| Intestinal disease as one of the three imprtant diseases has seriousely restricted the rapid development of modern rabbit industry. Intestinal disease was classified into specific enteropathies and non-specific digestive disorders (NSDD). The pathogen of NSDD was so complex that poor nutritional condition and sanitary status chould induce the NSDD. The individual susceptibility to NSDD has also been proposed to be partially genetically determined. NOD2gene was the first idetified gene associated with inflammatory bowel disease (IBD) in human and played an important role in immune system and microorganism recognition. In this study, firstly the mRNA relative expression of NOD2gene was detected to associate with the severity of NSDD induced by low fiber diet in New Zealand Rabbits, then the variations of NOD2gene were scanned and futher associated with NSDD. The results were labled below:1. The mRNA relative expression of NOD2gene has tissue specificity. In sacculus rotundus, the mRNA relative expression of NOD2gene was significantly higer than expression in colon and ileum (P<0.01). Meanwhile, the mRNA relative expression of NOD2gradually increased following the severity of NSDD in different tissues. In sacculus rotundus, the mRNA relative expression of NOD2gene in sever NSDD group (2.2844±0.3094) was significantly higher than mild NSDD group (1.1874±0.0857, P<0.05) and health group (1.1517±0.0897, P<0.05). Which revealed NOD2gene chould be a candidate gene for NSDD.2. NOD2gene exsisted two alternative splicings NOD2-S1and NOD2-S2. NOD2-S1was lacked of the begenning of NACHT domain, which lead to the frameshift of animo acid, as a result, only two CARDs domains have been translated. While NOD2-S2was cut off the starting of LRR domain, caused early termination of the amino acid coding, and translated two CARD domains and NACHT domain.3. The mRNA relative expression of NOD2-S2had tissue specificty. In sacculus rotundus, the mRNA relative expression of NOD2gene was higer than expression in colon amd ileum. Meanwhile, the mRNA relative expression of NOD2gradually increased following the severity of NSDD in colon and ileum. That suggested mRNA relative expression of NOD2-S2was associatd with NSDD.4. In coding region of NOD2gene discovered four synonymous mutations, located on exon2(c.513G>A), exon4(c.1818C>A), exon10(c.2961T>C) and exon11(c.3039T>C), respectivly. c.513G>A mutation located on CARD domain,while c.2961T>C and c.3039T>C sites located on LRR domain.5. PCR-RFLP mothod was used to analysis the genotype of c.1818C>A site. Allele C was predominant in case and control groups (64.49%vs70.00%). Association analysis revealed that genotype AC increased the suscepetibility of NSDD with an odds ratio (OR) value of1.79(95%confidence interval (CI):1.11-2.86, P<0.05).6. HRM (High Resolution Melting) method was used for genotyping in c.2961T>C site. The frequencies in case and control groups were significant different for allele C (52.3%vs67.7%, P<0.01) and genotype CC (36.9%vs60.7%, P<0.01). The association analysis revealed that allele T increased the suscepetibility of NSDD with an OR value of1.92(95%CI:1.27-2.70, P<0.01). Five inheritance models were used to analyse the association between genotypes and NSDD. Dominant inheritance was revealed as the best fit model for c.2961T>C site (genotype CC vs genotypes CT and TT), genotypes CT and TT could increase the suscepetibility of NSDD with an OR value of2.63(95%CI:1.67-4.17, P<0.01). Revealing that c.2961T>C site chould be a molecular marker for NSDD.7. Haplotype Analysis of c.1818C>A and c.2961T>C sites consisted into four haplotypes:32.19%of haplotype H1(CC),29.91%of haplotype H2(AC),27.61%of haplotype H3(CT) and10.29%of haplotype H4(AT). Association analysis revealed that haplotype H4enhanced the suscepetibility of NSDD (OR=6.89,95%CI:4.48-7.65, P<0.0047).8. The association between NOD2gene mRNA relative expressions and the genotypes of c.1818C>A and c.2961T>C sites identified genotype CC expression of c.2961was highly significantly than genotypes TC and TT (P<0.01), suggested the association between c.2961site and the NOD2relative expression.The study identified the mRNA relative expression of NOD2gene and NOD2-S2, c.1818C>A and c.2961T>C mutation sites were associated with the susceptibility of NSDD. The results revealed that NOD2gene chould be a candidate gene for NSDD, and the mutation sites could be used as molecular markers of NSDD in rabbit. |
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