在前期的研究中,克隆到牙鲆变态早期和变态前差异表达的parvalbumin基因片断cDNA.为了克隆parvalbumin基因的全长cDNA, 构建了变态早期 (孵化后23 d仔鱼) 牙鲆头部的全长cDNA文库,并对文库的滴度和插入片段大小进行了评估.同时采用锚定PCR法从cDNA文库筛选出parvalbumin 基因的全长cDNA.结果表明,所构建的牙鲆变态早期头部cDNA文库的滴度为1.2?106 PFU?mL-1,插入片段的平均长度为1 000 bp左右.parvalbumin基因的全长cDNA的长度为603 bp, 编码109个氨基酸,含有两个EF手型钙离子结合域:DQDGSGFIEEEEL (52～64)和DSDGDGKIGVEEF(91～103),以及一个ATP/GTP结合位点模序:ACGLAGKS(33～40),是一种典型的钙离子结合蛋白.通过parvalbumin基因氨基酸序列的同源比较分析表明,牙鲆parvalbumin基因是比较保守的蛋白,与其它鱼类享有61.5%～68.8%序列同源.系统分析表明,牙鲆的parvalbumin基因与狭鳕(Theragra chalcogramma)最为接近.

A fragment cDNA of parvalbumin gene has been cloned, which expressed differently between pre-metamorphosis and pro-metamorphosis stages of Japanese flounder, Paralichthys olivaceus. For the requirement of further research on the role of parvalbumin in metamorphosis stage, it is necessary to clone the full-length cDNA of parvalbumin gene. Here we constructed a head tissue full-length cDNA library of pro-metamorphosing flounder （23 dph, day post hatch） using SMARTTM technique. The titer of cDNA library was estimated as 1.2 ? 10^6 PFU?mL^-1, which was about 1000 times coverage of gene expressional profile, and the inserted fragments in the library are about 1000 bp length. Through two-runs anchor-PCR with target gene specific primers and λ TripIEx2 primers, we obtained a full-length cDNA of parvalbumin gene from the cDNA library. The full-length cDNA of parvalbumin gene consisted of 603 bp nucleofides, and encodes 109 amino acids with two EF-hand calcium-binding domains： DQDGSGFIEEEL （52- 64） and DSDGDGKIGVEEF（91 - 103）, and an ATP/GTP-binding site motif A （P-loop）： ACGLAGKS（ 33- 40）. The structure of the sequence shows the parvalbumin is a typical calcium-binding protein. Further analysis with homologue sequence comparison shows the flounder parvalbumin is a conserved protein, which shares 61.5% - 68.8% similarity with other teleosts, such as 61.5% with channel catfish lctalurus punctatus, 68.8 % with zebrafish Danio rerio. The phylogenetic analysis shows the closest relationship existed between parvalbumins of Japanese flounder and cod Theragra chalcogramma. Parvalbumin plays biological role of relaxation of muscle after contraction by soaking free calcium in cell. The recent researches show the parvalbumin gene expressed differentially in GABA neurons. However, it is unclear about the role of parvalbumin in metamorphosing flatfish, including Japanese flounder. The results reported here should be helpful for the lhture work such ~s gene expression location, gene function research.

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