Effect Of NLRP12’s SNP And Expression Levels On Non-specific Digestive Disorder In Rabbit

As the quick development of rabbit production in large scare, digestive disorder become one of the dangerous diseases. Among them, the non-specific digestive disorder (NSDD), characterized with diarrhea symptom, is one of the high incidence diseases. At present, many measures to control the digestive disorder made effectiveness incompletely. In many previous reports, both the environmental and genetic factors are believed to determine the occurrence and development of digestive disorder. Many researches reported that NLRP12participated in the process of regulating inflammatory cytokines and diminished inflammation. In this study, direct sequencing was applied to search the genetic variation of NLRP12CDS region. In order to search the checkpoint SNP that influence the function of the NLRP12protein, we used data from PANTHER to predict the functional consequence of each of the missense mutation in NLRP12gene. For the coding SNP of interest (c.1682A>G), SSCP method was employed for genotyping analysis in539individuals including272cases and267controls. We performed the case-control study to explore the association between NLRP12polymorphisms and NSDD in New Zealand White rabbits. NLRP12mRNA expression was analyzed in different health status and genotypes in three enteric tissues. Analysis results were as following:1. The coding region of the NLRP12gene in rabbit was successfully amplified and sequenced in the study. A total of19SNPs including5non-synonymous SNPs and14synonymous were identified by direct sequencing. The five non-synonymous SNPs (c.223C>T, c.1279A>G, c.1682A>G, c.2267G>A, c.2800T>G) resulted in amino acid changes, which were p.Arg75Cys, p.Thr427A1a, p.His561Arg, p.Ser756Asn, p.Ser934A1a, respectively. Both p.Thr427Ala and p.His561Arg were located in exon3which encoded the nucleotide-binding (NBD) domain. Among the five detected missense mutations, in silico functional analysis revealed that there was difference between the risks of the five missense mutations. In order to search the key SNP responsible for NSDD in rabbit, c.1682A>G SNP was selected with the lowest subPSEC value (-5.6218) for the case-control study.2. For the c.1682A>G SNP of the NLRP12gene, SSCP method was applied to analysis the genotype of the case-control population (case:n=272, control:n=267). After statistics, gene frequencies of the allele A and allele G in the case and control group were48.53%and51.47%,54.87%and45.13%, respectively. Genotype frequencies of the GA had the highest values both in case (47.8%) and control (56.6%) group, so the genotype GA was the predominant genotype. In order to search the risk factor for NSDD, case-control association study was applied. The results revealed that the allele A was significantly protective against NSDD with an odds ratio value of0.884(95%confidence interval,0.788-0.993; P<0.05).3. NLRP12mRNA expression was analysed by real-time PCR using mRNA from three type tissues of rabbits feed with fibre-deficient diet. The three type tissues included sacculus rotundus, ileum and colon. Mean level of NLRP12mRNA expression was gradually decreased with the aggravation of NSDD, with the highest expression in healthy group and lowest expression in severe NSDD group. In one-way analysis of variance, the expression difference among healthy group and severe NSDD reached statistical significance both in ileum and sacculus rotundus (P<0.05). Among the three genotypes, NLRP12mRNA expression difference reached statistically significant between AA and GG genotype both in colon and sacculus rotundus. The highest expression level was found in AA genotype. These expression results indicated that the NLRP12participated in the regulatory process of rabbit NSDD, but the precise molecular mechanism of the process was still unknown.From the results of case-control association analysis and mRNA expression, there was association between NLRP12polymorphism and rabbit NSDD susceptivity. The association between NLRP12’s cSNP and NSDD susceptivity could provide theoretical foundation for further protein function research. Therefore, NLRP12could act as a candidate gene in rabbit breeding in order to accelerate the process of breeding.