Cloning And Functional Analysis Of Genes Related To Tlr Signaling Pathway In Nile Tilapia

Tilapia is one of the most important farmed fish species in China and even the world.However,the outbreak of streptococcal disease in recent years has seriously threatened the healthy development of its aquaculture industry and caused huge economic losses.Therefore,it is of great importance to explore the immune mechanism of disease resistance in tilapia.The recognition of pathogen-associated molecular patterns(PAMPs)by pattern recognition receptors(PRRs)is a key step in initiating an innate immune response.Toll-like receptor(TLR)family is the first family of pattern recognition receptors to be discovered.The activation of TLRs and their signaling pathways have been widely concerned long by researchers.Interleukin-1 receptor associated kinases(IRAKs)and tumor necrosis factor receptor associated factor(TRAFs)family members are involved in the TLR signaling pathway.In this study,the cloning,expression and functional analysis of IRAK1,IRAK4,TRAF3,TRAF4 and TRAF6 genes were provided,which provided basic data for the immune regulation of the five genes and the anti-infective immune mechanism of tilapia.The main results were as follows: 1.Cloning and expression analysis of IRAK1 and IRAK4 genes in Nile tilapiaIn this study,the IRAK1 and IRAK4 genes of Nile tilapia were cloned and their expression characteristics were analyzed.The results showed that the DNA sequence of IRAK1(GenBank accession no.MK431853)contained 15 exons and 14 introns.The cDNA sequence was 3495 bp long,encoding 748 amino acid residues.The DNA sequence of IRAK4(GenBank accession no.MK431854)contained 10 exons and 9 introns.The cDNA sequence was 3025 bp,encoding 449 amino acid residues.Sequence analysis indicated that both IRAK1 and IRAK4 contained a death domain(DD)and a serine/threonine protein kinase catalytic domain(STKc).Amino acid sequence analysis showed that Nile tilapia IRAK1 had the highest similarity(94%)to the Maylandia zebra IRAK1,and Nile tilapia IRAK4 had the highest similarity with Haplochromis burtoni IRAK4,which was 96%.The results of qRT-PCR showed that IRAK1 and IRAK4 were expressed in all tissues examined,with the highest expression in the blood,and were expressed at different developmental stages after fertilization.After infection with S.agalactiae,the expression levels of IRAK1 and IRAK4 in five tissues of Nile tilapia(intestine,gill,spleen,kidney and blood)were significantly induced.In Nile tilapia macrophages treated with LPS,Poly I: C,S.agalactiae WC1535 and ?CPS,IRAK1 and IRAK4 gene expression were also induced.These results indicated that IRAK1 and IRAK4 genes play important roles in the immune mechanism of Nile tilapia against pathogens.2.Cloning and expression analysis of TRAF3,TRAF4 and TRAF6 genes in Nile tilapiaIn this study,TRAF3,TRAF4 and TRAF6 genes were isolated from Nile tilapia and their expression characteristics were also analyzed.Results showed that the DNA sequence of TRAF3(GenBank accession no.MK227432)contained 14 exons and 13 introns.The cDNA sequence was 2506 bp long,encoding 591 amino acid residues.There were 7 exons and 6 introns among the TRAF4 DNA sequence,and the cDNA(GenBank No.MH986338)was 4483 bp in length which encoded 470 amino acid residues.The DNA sequence of TRAF6(GenBank accession no.MK227433)contained 8 exons and 7 introns.The cDNA sequence was 3191 bp long,encoding 571 amino acid residues.SMART analysis showed that both TRAF3 and TRAF6 proteins contained a RING finger,two zinc fingers,a coiled coil region and a MATH domain.TRAF4 protein contained a RING finger,three zinc fingers,and a MATH domain.Amino acid sequence analysis showed that Nile tilapia TRAF3,TRAF4 and TRAF6 had high similarity with corresponding amino acid sequences in other species,and had the highest with Maylandia zebra(98%,99% and 98%).Nile tilapia TRAF3,TRAF4 and TRAF6 genes were expressed in all the eleven tissues,with the highest expression of TRAF3 and TRAF6 in the blood,and the lowest in the liver,while TRAF4 had the highest expression in the brain,and the lowest in the spleen and blood.TRAF3,TRAF4 and TRAF6 genes were expressed at different developmental stages after fertilization.After intraperitoneal injection of S.agalactiae into Nile tilapia,the expression of TRAF3,TRAF4 and TRAF6 were induced to different degrees in various tissues.The expression of TRAF3 and TRAF6 were significantly induced by LPS,Poly I: C and WC1535 in Nile tilapia macrophages in vitro,while did not respond to ?CPS treatment.These studies indicated that TRAF3,TRAF4 and TRAF6 genes play important roles in the immune mechanism of Nile tilapia against pathogens.3.Functional analysis of IRAK1,IRAK4 and TRAF3,TRAF4 and TRAF6 genes in Nile tilapiaIn order to investigate the functions of IRAK1,IRAK4,TRAF3,TRAF4 and TRAF6 genes and their roles in signaling pathways,eukaryotic expression vectors of the above genes were constructed and transfected into 293 T cells for dual-luciferase reporter assay,subcellular localization and Co-immunoprecipitation(Co-IP)experiments.The results showed that pcDNA3.1-IRAK1,pcDNA3.1-IRAK4,pcDNA3.1-TRAF3,pcDNA3.1-TRAF4 and pcDNA3.1-TRAF6 were stably expressed in 293 T cells,and the overexpression of these genes could significantly enhance NF-?B activity.Co-transfection of Myd88 and IRAK1 significantly up-regulated Myd88-mediated NF-?B activity,while co-transfection of Myd88 and IRAK4 had no effect on Myd88-mediated NF-?B activity.Subcellular localization revealed that IRAK1,IRAK4,TRAF3,TRAF4 and TRAF6 distributed mainly in the cytoplasm in 293 T cells.The results of Co-IP showed that IRAK1 interacted with IRAK4 and TRAF6.These studies provide basic information for clarifying the immune regulation mechanism of tilapia.