| TLR7 subfamily, including TLR7, TLR8 and TLR9, localized in endosome. TLR7, TLR8 and TLR9 are type I transmembrane protein with extracellular region, transmembrane region and intracellular region. The extracellular leucine-rich repeats (LRRs) can recognize pathogen associated molecular patterns (PAMPs). The intracellular toll/IL-1 reporter (TIR) domain is responsible for recruit a downstream adaptor molecule MyD88 or TRIF, activating IRAK-4, activate the IRF and NF-κB, induce IFNs and proinflammatory factor expression, ultimately initiate the innate and acquired immune response against pathogen invasion.Trachinotus ovatus is an important marine culture fish in the South of China. In recent years, with the development of high-density breeding on Trachinotus ovatus, deseases often ocured due to bacterias or virus infection, caused Trachinotus ovatus death and brought huge economic losses to aquaculture industry. Therefore, cloning the Trachinotus ovatus immune-relevant genes and analyzing their immune response are important research fields with theoretical and practical significance. In this study, we cloned the full-length cDNA sequence of TLR7 subfamily members and TLR singnaling related genes from Trachinotus ovatus, then analyzed the structure and function of protein deduced from genes mentioned above by using bioinformatics software. Furthermore, the expression of genes mentioned above in normal tissues, as well as in head kidney and spleen post Poly I:C or Vibrio alginolyticus stimulation were analyzed by using qPCR. The results summarized as follows:1. The full-length cDNA of TLR7 (TrTLR7), TrTLR8 and TrTLR9 from Trachinotus ovatus were 4185 bp,3432 bp and 3756 bp, respectively.5’UTR were 99 bp,93 bp and 66 bp, respectively,3’UTR were 945 bp,267 bp and 519 bp, respectively. The open reading frame (ORF) of these genes were predicted as 3141 bp,3072 bp and 3171 bp, encoding 1046,1023 and 1056 amino acid residues, respectively. Predict protein sequences present extracellular region, transmembrane region and intracellular region, the number of their extracellular LRRs was 16,15 and 17 respectively. Intracellular region has a conservative TIR domain. TrTLR7,TrTLR8 and TrTLR9 protein shared 66.2%-80.4%,53.3%-76.3% and 53.2%-77.6% identities with that of other fish respectively, higher than that with other vertebrate. Phylogenetic trees constructed by using neighbor-joining method revealed that TrTLR7,TrTLR8 and TrTLR9 were clustered with their counterparts from other teleost fish reported previously. The results indicated that their phylogenetic relationship was closed.2. The full-length cDNA of TrMyD88, TrIRAK4, TrIRF3 and TrIRF7 from Trachinotus ovatus were 1213 bp,1606 bp,1611 bp and 2083 bp, respectively,5’UTR were 63 bp,62 bp,64 bp and 69 bp, respectively,3’UTR were 283 bp,134 bp,104 bp and 691 bp, respectively. The ORF of these genes were predicted as 867 bp,1410 bp,1431 bp and 1323bp, encoding 288,469, 476 and 440 amino acid residues, respectively.TrMyD88 contain a DD and a TIR domain.TrIRAK4 contain a DD and a S-TKc domain.TrIRF3 and TrIRF7 contain a IRF and a IRF-3 structure domains, respectively. TrMyD88, TrIRAK4, TrIRF3 and TrIRF7 protein shared 69.6%-84.7%,49.5%-77.3%, 42.8%%-82.4% and 52.0%-81.7% identities with that of other fish respectively. Higher identity than that with other vertebrate. Phylogenetic trees constructed by using neighbor-joining method revealed that TrMyD88, TrIRAK4, TrIRF3 and TrIRF7 were clustered with their counterparts from other teleost fish reported previously. The results indicated that their phylogenetic relationship was closed.3. TrTLR7 subfamily and TLR signaling related genes were constitutively expressed in all tissues tested by using qPCR. TrTLR7, TrTLR8 and TrTLR9 were mainly expressed in spleen, followed by the gills and kidneys. TrTLR7 and TrTLR8 were expressed lowest in intestine, but TrTLR9 was expressed lowest in muscle. TrMyD88 was mainly expressed in spleen, followed by the liver and gill, the lowest expression was in muscle. TrIRAK4 was mainly expressed in skin, followed by the spleen and liver, the lowest expression was in intestine. TrIRF3 and TrIRF7 were mainly expressed in spleen and liver, the lowest expression was in intestine.4. After trachinotus ovatus were stimulated by Vibrio alginolyticus, the mRNA expression of TrTLR7, TrTLR8, TrIRF3 and TrIRF7 in head kidney and spleen were up-regulated, but not significant. TrTLR9, TrMyD88 and TrIRAK4 in head kidney and spleen were up-regulated significantly (P<0.05). After Polyl:C stimulation, the mRNA expression of TrTLR7 and TrTLR8 in head kidney and spleen were up-regulated significant (P<0.05); TrTLR9 was up-regulated,but not significant. TrMyD88, TrIRAK4, TrIRF3 and TrIRF7 were up-regulated significantly (P<0.05). The results suggested that above genes strongly involved in immune responses during Vibrio alginolyticus and Polyl:C infection. |
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