The Association Analysis Between The ATG16L1Polymorphism And Non-specific Digestive Disorder In New Zealand Rabbits Directed

The specific enteropathies and non-specific digestive disorder (NSDD) have been diagnosed as the severest diseases that were confirmed harmful to rabbit industry. So far the etiological factor and pathogenesis of NSDD remain unclear for its diverse clinical manifestations, that is difficult to diagnosis in early stage and to control in early raising and management. The development of modern biotechnology provides a new research approach to explore the pathogenesis of NSDD in rabbit. Meanwhile the previous studies showed that NSDD was related to genetic factors, autophagy related16-like1(ATG16L1), which as the autophagy protein played an important role in immune response via autophagosome pathway.The polymorphisms of TLR4、NOD2and NLRP3have been revealed to be associated with NSDD of rabbit, while there were few studies about the association between polymorphism of ATG16L1autophagosome gene and NSDD in rabbit. In the present study, the Case-Control study was carried out in393New Zealand rabbit subjects (203cases and190controls), the genetic variation of entire coding region in ATG16L1was identified by direct sequencing and the SNP which might affect the function of ATG16L1was screened; genotyping the SNP locus by HRM and the association between the SNP and NSDD was subsequently performed; quantitative real-time RT-PCR was performed to investigate ATG16L1mRNA relative expression of ileum, colon and sacculus rotundus in fibre-deficient diet ration induced rabbit in order to analyze the effect of different severities of NSDD and genotypes in different tissues on mRNA relative expression. The results were as following:1. By sequence alignment with Ensembl, three SNPs were identified in ATG16L1coding regions, that were synonymous mutations and located in ex on1(c.33C>T),exon7 (c.777T>C) and exon15(c.1482G>A), respectively c.1482G>A was identified to have the most potential effect on function of ATG16L1undergo the prediction of protein structure domain and the analysis of using degrees of relative synonymous codon (RSCU) in this study.2. The synonymous c.1482G>A variant was further genotype analysis in Case-Control group by HRM, the statistics results showed that the allele G was the predominant allele with frequency of58.4%, meanwhile GG genotype frequency (39.5%) was also higher than the other two genotypes GA (37.9%) and AA (22.6%), and the frequencies of A allele and AA genotype were significant differences between Case and Control groups (P<0.05). The correlation analysis showed that the allele A (OR value=1.43,95%CI:1.08-1.92) and genotype AA (OR value=1.71;95%CI:1.05-2.76; P<0.05) of ATG16L1c.1482G>A could increase the risk of developing NSDD.3. Analysis of ATG16L1gene mRNA relative expression in ileum, colon and sacculus rotundus issue showed that ATG16L1gene mRNA relative expression tended to gradually increase in three tissues along with the increasing severity of NSDD in New Zealand Rabbits. In ileum, the ATG16L1gene mRNA relative expression of serious NSDD group was extremely significantly higher (P<0.01) than normal group; in colon, the ATG16L1gene mRNA relative expression of NSDD group was significantly higher (P<0.05) than normal group; in sacculus rotundus, the ATG16L1gene mRNA relative expression was not significantly difference with the severity of NSDD. The results also found that the ATG16L1gene mRNA relative expression was no significant difference in the three issues (P>0.05), the highest was in sacculus rotundus (1.4345±0.8621), the moderate was in ileum, and the lowest was in colon (1.1895±0.9446).4. Analysis the association between different genotypes and ATG16L1gene mRNA relative expression in ileum, colon and sacculus rotundus, the results showed that the genotype AA had the lowest ATG16L1gene mRNA relative expression and the genotype GG had the highest in three tissues. In ileum, the genotypes GG and GA had a significantly higher expression than genotype AA (P<0.05); while in colon, the expression of genotypes GG was significantly higher than genotype GA (P<0.05) and extremely significantly higher (P<0.01) than genotype AA, respectively; in sacculus rotundus, there was no significantly was confirmed in three genotypes (P>0.05).According to these results, it was possible to evidence that the mutation of c.1482G>A in ATG16L1gene had a significantly affectability on NSDD in New Zealand Rabbits, which could be as a gene marker to participate in marker assisted selection for NSDD. The ATG16L1gene mRNA relative expression in ileum and colon were significantly relevant to the severity of NSDD, it suggested that ATG16L1gene would be a candidate gene involved in the susceptibility to NSDD and provide a theoretical basis for the pathogenesis of NSDD which would further become the new target of non-specific digestive disorder gene therapy.