Transcriptome Analysis Of Immune Tissue And Immune Cells And Study Of Immune Regulation After Infection Of Tongue Sole With Different Pathogens

Cynoglossus semilaevis(half-smooth tongue sole)is easily infected by bacteria in the breeding process,which will lead to the death of many fish and bring great difficulties to its breeding.Therefore,it is of great significance to study the immune response to different bacteria in half-smooth tongue sole.In this study,differentially expressed genes in the liver,spleen,head kidney and PBMC were analyzed in half-smooth tongue sole after injected with Vibrio anguillarum,Edwardsiella trada and Streptococcus agalactiae,respectively.In addition,the effects of target genes mediated by miRNA in response to the stimulation of bacteria on some immune genes were also studied.The main research results are as follows:1.Analysis of differentially expressed genes(DEGs)of half-smooth tongue sole infected with V.anguillarum,E.tarda and S.agalactiae:(1)A total of 148,552 and 962 DEGs were identified in the liver,spleen and head kidney of half-smooth tongue sole infected with V.anguillarum.(2)A total of 361,875 and 962 DEGs were identified in the liver,spleen and head kidney of half-smooth tongue sole infected with E.trada.(3)A total of 307,637 and 1027 DEGs were identified in the liver,spleen and head kidney of half-smooth tongue sole infected with S.agalactiae.(4)Among these DEGs,12 common DEGs,they play a role in DNA damage and repair,inflammatory response2.GO annotations of specific DEGs of half-smooth tongue sole injected with three bactiria:(1)A total of 14,91 and 115 specifically DEGs in the liver,spleen and head kidney after V.anguillarum infection,respectively.(2)A total of 91,138 and 149 specifically DEGs in liver,spleen and head kidney after E.trada infection,respectively.(3)A total of 34,118 and 281 genes specifically DEGs in the liver,spleen and head kidney after S.agalactiae infection,respectively.(4)The more specific GO term in V.anguillarum infection group was related to chemotaxis,bioregulation,activities of various enzymes;The specific GO term in E.trada infection group was related to the negative regulation of metabolic process and organic metabolism.The specific GO term in the head kidney is mostly associated with receptor/ion binding.The more specific GO term in S.agalactiae infection group was related to signal transduction and metabolism.3.KEGG pathway analysis of DEGs of half-smooth tongue sole injected with three bactiria:(1)The expression of some metabolism-related genes in liver decreased,and more down-regulated metabolism-related genes were found in E.tarda and S.agalactis infection groups.More immune-related signal pathways were activated in the liver of V.anguillarum infection group,protein synthesis signal pathway was up-regulated in E.tarda infection group,and protein synthesis and carbohydrate metabolism-related signal pathways were down-regulated.(2)The immune signal pathways in the spleen were activated to exert the host defense function.In addition,the signal pathways related to lipid metabolism and cellular signal transduction were activated in V.anguillarum and S.agalactis infection group.The signal pathways related to lipid and glucose metabolism were activated in E.tarda infection group.(3)The expression of metabolism-related genes in head kidney decreased,there were more specific immune-related signal pathways in V.anguillarum infection group,and the genes in protein decomposition-related signal pathway were down-regulated in E.tarda infection group.The endocrine-related signal pathways were changed in S.agalactis infection group4.PBMC transcriptome analysis in half-smooth tongue sole with three bacteria injection:(1)A total of 453,585,547 DEGs were identified in PBMC after infection with V.anguillarum E.trada and S.agalactiae.Sixty genes were significantly differentially expressed in PBMC after V.anguillarum,E.tarda and S.agalactiae infection,respectively.(2)There are three specific DEGs co-enriched GO term in V.anguillarum,E.tarda and S.agalactis infection group: response stimulation,cell periphery and protoplast membrane.The specific GO term in V.anguillarum infection group was mostly related to ion transport and metabolic activity,the specific GO term in E.tarda infection group was mostly related to carbon-water binding and substance metabolism,and the specific GO term in S.agalactis infection group was mostly related to ion binding and phosphorylation.(3)There are three common signal pathways in the signal pathways of DEGs enrichment:the intestinal immune network that produces Ig A,the cytokine-cytokine receptor interaction signal pathway and the cell adhesion molecule signal pathway.In addition,metabolism-related signaling pathways were activated in V.anguillarum infection group,and PPAR,arginine and proline metabolic signal pathways were activated in E.tarda infection group.TLR and steroid hormone synthesis signal pathways were activated in S.agalactis infection group.5.qRT-PCR and western blot confirmed that Cse-miR-727 could silence CsC6 and inhibit the translated CsC6 protein.The expression of complement components C7,C8?,C8?,C8?and C9 in the membrane attack complex was significantly changed after injected with Cse-miR-727 agomirs and inhibitors,and showed an opposite expression trend.In addition,the expression levels of some key genes in the immune signaling pathway,such as MAPKs,NF-?B,AP-1,IL-1?,IL6 and TNF?,were also significantly altered.These evidence suggest that Cse-miR-727 may affect the assembly of the membrane attack complex and other immune signaling pathways through CsC6 mediation.6.A new miRNA,Cse-miR-33b-5p,was found,and IL-1? was proved to be its target gene by dual luciferase reporting assay.The expression of some immune genes(IL-1R,My D88,TRAF6,IRAK1,IL6,TNF? and IL8)changed after injected with Cse-miR-33b-5p agomirs and inhibitors and showed an opposite expression trend.In this study,we analyzed the similarities and differences of DEGs in the liver,spleen,head kidney and PBMC after V.anguillarum,E.tarda and S.agalactiae infection,and further analyzed their functions in the GO terms and KEGG pathways.Common genes were significantly differentially expressed in different tissues,and different genes responded to stimulation in the same signaling pathway in half-smooth tongue sole after different bacteria infection.This study also proves that Cse-miR-727 can mediate CsC6 to affect the complement components of the complement membrane enslavement complex and other genes of immune signaling pathway in half smooth tongue sole.Cse-miR-33b-5p can mediate IL-1? to affect the expression of some immune genes.