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| Cloning, Sequence Analysis and mRNA Expression Pattern of Antithrombin Gene (AT) in Ayu(Plecoglossus altivelis) |
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Abstract Antithrombin(AT) is the major inhibitor of thrombin and other serine proteinases comprising the coagulation cascade enzymes. Furthermore, it has anti-inflammatory properties. The nucleotide sequence of full-length cDNA clone of ayu(Plecoglossus altivelis) AT gene (EMBL accession: No. FN429980) was obtained from the constructed liver cDNA of ayu. The whole cDNA length of ayu AT gene was 1 487 bp, consisting of a 1 362 bp open reading frame(ORF). The predicted mature ayu AT consisted of 453 amino acids preceded by a signal peptide of 23 residues. The structure of mature AT in ayu was similar with those in the mammalian AT, including serine protease active site near the carboxyl-terminal, three disulfide bonds formed by six conserved Cys residues and the four N-glycosylation sites. Sequence comparison showed that ayu AT had the highest amino acid indentity to Atlantic salmon(Salmo salar), which was 81%. Phylogenetic analysis showed that the evolutionary relationship of AT among different species was identical with the classification relationship of different species accepted currently and ayu AT grouped constantly with those previously reported from Atlantic salmon, tiger pufferfish(Takifugu rubripes), and zebrafish(Danio rerio). In healthy ayu, AT mRNA was mainly expressed in the liver, and weakly in the spleen, the kidney and the brain. Following Listonella anguillarum infection, liver AT mRNA expression change was determined by quantitative Real-time PCR(qRT-PCR) method. AT transcripts were significantly down-regulated in ayu liver at 4 and 8 h post injection(hpi) (P<0.05). However, AT transcripts were significantly up-regulated at 12~36 h when bacteria developing(P<0.05), suggesting that AT may be involved in the immune response of Ayu.
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Received: 07 January 2010
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