Molecular cloning, functional characterization and expression analysis of P65 subunit in response to GCRV infection in rare minnow (Gobiocypris rarus).

P65, the all-important subunit of the transcription factor NF-κB, plays an important role in the regulation of immune response. In this study, the cDNA of P65 subunit of rare minnow Gobiocypris rarus (GrP65) was cloned, and its expression patterns and functional role in rare minnow were investigated. The GrP65cDNA encodes a polypeptide of 573 amino acids, containing a well-conserved Rel-homology domain (RHD). The amino acid sequence analysis showed that GrP65 shared 81% and 69% identity to the grass carp (Ctenopharyngodon idella) and human (Homo sapiens) orthologous, respectively. Phylogenetic analysis revealed that GrP65 clustered with homologues from other teleosts. Cellular distribution anallysis demonstrated that GrP65 was located in the cytoplasm and nucleus. Quantitative real-time PCR analysis showed that GrP65 was ubiquitously expressed in all examined tissues, but especially highly in liver. Temporal expression analysis in vivo showed that the expression levels of GrP65 were significantly up-regulated in liver in response to GCRV infection, which suggested that GrP65 might play a crucial role in recognition and responses to GCRV infection in fish. In addition, GrP65 activated several interferon (IFN) promoters and induced the expression of downstream IFN-stimulated genes (ISGs). Furthermore, overexpression of P65 remarkably decreased the GCRV proliferation, while knockdown of P65 obtained opposite effects. In summary, we systematically characterized GrP65 and demonstrated its role in the innate immune response to GCRV infections.