Cloning and Expression Pattern of MAVS Gene in Largemouth Bass (Micropterus salmoides) and Its Effect on NF-κB and IFN-β Promoter Activation
GAO Feng-Ying1, DONG Jun-Jian1, ZHANG He-Tong1, LI Jia-Xin1,2, ZHU Zhi-Lin1,2, SUN Cheng-Fei1,*, YE Xing1
1 Pearl River Fisheries Research Institute/Key Laboratory of Tropical & Subtropical Fishery Resource Application &Cultivation, Ministry of Agriculture, Chinese Academy of Fishery Science, Guangzhou 510380, China; 2 College of Fisheries, Tianjin Agricultural University, Tianjin 300384, China
Abstract：Mitochondrial antiviral signaling protein (MAVS) is a key molecule involved in innate antiviral response through the RIG-1 like receptor pathway. As an essential adaptor in signal transduction, MAVS plays crucial role in innate immunity against viruses in mammals and fish. To investigate the role of MAVS gene in the innate immunity of largemouth bass (Micropterus salmoides), the cDNA sequence of MAVS gene (GenBank No. PP740757) was obtained by reverse transcription PCR (RT-PCR) and cloning, and bioinformatics was also analyzed. qRT-PCR was used to detect the expression pattern of MAVS in healthy individuals and in individuals intraperitoneal injection with Nocardia seriolae and polyinosinic polycytidylic acid (Poly I:C). Subcellular localization of the MAVS protein was determined using immunofluorescence assay, and the effects of the MAVS gene on the promoter activities of nuclear factor kappa-B (NF-κB) and β-type interferon (IFN-β) were analyzed by dual-luciferase reporter assay. The results showed that the MAVS cDNA was 1 921 bp in length and encoded a peptide of 586 amino acid residues. The deduced protein of MAVS in largemouth bass had a N-terminal CARD domain and a proline rich region, which were known to be important functional domains of mammalian MAVS. The MAVS protein of largemouth bass had high homology with other fish species (48.7%~96.1%, and 29.6% homology with grass carp (Ctenopharyngodon idella)). Phylogenetic analysis showed that largemouth bass MAVS was grouped with other fish MAVS. The MAVS mRNA was expressed in all tested tissues of healthy largemouth bass, with the highest expression level in the gills. In all 4 tested tissues, both immunostimulants could up-regulate the transcription level of MAVS in largemouth bass. Among them, on the 9 d after Poly I:C stimulation, MAVS mRNA expression level in the gills was as high as 53.17 folds that of the control group (P<0.05). In kidney, MAVS showed the highest increase at 6 d after N. seriolae infection, and the expression level in the infected group was 14.88 folds that of the control group (P<0.05). Subcellular localization showed that MAVS was presented in the cytoplasm of HeLa cells. Overexpression of MAVS in HeLa cells could increase the NF-κB and IFN-β promoter activity. These results indicated that MAVS played important roles in the innate immunity of largemouth bass. This study lays the foundation for further elucidating the role of MAVS gene in the innate immunity of largemouth bass.
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