| Siniperca chuatsi is one of the carnivorous,benthic,ferocious and valuable freshwater fish specializing in China.For a long time,Infectious spleen and kidney necrosis virus disease,as the first killer of Siniperca chuatsi,has brought great challenges to the aquaculture industry of Siniperca chuatsi.Therefore,it’s urgent to explore the pathogenesis and resistance mechanism of ISKNV disease in Siniperca chuatsi.In this study,the spleen tissues of Siniperca chuatsi infected with ISKNV and resistant to ISKNV on the 8th day were analyzed by transcriptome and proteome for the first time,the candidate genes and proteins for pathogenesis and resistance was screened,TRIM21,the differentially expressed gene in transcriptome was picked up,the relationship between this gene and the replication of ISKNV virus and its effect on the expression of interferon stimulating factors were preliminary studied.The results of our study will provide new ideas for the pathogenesis and resistance molecular mechanism,and also provide a valuable reference for the prevention and treatment of ISKNV disease and germplasm breeding of Siniperca chuatsi.1 Transcriptomic analysis of pathogenic and resistant mechanism in Siniperca chuatsiIn this study,the spleen of Siniperca chuatsi infected with ISKNV on the 8th day were amplified by nested PCR and observed by histopathology.In the pathogenetic Siniperca chuatsi,the specific bands of ISKNV MCP virus fragment were obtained by nested PCR and typical pathological changes of ISKNV infection were observed.There was no virus specific band in resistant Siniperca chuatsi and the histopathological changes were not obvious.Illumina NovaSeq 6000 was used to analyze the spleen tissues of pathogenesis(B),resistance(K)and control group(C)on the 8th day after ISKNV infection,differential sequencing results of Siniperca chuatsi between TB-TC group and TK-TC group showed that,the number of DEGs in TB-TC group(4667)was significantly higher than that in TK-TC group(3417).The average number of viral sequences reads in TB-TC group(502146)was 31.2 times of that in TK-TC group(16097).Functional annotation and analysis of DEGs in immune pathway in TB-TC group showed that,the down-regulated expression of CD126,CD36 and EPOR genes in Hematopoietic cell lineage pathway may inhibit the involvement of IL-6 in inflammation and affect thrombosis;the up-regulated expression of Shc and VAV genes in Natural killer cell mediated cytotoxicity pathway may inhibit the recruitment of autologous cell receptors;the up-regulated expression of NFAT gene in B cell receptor signaling pathway activates excessive inflammation,which may cause spleen injury in Siniperca chuatsi;the up-regulated expression of TACI gene in IgA producing intestinal immune network signaling pathway may induce the secretion of cytokines that inhibit the proliferation of B cells.Functional annotation and analysis of DEGs in immune pathway in TK-TC group showed that,the up-regulated expression of IL-1,CD121,CD2 and TNF genes in Hematopoietic cell lineage pathway may promote the recruitment and activation of effector cells involved in innate and adaptive immunity and antigen-antibody recognition;the down-regulated expression of TRAILR gene expression in Natural killer cell mediated cytotoxicity pathway can prevent excessive inflammation;the down-regulated expression of SHIP and FcyRIIB genes in B cell receptor signaling pathway may promote the normal activation of B cells and produce antibodies;the up-regulated gene IL-10 of IgA producing intestinal immune network signaling pathway may promote the process of IgA class conversion;In the unique FcεRI signaling pathway,IgE,FcεRly,Rac1 and TNF genes were up-regulated,while SHIP and 5-LO genes were down-regulated,which may play an antiviral role through the following processes:FcεRIy binds with IgE to activate mast cell degranulation and release downstream cytokine Rac1,Rac1 indirectly promotes TNF to exert anti-ISKNV effect,and the down-regulation of SHIP gene can also promote TNF expression.The down-regulation of 5-LO may neutralize part of inflammatory response by down regulating the expression of TNF.In summary,compared with TK-TC group,TB-TC group had more DEGs,but the tolerance to virus and the regulation ability of enriched differentially expressed genes in inflammation were worse in TB-TC group.However,TK-TC group showed stronger ability in antigen recognition,immune response and neutralization inflammation.2 Proteomic analysis of pathogenic and resistant mechanism in Siniperca chuatsiIn this study,iTRAQ was used to analyze the spleen tissues of pathogenesis(B),resistance(K)and control group(C)on the 8th day after ISKNV infection,there were 258 and 54 DEPs in IB:IC group and IK:IC group,respectively.The KEGG enrichment analysis and functional annotation of the differential expression protein in IB:IC group and IK:IC group showed that,in IB:IC group,down-regulated expression of perforin protein and up-regulated expression of Granzyme protein in natural killer cell-mediated cytotoxicity pathway may inhibit programmed cell death of target cells infected by ISKNV;the up-regulated expression of C4,C8,C3,C7 and C9 proteins in the complement and coagulation cascades pathway may be adverse immune response to the body caused by the over-activation of complement;the down-regulated expression of cofilin and Arp2/3 proteins in Fc gamma R-mediated phagocytosis pathway may induce inflammatory diseases;the down-regulated expression of RhoA,ROCK and MLC proteins in the Leukocyte transendothelial migration pathway may lead to the destruction of the cytoskeleton in this group and accelerate the infection of the virus;However,there was no immune related pathway in IK:IC group,it may be that the immunity of Siniperca chuatsi tends to be stable gradually with the extension of infection time in the resistant group after the effective removal of the virus.Correlation analysis showed that the correlation coefficients of transcriptome and proteome were 0.103 and 0.0229 respectively,which were positive correlation.There are 45 and 7 DEG/Ps in pathogenesis group and resistance group,respectively,which are involved in the metabolism of fibrinolytic enzyme,glycine,serine and threonine as well as steroid hormone biosynthesis signaling pathway.Trypsinogen in the anti-disease group may play a role in the repair process after inflammation.3 Functional Analysis of TRIM21 based on transcriptome in Siniperca chuatsiTRIM21(tripartite motif 21)gene has been studied extensively in mammals,affecting viral replication through its direct and indirect effects on the expression of interferon stimulating factors.However,the interaction between TRIM21 gene and virus in bony fish has not been studied.In order to verify the interaction between TRIM21 gene and ISKNV of Siniperca chuatsi,it was found that TRIM21 was significantly down-regulated in TK-TC group of above transcriptome database.The sequence of open reading frame was obtained from the whole genome database of Siniperca chuatsi.Its open reading frame sequence is 1841bp,which encodes 563 amino acids.The domain consists of one RING structure,two BBOX structures and one PRY-SPRY structure.Multiple sequence alignment and phylogenetic tree showed that the TRIM21 gene had the highest homology with Pseudosciaena Crocea and Epinephelus coioides.The TRIM21 gene of Siniperca chuatsi was up-regulated in spleen,head kidney,liver and intestine at 0 h,6 h,12 h,24 h,48 h and 96 h after infected with ISKNV virus,especially in spleen.The vitro experiments showed that over expression of TRIM21 could promote the replication of ISKNV MCP in MFF cells infected by ISKNV,and the expression of IRF3/7/8,IL-8,LGP2 and MDA5 was significantly inhibited,indicating that TRIM21 gene was positively correlated with ISKNV virus replication and negatively correlated with interferon-stimulated gene expression.This study provides a theoretical basis for the interaction between Siniperca chuatsi TRIM21 and ISKNV virus. |
PDF Full Text Request