The Polymorphisms And Expression Of α,β-defensins Genes In Non-specific Digestive Disorder Of New Zealand White Rabbits

With the development of large-scale rabbit industry, the rabbit’s gastrointestinal diseases, especially the non-specific digestive disorders impacted on the development of the whole rabbit farm. As a cationic antimicrobial, defensins peptide plays a very important role in the innate and acquired immune. In this experiment, the New Zealand White rabbits were selected to seek DEFB1, DEFB135and NP-5gene polymorphisms by direct sequencing. Then, the mutation sites were further genotyped to analyze the association with non-specific digestive disorder using Case-Control design by PCR-RFLP and HRM technology. According to the results and the importance of mutations, this experiment screened DEFB135, NP-5gene and tested their intetinal mRNA expression. The rabbits were induced illness by using fibre-deficient diet (9%). Lastly, they were divided into health group, mild group and severe group. DEFB135, NP-5gene mRNA expression levels were analyzed in ileum, colon, and sacculus rotundus; DEFB135, NP-5gene expression levels were analyzed in different groups, eventually found the genetic markers was associated with rabbit non-specific digestive disorder traits. Furthermore, it provided the evidence for rabbit’s molecular marker-assisted selection and breeding for disease resistance. The experiment results were as following:1By dierect sequencing the DEFBl gene exon2sequence, DEFB135gene untranslated region and translated region, NP-5gene mRNA sequence. It found that DEFB1gene had one single nucleotide polymorphisms (SNP, T>G) at-20bp from Exon2, DEFBl35gene had two SNPs (c-47T>A, c.-40C>A) in5’-UTR and one SNP (c.*42C>G) in3’-UTR, which were detected with100%linkage, NP-5gene had one SNP (r.154C>A), it was nonsynonymous.2Based on the Case-Control design, DEFB1gene and NP-5gene were genotyped by HRM technology. The results suggested that DEFBl gene T>G mutation in exon2from-20bp, T allele was the predominant allele. The allele T frequency of66%in Case group and69%in Control group, respectively. Different genotype frequencies TT(39vs45%), TG(53vs45%), GG(8%vs6%) in Case-Control groups had no significant difference (P> 0.05). The G allele OR was1.29(95%CI0.878-1.890, P=0.21), this suggested that DEFB1gene T>G mutation in exon2from-20bp, which had no significantly associated with non-specific digestive disorder in New Zealand Rabbits. NP-5gene r.154C>A, A allele in Case-Control were22%and33%, respectively. The A allele distributed significantly different between Case and Control groups, the A allele OR was0.577(95%CI0.39-0.085, P=0.0046) and AA genotype OR was0.16(95%CI0.06-0.043, P<0.0001). It indicated that the A alleles and AA genotype had a reduced effect on non-specific digestive disorders in New Zealand White rabbits. Among five genetic models, the recessive model is the most suitable for the r.154C>A mutation. The DEFB135gene c.-40C>A locus was genotyped by PCR-RFLP technology, the results showed that:A allele in the Case and Control group had no significant proportion, the A allele OR=1.11(95%CI0.85-1.46, P=0.44).3In three tissues, the expression of DEFB135gene in colon was significantly higher than that in ileum and sacculus rotundus tissues, the expression in sacculus rotundus was the lowest; in ileum and colon tissue, as the aggravation of inflammatory status increasing gradually, the mRNA expression was decreased, while in sacculus rotundus, the expression of mild group was significantly lower than that of severe group (P<0.05). In three tissues, expression of NP-5gene in sacculus rotundus was the highest and lowest expression from the colon; in colon and sacculus rotundus tissues, the expression level had a rising trend with the inflammation increase, in ileum, the expression in sereve group was lower than that of mild group, but higher than that of health group. There no significant among different health atatus in three tissues. AA genotype expression level was significantly higher than that of the CC genotype (P<0.01) and genotype CA (P<0.05) in ileal, AA genotype was significantly higher than the CC genotype (P<0.05) in sacculus tissue. In three tissues, AA had a reduced effect on non-specific digestive disorders in New Zealand White rabbits with the highest expression while the expression of CC was lowest.These results suggested that DEFB1、 DEFB135gene mutation sites might be not significantly associated with non-specific digestive disorder in New Zealand White rabbits. While NP-5gene mutation site was significantly associated with non-specific digestive disorders in New Zealand White rabbits, which provided basis theory for further research on mechanism of rabbit non-specific digestive disorders and laid the foundation for disease resistance.