Cloning, Expression and Functional Analysis of CmaZAP-70 from Hybrid Snakehead (Channa maculata♀×Channa argus♂)
LIU Fu-Cui1, 2, ZHAO Fei1, *, TAN Ai-Ping1, KONG Lu-Lu1, 2, HE Shan1, 2, ZHANG Rui-Quan1, DENG Yu-Ting1, JIANG Lan1
1 Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs/Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province/Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China; 2 College of Fishery and Life Science, Shanghai Ocean University, Shanghai 201306, China
Abstract：As a key factor in the initiation of T cell receptor (TCR) signal transduction, tyrosine protein kinase ZAP-70 (70 kD zeta-associated-protein) is critical to the proliferation and activation of T cells, contributing to the fight against bacterial infection. However, current studies on ZAP-70 in fish are quite limited. In this study, a series of experiments were conducted to uncover the potential role of ZAP-70 in hybrid snakehead (Channa maculata ♀ ×Channa argus ♂)(CmaZAP-70), a feature economic species in China. The complete ORF of CmaZAP-70 gene (GenBank No. MK134563) was cloned and its response to Aeromonas schubertii and Nocardia seriolae infection was analyzed. The structure, homology and phylogenetic tree of CmaZAP-70 sequence were also analyzed. Moreover, the plasmid pEGFP-N1-ZAP70 was constructed to detect the effect of overexpression on the activity of nuclear factor kappa-B (NF-κB) and activating protein 1 (AP-1). Sequence analysis showed that the complete ORF of CmaZAP-70 gene was 1 818 bp, which encoded 605 aa with relative molecular weight and theoretical point of 68.2 kD and 8.06, respectively. SignalP 4.1 Server analysis showed that CmaZAP-70 was a non-secretory protein without signal peptide, which contained 2 tandem SH2 domains and 1 SH1 kinase domain. In addition, CmaZAP-70 was highly conserved with some phosphorylation sites and catalytic motifs of mammal ZAP-70. Phylogenetic analysis showed that CmaZAP-70 was clustered into 1 group with other teleost and it was clustered closely with Dicentrarchus labrax, which indicated that 2 species had high homology. Moreover, the qRT-PCR results showed that CmaZAP-70 was widely expressed in 10 tissues of healthy hybrid snakehead, with the highest expression in spleen (P<0.05) and relatively highly in the thymus, head kidney and blood, whereas it was expressed at lowest levels in the skin (P<0.05). The expression changes of CmaZAP-70 were similar and mainly up-regulated in the liver, spleen, and head kidney at different time points after infection with the pathogens A. schubertii and N. seriolae, respectively. After infection with A. schubertii, CmaZAP-70 showed the highest expression in the liver, spleen and head kidney at 1 d, 1 d and 2 d, respectively (P<0.05), while it all displayed the highest expression in the liver, spleen, and head kidney 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 NF-κB and AP-1, which were 4.3 and 3.5 times higher than that of control, respectively (P<0.05). The results conclude that CmaZAP-70 might play a crucial role in the defense against pathogen infection, and participates in the regulation of signal transduction. This study would lay a solid foundation for further revealing the anti-infection mechanism of TCR signaling pathway in fish.
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