Functional Analysis Of TBK1L In Zebrafish And Cloning P47PHOX And P40PHOX In Grass Carp

TANK-binding kinase 1(TBK1) is an essential serine/threonine-protein kinase required for Toll-like receptor(TLR) and retinoic acid-inducible gene I(RIG-I)-mediated induction of type I IFN and host antiviral defense. In the present study,TBK1-like transcript, namely TBK1 L, was cloned from zebrafish. Compared with TBK1, TBK1 L contains an incomplete S_TKc domain, and lacks UBL_TBK1_like domain. Realtime PCR showed that TBK1 L was constitutively produced in embryos, early larvae and ZF4 cells, and unchanged in ZF4 cells following SVCV infection. Overexpression of TBK1 but not TBK1 L resulted in significant activation of zebrafish IFN1 and IFN3 promoters. Similarily, TBK1 L had litter impact on the antiviral state of the cells. However, the overexpression of TBK1 L negatively regulated the induction of zebrafish IFN1 and/or IFN3 promoters mediated by the retinoic acid-inducible gene I-like receptors(RLRs), MAVS and TBK1. In addition, the overexpression of TBK1 L in zebrafish embryos led to the decreased production of many IFN-stimulated genes induced by TBK1. Collectively, these data suggest that zebrafish TBK1 L negatively regulates RLRs-MAVS-TBK1 pathway.NADPH oxidase, located specially in plasma membrane of phagocytes, can generate reactive oxygen species(ROS) to participate in host defense by killing or damaging invading microbes. The oxidase is the precursor of a variety of microbicidal oxidants such as hydrogenperoxide(H2O2). The expression pattern of p47 phox and p40 phox, and their immune function in fish are less investigated.In the present study,full-length cDNA sequences of two regulatory subunits of NADPH oxidase, including p47 phox and p40 phox, were cloned from grass carp Ctenopharyngodon idella, utilizing the reverse transcription-PCR and rapid amplification of cDNA ends.The results showed that the full length p47 phox and p40 phox sequences are 1589 bp and 2103 bp, respectively. Their open reading frame(ORF) is 1233 bp and 1068 bp, encoding 410 aa and 355 aa, respectively. Using SMART and Scanprosite tool software for protein structure prediction, the grass carp p47 phox has a PX domain, two SH3 domains and a PC motif, and p40 phox has PX domain, SH3 domain and PB1 domain. Alignment of amino acid sequences and phylogenetic tree analysis suggested a high homology between grass carp and the bream Megalobrama amblycephala. Real time quantitative PCR(rt-qPCR) result revealed the different tissue distribution of p47 phox and p40 phox expressed in gills, heart, intestines, head-kidney, kidney, liver, spleen, skin and thymus. The highest level was found in thymus, heart and skin. High expression of p47 phox was found in skin,heart and thymus, then gills, liver, spleen and head-kidney. Similarly, high expression of p40 phox was found in heart, thymus and skin. However, lower level expression was observed for p40 phox in gills, liver and kidney.