| Liver-expressed antimicrobial peptide1(LEAP-1), LEAP-2and β-defensin are a group of cysteine-rich, cationic antimicrobial peptides, widely exist in fish. They are important components of innate immune system and play important roles in defending against pathogenic microbes invading. LEAP-1can inhibit the growth of bacteria, fungi, viruses and protozoa in vivo and in vitro. Furthermore, LEAP-1has been reported to play an important role in the control of body iron homeostasis. LEAP-2is another important antimicrobial peptide and has the antimicrobial effect. Besides the function of killing bacteria and viruses directly,(3-defensins are able to indirectly promote recruitment of effector cells, induce the expression of diverse cytokines and chemokinesas, as well as to promote wound healing and angiogenesis.Blunt snout bream (Megalobrama amblycephala) is a commercially important fish in our country. In order to elucidate the role of LEAP-1, LEAP-2and β-defensin in early development stage and in response to bacterial challenge in blunt snout bream, and provide the theoretical basis for investigating their biological activity in innate immune system, we cloned the full-length cDNA sequences of LEAP-1, LEAP-2and partial length cDNA sequences of β-defensin by gene cloning methods, constructed the temporal and spatial expression profile, and assessed the effect of bacterial challenge on gene expressions of these peptdie. The main results are as follows:1. Molecular cloning and expression analysis of LEAP-1and LEAP-2genes in blunt snout breamThe full-length cDNA sequence of blunt snout bream LEAP-1(GeneBank accession no. JQ308841) was779bp. It contained a285bp open reading frame (ORF), a115bp5’-untranslated region (UTR) and a379bp3’-UTR. The ORF encoded a peptide of94amino acids, with the first24residues predicted to be a signal peptide, a45amino acid predomain region and a25amino acid mature peptide. The deduced amino acid sequence of blunt snout bream LEAP-1based on ORF was highly similar to grass carp (95%), bighead carp (95%). Real-time PCR results indicate that after a decrease at mid-blastula stage during the embryonic development, the expression level of LEAP-1mRNA increased significantly to the highest value at eye vesicle appearance stage, and then sharply decreased again to a moderate level at caudal fin anlage stage. After hatching, it increased gradually to the moderate level at1day post hatching and3dph, and then decreased to the low level at7dph. In adult individuals, the expression level of LEAP-1mRNA was the highest in the liver. In liver tissues of blunt snout bream, the levels of LEAP-1mRNA expression increased gradually and reached the highest value at144h post-injection, but had no significant differences at different time points (P>0.05). A similar tendency to increase in expression was observed in the brain, with the level of LEAP-1mRNA expression reaching the highest value at72h post-injection (P>0.05). However, bacterial challenge caused a significant upregulation of LEAP-1mRNA expression in the spleen at24h post-injection and a significant upregulation in the gill at6h post-injection, respectively(P<0.05).The full-length cDNA sequence of blunt snout bream LEAP-2(GeneBank accession no. JQ344324) was564bp. It contained a279bp open reading frame (ORF), a35bp5’-untranslated region (UTR) and a250bp3’-UTR. The ORF encoded a peptide of92amino acids, with the first26residues predicted to be a signal peptide, a25amino acid predomain region and a41amino acid mature peptide. The deduced amino acid sequence of blunt snout bream LEAP-1based on ORF was highly similar to grass carp (95%), zebrafish (84%). During embryonic development, the expression level of LEAP-2mRNA increased significantly to its highest value at mid-gastrula stage, and then the expression level decreased sharply to a low level at which it was maintained until heart beat stage. Subsequently, the expression level increased significantly at prophase of hatching and reached the second highest value at newly hatching stage. And then it decreased significantly to a low level at3dph and then fluctuated until31dph. The expression level of LEAP-2mRNA was highest in the midgut. After induction by A. hydrophila, the levels of LEAP-2mRNA expression in the liver were significantly up-regulated at72h and144h post-injection (P<0.05). Bacterial challenge caused significant upregulation of LEAP-2mRNA expression in the spleen, brain and gill at6h,24h and6h post-injection, respectively (P<0.05).2. Molecular cloning and expression analysis of two β-defensin genes in blunt snout breamThe part-length cDNA sequences of two β-defensin genes (maBD-1, maBD-2) in blunt snout bream (Megalobrama amblycephala) were201bp and207bp. The deduced amino acid sequence of blunt snout bream maBD-1, maBD-2was highly similar to common carp (98%) and zebrafish (97%) The deduced amino acid sequence of maBD-2was highly similar to common carp (90%) and zebrafish (87%). During embryonic development, the expression of maBD-1mRNA was kept at a low level until heart appearance stage, and then it increased to a moderate level at heart beat stage. After hatching, the expression level of maBD-1mRNA increased significantly to the highest value at1dph, and then decreased to the moderate level at3dph and further decreased to a low level until31dph. The expression of maBD-2mRNA showed the highest value in fertilized eggs, and then the expression level decreased gradually to a low level at which it was maintained until31dph except a moderate increase at muscular contraction stage. The expression of maBD-1mRNA had the highest level in skin. The mean expression level of maBD-2mRNA was higher in liver than those in kidney, brain and foregut (P>0.05), but the expression levels were very low in these tissues. Following bacterial challenge in vivo by Aeromonas sobria, maBD-1expressions were significantly up-regulated in liver and skin, and maBD-2expressions were significantly up-regulated in liver, skin, gill and foregut. |
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