Molecular Characterization,Expression Analysis And Functional Study Of Key Genes In TLR Signaling Pathway Of Larimichthys Crocea

Large yellow croaker,Larimichthys crocea(L.crocea),is one of the most economically important marine-culture species endemic to China.However,its wild stocks have been severely suffering from overfishing while its aquaculture species are vulnerable to various pathogenic diseases.Toll-like receptors(TLRs)recognize a variety of pathogens and are involved not only in innate immunity but also in acquired immunity.We studied the immune function of toll-like receptor 5(TLR5)and its family genes as well as key genes of TLR signaling pathways,thereby deepening the understanding of fish physiological and pathological processes,and providing basic information for the preventive treatmeant and diagnosis of diseases in marine aquaculture.We studied the immune function of L.crocea TLR5 M,TLR13,TAK1,TAB1-3,IL12 A,IL16 and IL34 from gene and protein levels.The main research contents were molecular characterization of target genes,analysis of their transcript expression,and functional studies of TAB1-3 and TAK1 proteins in TLR signaling pathway of L.crocea.The research methods and results are as follows:⒈ Molecular characterization analysis.The DNA sequence of the target genes was downloaded from the L.crocea gene library in the laboratory by secondary search.Then the coding sequence(CDS)of target genes was predicted using online softwares such as ORF finder,from which their amino acid sequences were translated.By analyzing the amino acid sequences,their functional domain structures,three-dimensional structures,multiple sequence alignment and evolutionary relationships with homologous genes were obtained.We found the functional domain structure and special motifs of the target genes were conserved with their homologous genes in other species,which indirectly proved that these genes have immune function.⒉ Transcript expression analysis.CDS were used to predict the primers which were necessary to measure the transcript levels by quantitative real-time PCR(qRT-PCR).The cDNA of each samples,the template for qRT-PCR,was obtained by reverse transcription of the totol RNA extracted from healthy L.crocea tissues(spleen,liver,kidney,heart,muscle,intestines,gill,skin,brain and fins)and tissues(spleen,liver and kideny)from L.crocea intraperitoneally injected with PBS or pathogen(Vibrio or poly I:C)according to the time gradient: 0h,6h,12 h,24h,48 h,72h,and 96 h.The immune tissues of healthy L.crocea were more likely to highly express the target genes,and pathogen infection could cause the up-regulation of mRNA level of the target genes,which proved that the target genes play roles in the immune system of L.crocea.⒊ Functional study of proteins in the pathway signal transduction.After the overexpression of L.crocea TAB1,TAB2,TAB3 and TAK1(lcTAB1,lcTAB2,lcTAB3,and lcTAK1),the immunofluorescence(IMC)staining was performed to determine whether these genes could be expressed successfully,understand the subcellular localization of their proteins,and detect the protein level of downstream NF-κB.Meanwhile,the total protein was extracted,and the effect of LPS on lcTAB1-3 and lcTAK1 protein levels as well as the effects of lcTAB1-3 and lcTAK1 overexpression on NF-κB protein levels were detected by Western blot.Finally,the co-immunoprecipitation(Co-IP)assay was performed to understand the interaction between lcTAB1-3 and lcTAK1.The results of subcellular localization showed that lcTAK1 and lcTAB1 proteins were mainly expressed in cytosol,and lcTAB2 and lcTAB3 were localized in endosomes.The results of IMC staining and Western blot showed that when lcTAB1-3 and lcTAK1 were co-overexpressed,the protein level of downstream NF-κB was significantly up-regulated,which indicated that lcTAB1-3 interact with lcTAK1 and the signal transduction of the pathway can be affected by their interaction.Co-IP assay further demonstrated that TAB1-3 can interact with TAK1 directly.In conclusion,the molecular characteristics of TLR5,TLR13,TAK1,TAB1-3,IL12 A,IL16 and IL34 in L.crocea indicated indirectly that they have similar immune functions with homologous genes in mammals.The transcript levels of the target genes in healthy L.crocea and L.crocea after challenge were detected quantitatively.The results showed that these genes were tend to be highly expressed in immune organs such as spleen,liver and kidney.Moreover,the transcriptional level of all genes were up-regulated after immune stimulation,which further proved that these genes play a role in the immune system of L.crocea.The study of the function of lcTAB1-3 and lcTAK1 proteins in TLR signaling pathway showed that the binding of lcTAB1-3 to TAK1 affects the signal transduction of the pathway,and ultimately leads to the activation of NF-κB.Our studies identified the immune functions of these key genes in the TLR pathway of L.crocea,and preliminarily determined the function of lcTAB1-3 and lcTAK1 in the TLR signaling pathway.