| Chinese sea perch(Lateolabrax maculates)is a Euryhaline Fishes,because of its easy breeding,disease-resistant ability and delicious meat,has become important aquaculture species in China.However,with the improvement of aquaculture and the increase of aquaculture density,the problem of L.maculates disease also occurred frequently.Therefore,it is increasingly important to research the way and method to enhance the antibacterial ability of the L.maculates by studying the anti-disease mechanism,and provide theoretical support for the treatment of diseases in the culture.Innate immunity in fish has received increasing attention in the context of aquaculture worldwide.This most ancient and widespread form of host immunity is an efficient first line of defense against invading microbial pathogens.Pattern recognition receptors(PRRs)recognize microbial components,known as pathogen-associated molecular patterns(PAMPs),and trigger signaling pathways that activate immune cells in response to infection with pathogens.As important PRRs,Toll-like receptors(TLRs)play an indispensable role in PAMP recognition.TRAF6 and IRAK4 are important component of toll-like receptor family signaling pathway.IRAK4 could be recruited to Myd88,following dissection from MyD88 in the TLR signal,that acts upstream of IRAK-1,and associate with TRAF6 to form a signal transduction complex,which then activates transcription factors,such as NF-κB,that regulate the expression of cytokine genes.Here,the TNF receptor associated factor 6(TRAF6)and Interleukin-1 receptor-associated kinase 4(IRAK4),from L.maculates were characterized and its intracellular localization and expression pattern analyzed.To check out the effects of TRAF6 overexpression in HEK293 T cells that stimulated by Vibrio harveyi and Streptococcus agalactiae.Cell cycle analysis was carried out with the help of the flow cytometry core facility.The activity of NF-κB activated by TRAF6 was detected by a Dual-luciferase reporter system.Moreover,in this paper,we have obtained the cultured cells of the L.maculatus primary kidney,which expressed the interference of LmTRAF6 and TRAF6 siRNA.A gene research platform based on L.maculatus primary kidney was initially established.The cloned full-length LmTRAF6 cDNA sequence was 2,388 bp,including an ORF of 1,707 bp encoding a peptide of 568 amino acids.The predicted isoelectric point and molecular weight of the encoded peptide were 6.01 and 64.01 kDa,respectively.SMART analysis showed that LmTRAF6 contains structural domains typical of TRAFs,as follows: a ring finger domain(residues 80–119),2 zinc fingers domain(residues 161–202,214–268),a coiled-coil region(residues 355–395),and a C-terminal TRAF homology(MATH)domain(residues 660–801).Our multiple sequence alignment analysis revealed high levels of sequence identity between LmTRAF6 and TRAF6 proteins from other fish,with the highest identity value(93%)being observed in relation to O.fasciatus TRAF6.Phylogenetic analysis provided further evidence that LmTRAF6 is closely related to the TRAF6 proteins of fish,especially those of E.coioides.Therefore,we concluded that LmTRAF6 is a typical member of the fish TRAF family and may have functions similar to those of teleost fish TRAF6 s.In this study,LmTRAF6 was mainly diffusely distributed in the cytoplasm of HEK293 T cells,a pattern similar to that observed with mammals and other fish TRAF6 in HEK-293 cells.LmTRAF6 transcripts were detected in different tissues and were most abundant in the gill,followed by the brain and intestines.To explore the immune effects of LmTRAF6 during bacterial infection,we used the bacteria V.harveyi and S.agalactiae.The spleen,head kidney,and liver were selected for this experiment due to their important roles in immunity.LmTRAF6 expression was found to be significantly upregulated in the detected tissue after V.harveyi or S.agalactiae infection,meanwhile,LmTRAF6 expression was more sensitive to V.harveyi than S.agalactiae.To further clarify the function and molecular organization of LmTRAF6,in the cell-immune model,cell apoptosis assay and fluorescence microscopy was used to confirm LmTRAF6 inhibit cell apoptosis by activating NF-κB.The results of cell apoptosis showed that LmTRAF6 could significantly inhibit the apoptosis of HEK293 T caused by V.harveyi and S.agalactiae,suggesting that LmTRAF6 inhibited apoptosis.The results of Dual-luciferase reporter system showed that LmTRAF6 could significantly activate the NF-κB reporter gene.Meanwhile,LmTRAF6 can further activate NF-κB to inhibit HEK293 T apoptosis after stimulated by V.harveyi and S.agalactiae.Different domains of TRAF6 were mutated,and the date of dual-luciferase reporter system showed RING finger mutants and ZINC finger mutants had obvious declines in luciferase activities.The effect of MATH mutant on the activation of NF-kB was not obvious.It indicates that RING finger domain or ZINC finger domain is the key function domain for LmTRAF6 to transmit signals downward.The total cDNA sequence of L.maculatus IRAK4(LmIRAK4)CDS is 729 bp,which encoding 313 amino acids with an N-terminal death domain(residues 8–104)and a central protein kinase domain(residues 185–312).The predicted isoelectric point and molecular weight of the encoded peptide were 5.65 and 34.97 kDa,respectively.Blast analysis showed that deduced amino acid sequence of LmIRAK4 share high identity with IRAK4 of other fishes.Tissues distribution analysis indicated that LmIRAK4 express in all examined tissues with various expression levels and the highest expression was detected in head-kidney.To check out LmIRAK4 functions against bacterial infection,the pathogenic bacteria Vibrio harveyi and Streptococcus agalactiae were selected and the results showed that LmIRAK4 is significant up-regulated after bacteria stimulation in the three immune tissues(head-kidney,spleen and liver).The eukaryotic expression plasmid of Lmirak4 is primarily expressed in the cytoplasm and membrane of HEK293 T cells.In order to establish a gene research platform with L.maculatu cells as the carrier,the primary cells of the head kidney of L.maculatu were cultured,and the LmTRAF6 overexpression and interference experiments were carried out.The results showed that the expression of exogenous LmTRAF6 was successful,but the amount of expression was less.It was necessary to further explore the related factors,such as promoter.Endogenous LmTRAF6 was successfully disturbed by siRNA,and the expression decreased to 30%.This experiment provides a basic platform for further research on the preliminary gene structure,expression pattern and its downstream factor in Lateolabrax maculatus. |
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