| Grass carp(Ctenopharyngodon idella)is an important freshwater aquaculture fish in China.Grass carp hemorrhagic disease caused by Grass carp reovirus(GCRV)has long affected the healthy development of the grass carp aquaculture industry.Screening based on molecular markers to breed new strains of highly resistant grass carp is one of the effective ways to prevent and control this disease.The TRIM(Tripartite-motif,TRIM)gene family is conserved in various vertebrates and has been shown to be involved in various biological processes such as apoptosis,cycle regulation and immune response.TRIM-B30.2(TRIM protein containing the B30.2 domain)has important antiviral functions.In recent years,it has been shown that TRIM-B30.2 expands and replicates in large numbers in teleosts which has stimulated extensive research interest from fish antiviral experts.In this study,we firstly screened the TRIM-B30.2 in grass carp based on available genomic and transcriptomic data,and identified potential antiviral-related TRIMs,and then investigated the structure and evolutionary characteristics of antiviral-related TRIMs and the functional mechanism of anti-GCRV.The study aimed to reveal the role of TRIM gene family in the immune response process of grass carp against GCRV and enrich the theoretical content of the molecular basis of GCRV resistance,with the following main results:(1)Genome-wide identification of grass carp antiviral-related TRIM.The 42 Ci TRIMs contained in the grass carp genome were identified by Hidden Markov Model Biological Sequence Analysis software(HMMER)and protein domain structure,and their physicochemical properties,protein structure,evolutionary features and expression characteristics were analyzed,and a total of 17 TRIM-B30.2 were identified;based on two transcriptomic data during GCRV infection in grass carp,the expression characteristics of the Ci TRIM gene family under viral stimulation were revealed,and three potential antiviral Ci TRIMs were screened using weighted correlation network analysis(WGCNA)and gene expression trend analysis,namely Cibtr40,Ci TRIM103 and Ci TRIM109.(2)Structural and expression characterization of grass carp antiviral-related gene Ci TRIM103.By constructing a phylogenetic tree of TRIM-B30.2 family,we found that Ci TRIM103 was independent of the three subfamilies of btrs,hltrs and ftrs;the collinearity analysis revealed that Ci TRIM103 had large collinearity differences in the teleosts genome compared to mammals and clustered with other TRIM genes and underwent new gene duplication.Structurally,the B30.2 domain of Ci TRIM103 was found to be highly similar to the mammalian B30.2-containing TRIM5α,TRIM21 and TRIM34 proteins by protein structure comparison and molecular docking,and had a more compatible docking with the GCRV outer capsid proteins VP5 and VP7,suggesting that it has a virus recognition function.The quantitative fluorescence assay showed that Ci TRIM103 was expressed in 10 tissues,including skin,muscle,gills,brain,headkidney,heart,liver,spleen,kidney and intestine,with the highest expression in headkidney.Pierson correlation analysis further showed that GCRV proliferation and Ci TRIM103 expression were strongly correlated,namely,as GCRV replication increased,Ci TRIM103 expression also increased.(3)Preliminary analysis of the functional mechanism of Ci TRIM103 against GCRV.The subcellular localization analysis of Ci TRIM103 revealed that it was expressed in the cytoplasm.A combination of overexpression and RNA interference experiments showed that Ci TRIM103 regulates type I and type II interferon expression through the IRF3/7 signaling pathway,and also affects the RLR antiviral immune response by regulating RIG-I,MDA5 and LGP2.Under GCRV attack conditions,Ci TRIM103 expression increased significantly after MDA5 and LGP2 overexpression,indicating that RLR also regulates Ci TRIM103.Mutual regulation of Ci TRIM103 and RLR may play an important role in the anti-GCRV process of grass carp. |
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