Characterization of MMP-9 gene from grass carp (Ctenopharyngodon idella): an Aeromonas hydrophila-inducible factor in grass carp immune system.

Matrix metalloproteinase-9 (MMP-9) belongs to a family of zinc-dependent endopeptidases and is associated with vital inflammatory processes. Here, we isolated and characterized MMP-9 cDNA from grass carp (Ctenopharyngodon idella) (designated as CiMMP-9). The cDNA was 2880 bp long and encoded a putative protein of 675 amino acids, with a predicted molecular mass of 75.816 kDa and an isoelectric point (pI) of 5.25. CiMMP-9 contained all three classical MMP-9 family signatures. The mRNA of CiMMP-9 was constitutively expressed in all tested tissues of untreated grass carp, with the highest expression levels in the blood, trunk kidney, head kidney and spleen. CiMMP9 transcript was present in unfertilized eggs, which suggests that CiMMP9 transcription is maternally inherited. Fluorescent real-time quantitative RT-PCR was used to examine the expression of the CiMMP-9 gene in C. idella after being challenged with Aeromonas hydrophila. A clear time-dependent expression pattern of CiMMP-9 was found after the bacterial challenge, and mRNA expression reached a maximum level at 7 days post challenge. This indicates that MMP-9 is inducible and is involved in immune responses, thus suggesting that CiMMP-9 plays an important role in A. hydrophila-related diseases and in early embryonic development stages in C. idella.

Molecular characterization and expression analysis of an insulin-like gene from the androgenic gland of the oriental river prawn, Macrobrachium nipponense.

The androgenic gland (AG), a male-specific endocrine organ in crustacean, is responsible for the maintenance of male characteristics and gender differentiation. In this study, an AG-specific gene, the Macrobrachium nipponesne insulin-like androgenic gland factor (MnIAG) was isolated from a transcriptome library of M. nipponesne and its full-length cDNA sequences were obtained by RACE method. The cDNA was 1,547 bp in length and encoded a precursor protein of 175 amino acids. The deduced precursor protein consisted of a signal peptide, B chain, C peptide and an A chain, which exhibited the same structural organization as that of previously identified insulin-like androgenic gland in crustacean. The mature peptide of the MnIAG owned two additional conserved cysteine residues, which were also found in the Palaemonidae species reported. Results of the tissue distribution and in situ hybridization showed the MnIAG expressed exclusively in androgenic gland. The quantitative RT-PCR results demonstrated that the MnIAG transcript was present at blastula stage and later developmental stages with low levels, which suggested that the primordial cells of the AG might form at these stages.

Matrix metalloproteinase 2 of grass carp Ctenopharyngodon idella (CiMMP2) is involved in the immune response against bacterial infection.

The gene encoding matrix metalloproteinase 2 (MMP2) was cloned from grass carp (Ctenopharyngodon idella), and its expression levels during Aeromonas hydrophila infection and embryonic development stages were evaluated. The complete open reading frame of CiMMP2 was 1974 bp in length, encoding a 658-amino acid polypeptide. The deduced MMP2 protein contained four conserved domain structures, including an N-terminal signal sequence, a propeptide domain, three repeats of fibronectin-type II domain inserted in the catalytic domain and a C-terminal hemopexin-like domain. Phylogenetic analysis of MMP2s grouped grass carp with other teleosts. Detected in all fish tissues examined, CiMMP2 expression increased in the spleen and head kidney at 4 h and was significantly downregulated at 1 d after A. hydrophila infection. CiMMP2 transcripts were present in unfertilized eggs, suggesting its maternal origin. These findings implicate an important role for CiMMP2 in A. hydrophila-related diseases and early embryonic developmental stages of grass carp.

Molecular cloning, characterization and expression patterns of HSP60 in the grass carp (Ctenopharyngodon idella).

HSP60 is a highly immunogenic molecule, which is able to activate a large number of T cell types and is implicated in a variety of autoimmune diseases. The grass carp (Ctenopharyngodon idella), a freshwater fish species of the family Cyprinidae, accounts for the third biggest value (USD 4.8 billion) at single species level of major cultured fish species in the world. Here, we isolated and characterized the HSP60 cDNA from grass carp (designated as CiHSP60). Its cDNA was 2434 bp in length and encoded a putative protein of 575 amino acids. BLAST analysis revealed that the CiHSP60 gene shared a high similarity with other known HSP60 sequences. CiHSP60 contained all three classical HSP60 family signatures. The mRNA of CiHSP60 was constitutively expressed in all tested tissues of untreated grass carp, including brain, muscle, trunk kidney, liver, head kidney, skin, spleen, heart, gill, intestine, and fin, with the highest expression level in the blood. CiHSP60 transcript was present in unfertilized eggs, which suggests that CiHSP60 transcription is maternally inherited. Fluorescent real-time quantitative RT-PCR was used to examine the expression of the CiHSP60 gene in grass carp after the challenge with the bacterium Aeromonas hydrophila. A clear time-dependent expression pattern of CiHSP60 was found after the bacterial challenge, and the mRNA expression reached a maximum level at three days post challenge, and returned to control levels after seven days. The upregulated mRNA expression of CiHSP60 in grass carp after bacterial challenge indicates that the HSP60 gene is inducible and involved in immune responses. These results suggest that CiHSP60 plays an important role in A. hydrophila-related diseases and in early embryonic development stages in grass carp.