| Takifugu fasciatus is a typical anadromous fish with high economic value.Current researches of T. fasciatus mainly focus on the growth,salinity adaptaion and migrant behavior. Studies on the immune related genes involved in immune response are scarcely reported. Toll-like receptors (TLRs) are a class of the most important germline-encoded pattern recognition receptors (PRRs) that discriminate and combine with the conserved pathogen-associated molecular Patterns (PAMPs) to activate the innate immune responses, and play an important role in resisting the invasion of external pathogens. However,superoxide dismutases (SODs), as the primary antioxidant metalloenzymes, play a first role in the process of preventing oxidative stress caused by excessive superoxide anion (O2-) in living organisms. In this study,we analyzed the expression patterms of TLRs and SOD genes of T. fasciatus under injection of Aeromonas hydrophila or Lipopolysaccharide(LPS), it can provide theory basis for further understand the regulator mechanism in the innate immune system of T. fasciatus. The specific results as follows:(1) Preliminary analysis of molecular characteristics and immune response of T. fasciatus TLRs geneThe full-length TLR2, TLR7 and TLR21 cDNA of T. fasciatus were cloned by using homology cloning and rapid amplification of cDNA ends (RACE). Sequence analysis revealed that the full length of TfTLR2, TfTLR7 and TfTLR21 cDNA were 3666 bp, 3359 bp and 3259 bp, respectively. And the putative length of open reading frame (ORF) were predicted as 2616 bp, 3144 bp and 2898 bp respectively, that encoded 871, 1047 and 965 amino acids respectively. The length of the 5 were 85 bp,47 bp and 104 bp respectively, and the length of 3’ UTR were 965 bp,168 bp and 257 bp, respectively. The 3’ UTR of TfTLR2 and TfTLR7 gene both possessed a polyadenylation signal sequences of AATAAA and a mRNA instability motifs ATTTA.However, the 3’ UTR of TfTLR21 gene only possessed a mRNA instability motifs ATTTA. The structural analysis revealed TfTLR2, TfTLR7 and TfTLR21 gene all contained a typical TLR domain architecture including a signal peptide, LRRs at the extracellular region,transmembrane regions and a TIR domain in the cytoplasmic region. The homology comparison revealed that consistencies of TfTLR2, TfTLR7 and TfTLR21 gene in T. fasciatus with other species were 25-98%, 54-99% and 40-99%, respectively. Takifugu rubripes have the highest homology. Phylogenic analysis indicated that TfTLR2, TfTLR7 and TfTLR21 proteins were all within fish monophyletic clade.Quantitative real-time PCR was used to quantify the expressions of TLR2, TLR7 and TLR21 genes in eigh tissues of healthy T. fasciatus. The results showed that liver,kidney, muscle, brain, spleen, gill, intestine, heart all have TLR2, TLR7 and TLR21 gene expressions. Wherein the expressions of liver, kidney and spleen were high. The time-course dynamic expressions of TfTLR2, TJTLR7 and TJTLR21 were observed through the experimental challenges with A. hydrophila or LPS, which were all significantly altered in the three distinct tissues (liver, kidney and spleen). In addition,western blot analysis also showed that the dynamic changes of TfTLR2 in liver and TfTLR7 in spleen at the protein levels were all significantly regulated by A.hydrophila or LPS challenge (P< 0.05). Moreover,the experimental results show that A. hydrophila can quickly produce immune response of T. fasciatus compared with LPS.(2) Preliminary research of SOD gene of T. fasciatusQuantitative real-time PCR was used to quantify the expressions of TJMnSOD and TfCu/ZnSOD genes in eigh tissues of healthy T. fasciatus. The results showed that TfMnSOD and TfCu/ZnSOD genes were widely expressed in all selected tissues (liver,kidney, muscle, brain, spleen, gill, intestine, heart). In addition, both TfMnSOD and TfCu/ZnSOD were highly expressed in liver, followed by heart and brain, while their expressions in rest of the tissues were significantly low. We analysed the mRNA,ezymatic activities and protein expression patterns of two TfSODs genes under injection of A. hydrophila or LPS in three distinct tissues (liver, kidney and gill) of T.fasciatus. All the results indicated that the dynamic changes of AmSODs at the mRNA,enzyme activity and protein levels were all significantly regulated by A.hydrophila or LPS challenge (P < 0.05). In addition, the experimental results show that gill might possess more sensitive immune response compared with liver and kidney. All the result indicated the siginificant role of the two TfSODs genes in innate immune response of T. fasciatus. |
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