| Non-specificity digestive disorders of rabbit are caused by the disorder of the immune response which is characterized by chronic inflammation in the gut. It is difficult to cure and its morbidity is increasingyear by year. It has become one of the three major diseases that lead the deaths of rabbit now. NLRP3(NLR family pyrin domain containing3) plays an important role in the process of immune regulation and anti-microbial function.To study the possibility that NLRP3as a candidate genes of rabbit intestinal disease resistance breeding, present study use New Zealand rabbit as the research object. First NLRP3gene exon3of genetic variation was identified by sequencing method to find NLRP3functional SNPS loci. Then HRM technology was used to evaluate the genetic polymorphism of305samples (includinghealth group102, enteritis group162, low fiber group41). Next SNPs, haploid type and correlation analysis of non-specificity digestive disorders susceptibility was done based on the case-control design. Meanwhile the relationship between expression of NLRP3to severity of non-specificity digestive disorders and its downstream cytokines IL-18expression quantity was analysed. The results are as follows:1. After comparing the NLRP3gene exon3sequencing and the NCBI reference NLRP3gene sequences in the rabbit, five SNP loci were discovered:c.456c> G, c.594G> T, c.1224G> T, c.1248G> A, c.1627G> A, all of which were synonymous mutations. Accordingto relative synonymous codon use degree (RSCU) analysis, two SNP loci (c.456G> c, c.594G> T) that may affect NLRP3function were further genotyped for association analysis based on case-control design (n=162vs n=102).2. HRM (high resolution melting) technology was used to evaluate the genetic polymorphism of the305samples. Then SNPs, haploid type and correlation analysis of non-specificity digestive disorders susceptibility was done based on the case-control design. Result showed that allele C.456G (OR=0.4537,95%confidence interval0.31320.6573, P <0.01) and allele C.594T (OR=0.6975,95%confidence interval0.4892to0.9944, P<0.05) carried a potential protective role for digestive disorders; Haplotype GT also carried a potential protective role for digestive disorders (P<0.001). 3. In SNP c.456C>G the NLRP3gene expression level of CC genotype was highly significantly higher than GC genotype and GG genotype (P<0.01). In SNP c.594G>T the NLRP3gene expression level of TT genotype was significantly higher than GG genotype (P<0.05).4. The assay of colon, ileum, the round sacculus’NLRP3and IL-18expression of mRNA from genetic differences of low-fiber enteritis groups shows that the relative transcript level of NLRP3mRNA and IL-18mRNA correlated with the severity of inflammation in the colon. NLRP3mRNA expression in severe group was significantly higher than normal group (P<0.01), significantly higher than that in mild group (P<0.05), no significant differences was found between mild group and normal group (P>0.05); IL18mRNA expression in the severe group is significantly higher than normal group (P<0.05) and there was extremely significant correlation between NLRP3and IL-18mRNA expression (P<0.01). In ileum there is negative correlation between IL-18mRNA expression and the severity of inflammation, but there is no significant difference. In sacculus rotundus the relative transcript level of NLRP3mRNA and IL-18mRNA correlated with the severity of inflammationResults showed that New Zealand Rabbits NLRP3gene exon3region c.456and c.594mutation loci polymorphism loci can be used as genetic markers. New Zealand Rabbits NLRP3genetic polymorphism was significantly associated with enteritis susceptibility. In colon NLRP3mRNA expression is correlated with the severity of inflammation and IL-18mRNA expression, NLRP3and IL-18might play a positive role in promoting colon inflammation. This study may laying the groundwork for follow-up stud yand provide theoretical support for the role of NLRP3in non-specificity digestive disorders mechanism, disease-resistant breeding research. |
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