| Octopus ocellatus,belonging to Mollusca,Cephalopoda,octocarpa and octopus,is one of the main cultivated and released species in China.In recent years,due to the invasion of diseases,Octopus ocellatus breeding industry in China has suffered huge losses.O.ocellatus belongs to invertebrate,which lacks specific immunity and can only rely on innate immune barrier to resist diseases.Toll like receptors play an important role in the recognition and clearance of pathogenic microorganisms.In order to explore the function and signal transduction of Toll like receptors in O.ocellatus,and to better understand the innate immune mechanism of O.ocellatus,we obtained the sequences of TLR2,TLR4 and TLR6 from the transcriptome of O.ocellatus,and analyzed the relative expression levels of the three TLR genes in different tissues of normal O.ocellatus and the white body and blood at different time points after Vibrio anguillarum infection by real-time quantitative PCR The relative expression in cells.Objective to construct eukaryotic expression vector and detect the localization of TLR2 and TLR6 in HeLa cells.We also studied the TLR pathway related adaptor molecules(my D88 and TRAF6)and the downstream transcription factor NF-?B family members.The results are as follows:The full-length cDNA of TLR2,TLR4 and TLR6 were 3014 bp,1717bp and 3988 bp,respectively,containing 2112 bp,891bp and 3363 bp ORF,encoding 703、296 and 1120 amino acids,respectively.TLR2,TLR4 and TLR6 proteins had an N-terminal signal peptide,several repeated LRR sequences,a conserved TIR domain and transmembrane region.TLR2,TLR4 and TLR6 shared the highest homology with Octopus sinensis,octopus bimaculoides and Octopus vulgaris,respectively.they all clustered with toll-like receptors of cephalopods.Based on the cDNA sequence of TLR gene from the transcriptome of O.ocellatus,fluorescent quantitative primers were designed for fluorescent quantitative PCR,the results showed that TLR gene was expressed in all tissues of O.ocellatus.The expression levels of the three genes were the highest in the white body of O.ocellatus,and they were also highly expressed in the blood cells.After V.anguillarum stimulation,the relative expression levels of TLRs in white body and blood cells were changed.The results showed that TLR gene could be induced to express by bacteria,and the expression levels of TLRs in white body and blood cells were significantly up-regulated at 12 h and 24 h,respectively.The results of subcellular localization showed that TLR2 and TLR6 were located in the cytoplasm and cell membrane of cells,belonging to transmembrane proteins.The results of prokaryotic expression showed that TLR2 and TLR6 proteins were successfully induced with clear bands,which could be used for subsequent experiments.MyD88 and TRAF6 cDNA sequences were obtained from the full-length transcriptome of Octopus ocellatus.The open reading frame of MyD88 was 1035 bp,encoding 344 amino acids.The structure of MyD88 was predicted to be an N-terminal death domain and a C-terminal TIR domain by smart software,without signal peptide and transmembrane region.The open reading frame of TRAF6 gene is 2633 bp,encoding 569 amino acids.The structure of TRAF6 gene is predicted by smart as one ring domain at Nterminal,two zinc finger structures,one ring ring α helix structure at C-terminal and TRAF homologous domain math.MyD88 and TRAF6 were clustered with invertebrates and cephalopods respectively.Based on the cDNA sequences of MyD88 and TRAF6 genes in transcriptome,quantitative primers were designed for fluorescent quantitative PCR.The results showed that MyD88 and TRAF6 were expressed in all tissues of O.ocellatus,and were highly expressed in white body,gill and blood cells.After V.anguillarum stimulation,the expression of MyD88 in white body and blood cells was significantly up-regulated at 12 h and 24 h,respectively,and the expression of TRAF6 in white body and blood cells was significantly up-regulated at 12 h and 24 h,and then down regulated.The open reading frame of P105 was 2337 bp,encoding 778 amino acids,including an N-terminal RHD domain,an IPT domain,multiple ankyrin sequences and a C-terminal death domain.The open reading frame of p65 is 1494 bp,encoding 497 amino acids,including an N-terminal RHD domain and a C-terminal IPT domain.Phylogenetic tree showed that P105 and p65 genes were clustered with cephalopods.Based on the obtained cDNA sequences of P105 and p65 genes,quantitative primers were designed.The results of fluorescence quantitative PCR showed that P105 and p65 were highly expressed in the white body,blood cells,skin and muscle of O.ocellatus.After V.anguillarum stimulation,the mRNA expression in white body was significantly up-regulated at 24 h.In conclusion,three TLR genes,signal pathway related adaptor molecules(MyD88 and TRAF6)and downstream transcription factors(P105 and p65)were obtained from O.ocellatus transcriptome.Their relative expression levels in normal immune organs and V.anguillarum stimulated immune organs were analyzed,and the localization of TLR2 and TLR6 in HeLa cells was identified.The results showed that these genes were involved in the immune response of O.ocellatus.This study provides a theoretical basis for studying the function of TLR gene and its role in TLR signal transduction in O.ocellatus. |
