Cloning,expression And Functional Characterization Of Toll Like Receptors And Interferon Regulatory Factors In The Blunt Snout Bream(megalobrama Amblycephala)

The blunt snout bream(Megalobrama amblycephala)is one of the most economically important freshwater fishes in China.However,with the increasing of the stocking density and the deterioration of the fish culture environment,it leds to the decreased in disease resistance and the frequently of disease outbreak,especially disease which associated with Aeromonas hydrophila infection.A.hydrophila infection has seriously harmed the development of the blunt snout bream breeding and resulting in severe economic losses.In order to solve the problem of diseases in fish farming,researchers began to study and discovered the immune response and regulatory mechanism in fish.Therefore,the sequence and function of toll-like receptors(tlrs)and interferon regulatory factors(irfs)genes in blunt snout bream were analyzed to distinguish the role of tlrs and irfs in regulating the anti-bacterial immune response to provide the basic reference for the prevention and treatment of A.hydrophila infected disease in fish.The main results of this experiment are as following:1.In this study,four Matlrs genes which specifically recognize bacterial components were cloned and identified,named as Megalobrama amblycephala tlr5a(Matlr5a),Matlr5 b,Matlr9 and Matlr21.Sequence analysis showed that all MaTlrs had typical TLR protein structure,extracellular LRR(Leucine-rich repeat)domain,intracellular TIR(Toll/interleukin-1 receptor)domain and transmembrane TM(Transmembrane)domain.LRRCT domain and intracellular TIR domain were used for tertiary structures.The analysis results show that both of them exhibit more conservative structural features,and the LRRCT domain conforms to the characteristics of α-helix and β-fold tandem structure.Phylogenetic analysis showed that MaTlr5 a,MaTlr5b,MaTlr9 and MaTlr21 belonged to TLR5 subfamily,TLR7 subfamily and TLR11 subfamily respectively.The expression of Matlr5 a,Matlr5b,Matlr9 and Matlr21 genes was detected by quantitative PCR.The expression of Matlr5 a,Matlr5b,Matlr9 and Matlr21 genes was widely expressed in all detected tissues,and relatively high in immune-related tissues and lymphoid tissues.After infection with A.hydrophila,the expression of Matlr5 a,Matlr5b,Matlr9 and Matlr21 in liver,spleen,kidney,intestine and gill were up-regulated.2.Eleven M.amblycephala irfs(Mairfs)genes of blunt snout bream were cloned and identified.Using Clustal W and SMART analysis showed that all 11 proteins belonged to non-transmembrane proteins and had no signal peptide,except MaIrf1 and MaIrf2,the remaining MaIrfs contained two conservative functional domains: IRF and IRF3.N-terminal domain and C-terminal domain were used for tertiary structures.The structure of N-terminal domain in the same subfamily of Irfs factors was almost the same,showing high conservation.Phylogenetic analysis showed that 11 MaIrfs belonged to IRF1 subfamily,IRF3 subfamily,IRF4 subfamily and IRF5 subfamily respectively.The transcriptional expression of 11 Mairfs genes was detected by qPCR and expressed in all detected tissues.The expression of Mairfs genes was relatively high in immune-related tissues and lymphoid tissues.A.hydrophila infection can effectively regulate the expression of these genes in liver,spleen,kidney,intestine and gill.And these genes participate in the immune response of the body against A.hydrophila.M.amblycephala inhibitor of nuclear factor-kappa B β(MaIκB-β)soluble protein and MaIrf1 and MaIrf2 insoluble protein were obtained by prokaryotic expression.The results of antibacterial experiment showed that MaIκB-β soluble protein had no antibacterial effect.However,Western-Blot(WB)and ImmunoFluorescence(IF)experiments showed that interferon regulatory factors and inflammatory factors could participate in the antimicrobial immune response during A.hydrophila infection.3.We constructed eukaryotic expression plasmids to study the similarities and differences of the functions of four Matlrs in regulation and signal transduction.Using GFP fusion protein for subcellular localization,the results showed that MaTlr5 a,MaTlr5b,MaTlr9 and MaTlr21 proteins are located in the cytoplasm,where MaTlr5 a and MaTlr5 b are located at different positions with the early endosomes and late endosomes,while MaTlr9 and MaTlr21 is located in the same position as the early endosomes and the late endosomes.In carp EPC cells,overexpression full-length of Matlr5 a,Matlr5b,Matlr9 and Matlr21 genes could up-regulated the expression of irf3,irf7,isg15,mx1,pkr and viperin,indicated that the signal transduction of four Matlrs was similar.However,the up-regulation time and the up-regulation of gene expression level was different,the up-regulation of viperin,isg15 and irf7 was higher,while the up-regulation of pkr was smaller,the first regulates interferon pathway was Matlr5 b,while Matlr21 was the latest.Matlr5 b may play a major role in signal transduction process,while Matlr21 may exertive an assistance function.So,they may differences in the process of performing function,but they are both involved in the body’s immune response against pathogens.4.Six Mairfs promoter sequences were cloned and NF-κB loci were found in their promoter sequences.The signal transduction between Matlrs and Mairfs was studied by dual-luciferase activity assay,similar results were obtained in human 293 T cells and EPC cells of carp.The results showed that the signal transduction process was not limited by species.Matlr5 a and Matlr9 of blunt snout bream could promote the active of Mairf7 promoter.Mairf5 b can promote the activity of Mairf1 and Mairf7 promoters,while Matlr21 could not promote the activity of any Mairfs promoters.This indicates that Matlrs regulation of the Mairfs promoter is complicated.The function of overexpression of Matlr5 a,Matlr5b,Matlr9 and Matlr21 in cell apoptosis was studied by using cell apoptosis experiment.The results showed that MaTlr5 a,MaTlr9 and MaTlr21 could inhibit cell apoptosis to some extent,while MaTlr5 b promoted cell apoptosis.In summary,Matlr5 a,Matlr5b,Matlr9,Matlr21,Mairf1,Mairf2,Mairf3,Mairf4 a,Mairf4b,Mairf5,Mairf6,Mairf7,Mairf8,Mairf9 and Mairf10 can regulate immunity in the process of resisting A.hydrophila infection in the blunt snout bream,and through the interaction of the two gene families,it can protect the body from the invasion of pathogens,but their functions are different.This study laying a foundation for further exploring the regulation of TLR and IRF signaling pathways in the process of antibacterial.