Identification Of IL-1β And TLR4 And Effect Of Bacillus Subtilis On Intestine Immunity In Chinese Soft-shelled Turtle Pelodiscus Sinensis

Chinese soft-shelled turtle(Pelodiscus sinensis) is semi-aquatic reptile, as a ectothermic amniotes, it provide the key link between ectothermic anamniotic fishes and amphibians, and endothermic amniotic birds and mammals and have a high scientific value for system biology and developmental biology. The fresh water turtle is known as health-promoting food in Asian countries such as China, Japan, and Korea. Concurrent with recent large-scale farming of Chinese soft-shelled turtle is the emergence of a number of infectious diseases. However, little information is available in turtle immunity or immune responses in infection and this result loss control of the diseases and abuse of antibiotics, In the study, we aim to present gene sequence and function characterization of IL-1β and Toll-like receptor 4 from Chinese soft-shelled turtle and evaluate the effect of dietary supplementation of probiotic on performance, immune gene expression and intestinal microflora of Chinese soft-shelled turtle.1. Identification and function characterization of Interleukin-ipInterleukin-1β (IL-1β) belongs to IL-1F family, as an inflammatory cytokine, it is induced in tissue injury and pathogen infection and plays an important role in inflammatory response of vertebrates. In this study, IL-1β gene (tIL-1β) from Chinese soft-shelled turtle (Pelodiscus sinensis) was cloned and charcterized, which is first time for reptile. The full length cDNA of tIL-1β consists of 1529 bp nucleotide sequence, contains an ORF of 831 bp. Under bacterial infection, tIL-1β mRNA expression level increased significantly, however, GC content of tIL-1β in coding sequence and 3’-UTR is considerably higher than other IL-1βs. As others non-mammal vertebrate IL-1β, tIL-1β lacks typical IL-1β converting enzyme (ICE) cut site, but could be cleaved by turtle caspase-1 (tcaspase-1). By mutation of potential cleavage site, D130 was identified as ICE site in tIL-1β. To examine the activity of this mature peptide, truncted tIL-1β was expressed using baculovirus system, and it was used to stimulate the ioslated peripheral blood monocytes and macrophages. The results suggested that tIL-1 P expressed is bioactive in immune defense and inflammatory responses.2. Characterization and functional analysis of Toll-like receptor 4Toll-like receptors (TLRs) are pivot molecule for triggering the innate immune responses. Among them, TLR4 is most well characterized member; it is responsible for LPS recognition in mammals. Compared to mammals, lower vertebrate are resistance to LPS, it raised whether existence of functional TLR4 gene in these vertebrates. In this study, the toll-like receptor 4 gene from Chinese soft-shelled turtle(Pelodiscus sinensis) (tTLR4) was identified and characterized, representing the first TLR4 report in retile species. In silico analysis reveal that tTLR4 has same structure with known TLR4, investigation of its expression pattern indicated that it is inducible after bacterial infection. PBMo stimulated with LPS resulted in inflammatory related gene expression concluded that turtle could recognize LPS and active immunity system. To figure out whether tTLR4 is involving LPS recognition in turtle, tTLR4 stable expressed HEK 293 cell was constructed and dual luciferase assay was used to examine the NF-κB/IFN-β promoter activity. Result indicated that NF-κB promoter was active with assistance of turtle MD2 and CD 14, while INF-β was not after LPS stimulation. These studies provided the evidence of functional TLR4 in reptile.3. Effect of dietary supplementation of probiotic on performance, intestinal microflora and immune gene expressionThis experiment was conducted to study the effect of dietary supplementation of probiotic on performance, and intestinal microflora of Chinese soft-shelled turtle. De novo assembled transcrpt from peripheral blood transcrptome was used for Toll-like receptor search. Healthy Chinese soft-shelled turtle (6000 pieces with average weight of 5g) were selected and randomly divided into two groups:control and treatment with three replicates each. The control was fed on normal basal diet whereas treatment was fed on basal diet supplemented with 1×108 cfu.g-1 Bacillus subtilis preparation. The results showed that the final mean weight of treatment (265.84±3.48 g) was significantly higher (P< 0.05) than that of control (240.45 ± 4.32 g). There was significant difference (P< 0.05) in FCR and DG were obtained in probiotics fed group as compared to control. The activties of sucrase, maltase, amylase, lipase and ATPase of treatment were significantly higher (P< 0.05) than that of control. Pyrosequencing showed that the dietary supplementation of probiotic Bacillus strain decreased microbial diversity. Phylum Firmicutes was dominant in both groups. Compared with the control, Firmicutes was increased, while others were decreased in treatment group. In addiotion, it was found that lots of inflammatory gene and TLR gens were down-regulated. Pre-treatment of PBMo with Bacillus subtilis showed anti-inflammatory effect on pathogen induced inflammatory response. The results indicated that dietary supplementation of probiotic could improve growth performance, digestibility and intestinal microflora in Chinese soft-shelled turtle.