and pGL3-377) were built into the pGL3-Basic luciferase expression vector, and then transiented transfection and detected dual luciferase activity determination. We identified CXCL10 gene core promoter region is located in g.- 377~g.-1. We found that there are many transcription factor binding sites on the core promoter regions, such as OCT1, E2 F and HNF1 by carried out bioinformatics analysis. These transcription factors may in play a crucial role in CXCL10 gene transcription process. After PCR amplification and sequence alignment,we found that CXCL10 gene core promoter regions existed two complete chain of SNPs(g.-324 C>A and g.-163 C>T). And we also found that mutant promoter activity is significantly higher than the wild type(P < 0.05) through the carrier construction, transient transfection and dual luciferase activity determination. Software analysis found wild type increased HNF1 transcription factor binding sites. For SNPs(g.-324 C>A and g.-163 C>T) carried out correlation analysis of genotypes and SCS, it found that the mutant type individual has lower SCS. 3. Relevant research between CXCL10 3'UTR and bta-miR-339Using the methods of PCR and sequence alignment, we found that CXCL10 gene in 3'UTR region existed A SNP(g. +1642 A>G). By RNA hybrid and RNA 22 target prediction software we found that bta-miR-339 could be combined into CXCL10 gene 3'UTR region, and that the bta-miR-339 had obvious different binding capacities on the wild and mutant 3'UTR of CXCL10 gene. For relevance of this SNP analysis, we found that mutant(GG) has lower scores of somatic cells(SCS)(P < 0.05). Wild(AA) genotype and mutant(GG) genotype 3'UTR of 355 bp fragment respectively were connected into the PMIR-ReportTM luciferase expression vector, and then were co-transfected into 293 T cells with bta-miR-339 eukaryotic expression vector. Through the dual luciferase expression quantity analysis, we found mutant(GG) genotype significantly reduced the bta-miR-339 and CXCL10 gene 3'UTR region combining ability, and that bta-miR-339 could reduce CXCL10 gene expression. We respectively extracted total RNA with GG and AG and AA genotype individuals in breast tissue. Through the RT-PCR and RT- qPCR, we found the expression quantity of CXCL10 mRNA with AA genotype individuals is significantly lower than other genotypes(P < 0.05). For SNP g. +1642 A>G carried out correlation analysis of genotypes and SCS, it found that the AA type individual has lower SCS. In addition, SNPs found in the core promoter region and 3'UTR region were carried out correlation analysis between different haplotype and SCS in this study. We found that H1H1,H2H4 and H3H4 haplotype combination has lower SCS.In summary, this study selected CXCL10 as cow mastitis resistance candidate genes. We cloned target genes and regulatory elements of cloning by PCR and PCR-RFLP method, screened SNPs and correlation analysis. We had screened significantly the molecular markers of dairy cow mastitis. Bioinformatics is applied to forecast the promoter region and miRNA of target genes. And through the cell transfection experiment, the level of preliminary confirmed that the mechanism of the discovery of new molecular markers, for mastitis resistance marker-assisted selection to lay a theoretical foundation.