| Dojo loach(Misgurnus anguillicaudatus)is a small commercial fish in our country.In the aquaculture process,diseases caused by pathogens in the water pose a serious threat to the culture of dojo loach.T cell receptor(TCR)is a specific receptor on the surface of T cells,which plays an important role in the recognition and presentation of antigens,and the activation of immune responses.So far,little has been known about the role of TCRα and TCRβ in pathogen infection of teleost fishes.In this study,dojo loach was infected by Flavobacterium columnare,Ichthyophthirius multifiliis and Saprolegnia parasitica,and the morphological changes of skin and gill tissues of dojo loach infected with the three types of pathogens were analyzed,as well as the changes of TCRα and TCRβ expression levels of TCRα and TCRβ in spleen,kidney,skin and gill,and the effects of three pathogens on the expression levels of TCR-related pathway genes of dojo loach were studied by transcriptome sequencing.Furthermore,how TCR responds to the infection of three pathogens to exert immune function was discussed.The main experimental results of this study are as follows:1.The TCRα and TCRβ genes of loach were cloned,and the gene structure and temporal expression characteristics were analyzed:(1)the results of gene cloning showed that the full-length c DNA sequences of dojo loach TCRα and TCRβ were 859 bp and 1127 bp,encoding 241 and 293 amino acids,respectively;(2)gene structure analysis found that TCRαand TCRβ have typical TCR domains,namely signal peptide,immunoglobulin region(Ig V,Ig C),connecting peptide(CPS),transmembrane region(TM)and cytoplasmic(CYT);(3)phylogenetic analysis showed that TCRα and TCRβ of dojo loach were most closely related to carp;(4)TCRα and TCRβ were widely expressed in 13 tissues including spleen,kidney and gill,and the expression level of TCRα in each tissue was significantly higher than that of TCRβ;both TCRα and TCRβ were expressed in the early developmental processes of embryos and larvae,and the expression levels of the two changed in the same trend with the development of embryos.2.Construction of an experimental model for Flavobacterium columnare infection of dojo loach:(1)the histopathological results showed that the mucous cells in the skin of dojo loach were significantly increased,the epidermal layer was thickened,and the gill lamella became shorter and wider;(2)q RT-PCR results showed that the expression levels of TCRα and TCRβ in spleen,kidney,skin and gill were significantly up-regulated and peaked at 3 days after infection,maintained at high levels at 4 days,and then gradually decreased,the expression level of TCRα is much higher than that of TCRβ;(3)KEGG enrichment showed that 23.1%(3/13)of spleen genes,90.0%(9/10)of skin genes,and 60.3%(32/53)of gill genes were significantly enriched in pathways related to signal transduction,immune system,immune diseases and infectious diseases.Analysis of genes related to TCR signaling pathway found that the upstream genes MHC IIβ and CD4 were up-regulated in the spleen,and then the signal was transmitted to the kidney to up-regulate TCRα and TCRβ,activating the early TCR signaling activators ZAP70,GADS,LCK and SLP-76,which in turn made TCR downstream Ras-MAPK pathway molecules(Ras,Raf,MEK2 a and Erk)are up-regulated by presentation.At the same time,TLR4 and TLR5 b cooperate with TCR to recognize pathogens and activate the up-regulation of transcription factor c-FOS,and activate pro-inflammatory cytokines IL-1β,IL-8 and CXCL10 Upregulation of IL-17 F in the skin promotes tissue damage in the skin and gills.3.Construction of an experimental model for Ichthyophthirius multifiliis infection of dojo loach:(1)the histopathological results showed that ich trophozoite could be observed in the skin and gills;(2)q RT-PCR results showed that the expression trends of TCRα and TCRβin the spleen,kidney,skin and gill were quite different,it is worth noting that the expression levels of TCRα and TCRβ in the spleen were significantly up-regulated 21 days after infection,and each The expression level of TCRα in tissues is higher than that of TCRβ;(3)KEGG enrichment found that in pathways related to signal transduction,immune system,immune diseases and infectious diseases,The proportion of differential genes was 6.5%(3/46)in spleen,22.2%(8/36)in kidney,23.8%(5/21)in skin and 53.7%(29/54)in gill.Analysis of genes related to TCR signaling pathway found that upstream genes MHCⅡβ and CD4 were up-regulated in the kidney,and then the signal was transmitted to the spleen to up-regulate TCRα and TCRβ,activating the early TCR signaling activators ZAP70,GADS,LCK and SLP-76 in the kidney,and then TCR up-regulation of downstream PI3K-Akt pathway genes(PI3K and Akt).At the same time,TLR4 and TLR5 b cooperate with TCR to recognize pathogens and activate the transcription factor NF-κB to up-regulate,activate the pro-inflammatory cytokine IL-1β and the anti-inflammatory cytokine TGF-β1 and other up-regulated production Inflammation,upregulation of IL-17 F promotes tissue damage in skin and gills.4.Construction of an experimental model for Saprolegnia parasitica infection of dojo loach:(1)the skin surface of dojo loach was covered with white floc mycelia,and the life cycle of mycelia was determined as Saprolegnia under microscope;(2)q RT-PCR results showed that the expression trends of TCRα and TCRβ in the spleen,kidney,skin and gill were significantly different;the expression levels of TCRα and TCRβ in the gill were significantly up-regulated at 14 d and 21 d after infection,respectively,and the expression levels of each tissue were significantly different,the expression level of TCRβ was higher than that of TCRα;(3)KEGG enrichment found that in pathways related to signal transduction,immune system,immune diseases and infectious diseases,The differential gene proportion was 23.9%(11/46)in spleen,20.8%(5/24)in kidney,10.8%(4/37)in skin and 30.8%(12/39)in gill.Analysis of genes related to the TCR signaling pathway found that the upstream molecules MHC IIβ and CD4 were down-regulated in the spleen,and then the signal was transmitted to the kidney to up-regulate TCRα and TCRβ,activating the early TCR signaling activators ZAP70,GADS and SLP-76,which in turn made TCR downstream Ras-MAPK pathway genes(Ras GRP1,Ras,MEK2 b,and Erk)are up-regulated by presentation.At the same time,TLR5 b cooperates with TCR to recognize pathogens and activate the up-regulation of transcription factor c-FOS,which activates pro-inflammatory cytokines such as IL-1β and IL-8 to produce inflammation,while the down-regulation of IL-17 F promotes tissue damage in the skin. |
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