Cloning, Characterization And Temporal Spatial Transcription Analysis Of The Proteasome Activator PA28-α Subunit Gene In Common Carp (Cyprinus Carpio L.)

PA28-αand PA28-βinduced by interferon-γ(IFN-γ) are two key activators and activate the latent 20S proteasome which generate antigenic peptides presented on MHC class I molecules to cytotoxic T-cells, thus playing an important role in the processing of MHC class I antigen. PMSE1 and PMSE2 genes encoding these activators have been characterized and documented in mammals, whereas, few reported among fishes. In the present study, we reported the cloning of a PA28-αgene homologue from the cDNA library of common carp peripheral blood leucocytes stimulated with mitogen LPS, PHA and ConA. The full-length cDNA of common carp PA28-αencompasses 1097 nucleotides (nt) and encodes a protein of 249 amino acids (aa). The deduced protein sequence shares 92, 56, 54, 53 and 54% sequence identity to sequences found in zebrafish, mouse, pig, cattle and human, respectively. The deduced common carp PA28-αcontains a PA28-αsubunit-specific insert corresponding to the KEKE motif of the known PA28-β(Region B), a characteristic proline-rich motif (Region A), a potential protein kinase C recognition site (Region D), a conserved activation loop (Region C) and a highly homologous C-terminal region among all three PA28 subunits (Region E). Genomic DNA (PSME1 gene) of common carp PA28-αwas also obtained in this study. It encompasses 3583 nucleotides, 11 exons, 10 introns which observe the regulation of GT-AG completely. Comparing with human, pig, mouse, zebrafish, the structure of common carp PMSE1 gene has been well conserved through evolution. RT-PCR was used to analyze the temporal spatial transcription of common carp PA28-α. Transcription analysis in leucocytes indicated that the transcription of PA28-αin stimulated leucocytes were higher than that in normal leucocytes all the time (LPS, PHA: 4h (P<0.05), 12h (P<0.05); ConA: 4h (P<0.05), 24h (P<0.05)), but gradually decreased in stimulated leucocytes as the stimulation time going (LPS, PHA: 4h>12h (P<0.05), ConA: 4h>24h (P<0.05)). Transcription profiles analysis of tissues indicated that the transcription of common carp PA28-αin various tissues from stimulated common carp were higher than that in corresponding tissues from normal common carp (P₁, P₂, P₃, P₄, P₅, P₆, P₇ <0.001), as well as, the transcription of PA28-αin induced common carp were significantly up-regulated in these tissues association with immunity (gill, spleen, kidney and liver), however, weakly transcribed in muscle, brain and heart.