Abstract:Abstract Tyrosine protein ZAP-70 (70 kDa zeta-associated-protein, ZAP-70) is a key factor in the initiation of T cell receptor (TCR) signal transduction, which plays an important role in the proliferation and activation of T cells. In order to explore the role of hybrid snakehead (Channa maculata ♀ ×Channa argus ♂) ZAP-70 (CmaZAP-70) in antibacterial infection and the function in signal transduction, the complete open reading frame (ORF) of CmaZAP-70 gene was cloned and its response to Aeromonas schubertii and Nocardia seriolae infection was analyzed. Moreover, the plasmid pEGFP-N1-ZAP70 was constructed to detect the effect of overexpression on the activity of NF-κB and AP-1. Sequence analysis showed that the complete ORF of CmaZAP-70 gene was 1818 bp, encoding 605 amino acids. CmaZAP-70 contained two tandem SH2 domains and one SH1 kinase domain. In addition, CmaZAP-70 was highly conserved with some phosphorylation sites and catalytic motifs of mammal ZAP-70. CmaZAP-70 was clustered into one group with ZAP-70 proteins from other teleost and had the closest affinity with Dicentrarchus labrax ZAP-70 in the evolutionary tree. The qRT-PCR results showed that CmaZAP-70 was widely expressed in ten tissues of healthy hybrid snakehead, with the highest expression in spleen and the lowest in skin. CmaZAP-70 expressed abundantly and mostly up-regulated in spleen and head kidney after infection with two pathogenic bacteria, respectively. After infection with A. schubertii, CmaZAP-70 peaked in liver, spleen and head kidney at 1 d, 1 d and 2 d, respectively (P<0.05), while its expression peaked at 3 d after infection with N. seriolae (P<0.05). Furthermore, the dual-luciferase reporter assay showed that overexpression of pEGFP-N1-ZAP70 significantly enhanced the activity of transcription factors NF-κB and AP-1. Taken together, the above results indicate that CmaZAP-70 plays a crucial role in the defense against pathogen infection response, and participates in the regulation of signal transduction, which provides a theoretical basis for further revealing the anti-infection mechanism of TCR signaling pathway in fish.
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