Cloning,expression And Functional Study Of TCR Signaling Pathway Related Genes In Hybrid Snakehead(Channa Maculata ♀×Channa Argus ♂)

Immunocompetent T cells,mainly generated by the activation of T cells accompanied by a series of complex processes of TCR signaling pathway activation,are critical to the adaptive immunity of mammals.However,current researches on TCR signaling pathway in fish are quite limited.The initiation of TCR signaling pathway depends on the transduction of extracellular activation signals into intracellular by membrane TCR co-receptors’(such as CD4/8,CD3 and CD45)recognition of antigen.Actually,TCR itself has no inherent enzyme activity,but relies on the interaction of a series of protein tyrosine kinases(such as LCK,PKCθ,ZAP70)and other downstream signaling molecules to initiate subordinate signal transduction.Hybrid snakehead(Channa maculata ♀ × Channa argus ♂)is a feature economic species in China,whose culture has been severely damaged by “sarcoidosis” and “sarcoidosis-like” caused by Nocardia seriolae and Aeromonas schubertii.Massive experiments have shown that hybrid snakehead can generate acquired immunity after pathogenic bacteria infection,but the specific immune response mechanism has not yet been elucidated.In this study,we cloned six TCR-related genes(CmaLCK,CmaZAP-70,CmaGRB2,CmaRasGRP1,CmaRAF and CmaPKCθ)in a complete open reading frame(ORF).Real-time fluorescence quantitative PCR was employed to detect and analyze the distribution of six genes in healthy hybrid snakehead tissues and their response characteristics to the infection of A.schubertii and N.seriolae respectively.Six eukaryotic expression recombinant plasmids pEGFP-N1-(LCK,ZAP-70,GRB2,RasGRP1,RAF and PKCθ)were constructed and transfected into HEK293 T cells respectively.The subcellular localization was observed by confocal microscopy.Then the five eukaryotic recombinant plasmids pEGFP-N1-(LCK#,ZAP-70#,GRB2#,RAF# and PKCθ#)wereconstructed and the dual luciferase report gene test was used to detect the effect of over-expression on the activity of NF-κB and AP-1 respectively.The main results were as follow:CmaLCK,CmaZAP-70,CmaGRB2,CmaRasGRP1,CmaRAF and CmaPKCθgenes were cloned successfully.Their ORF sequences were 1506 bp,1818 bp,654 bp,2394 bp,1914 bp and 2151 bp,encoding 501,605,217,797,637 and 716 amino acids,respectively.The coding protein sequences,sites and structural characteristics of the six genes were highly homologous with the corresponding proteins of mammals and teleosts,among which CmaGRB2 was highly conserved in the evolutionary process with GRB2 of mammals and teleosts.The results of quantitative fluorescence PCR showed that CmaLCK,CmaZAP-70,CmaGRB2,CmaRasGRP1,CmaRAF and CmaPKCθ genes could be expressed widely in ten tissues of healthy hybrid snakehead,including spleen,head kidney,liver,thymus,heart,intestine,muscle,skin,gill and blood.The expression of CmaLCK,CmaZAP-70,CmaGRB2,CmaRasGRP1,CmaRAF and CmaPKCθ genes was abundant in immune tissues,thymus,spleen and head kidney,and also high in liver.The expression of six genes CmaLCK,CmaZAP-70,CmaGRB2,CmaRasGRP1,CmaRAF and CmaPKCθ in spleen,head kidney and liver tissues were up-regulated in different degrees after infection by A.schubertii and N.seriolae,respectively.The expression of CmaLCK,CmaZAP-70,CmaGRB2 and CmaRasGRP1 in spleen and head kidney of immune tissues changed more significantly.The expression of CmaPKCθ and CmaRAF in liver also changed significantly.The response of hybrid snakehead to A.schubertii infection is faster and stronger than that of N.seriolae infection,which may be related to the physiological characteristics of the two pathogenic bacteria.It was suggested that the six TCR-related genes of hybrid snakehead may play a key role in T cell proliferation and activation,signal transduction and resistance to pathogenic bacterial infection.Sublocalization of HEK293 T cells showed that LCK,ZAP-70,GRB2,RasGRP1,RAF and PKCθ were scattered in the cytoplasm,ZAP-70 was also distributed in the cell nucleus.The results of dual luciferase reporter genes showed that the overexpression of pEGFP-N1-LCK#,pEGFP-N1-ZAP70# and pEGFP-N1-GRB2# in HEK293 T cells could significantly enhance the activity of transcription factors NF-κB and AP-1,whichsuggested that both NF-κB and AP-1 signals were activated.The overexpression of pEGFP-N1-RAF# had no significant regulatory effect on the activity of NF-κB,but promoted the activity of AP-1.pEGFP-N1-PKCθ# can promote the regulation of transcription factors NF-κB and AP-1,but the effect is not significant.The above results were concluded that these six genes maybe play an essential role in the defense of pathogenic bacterial infection response and the participation process of the regulation of signal transduction,which provided a theoretical basis for further understanding the anti-infection mechanism of TCR signaling pathway in fish.