報告書番号

V11008

タイトル（和文）

フジツボ類の着生フェロモンに関する研究-着生フェロモンの精製・構造解析と生産方法の確立-

タイトル（英文）

Studies on larval settlement-inducing pheromone of the barnacles -Purification, cDNA cloning and recombinant protein expression of the larval settlement-inducing pheromone-

概要 （図表や脚注は「報告書全文」に掲載しております）

フジツボ類付着期幼生は着生フェロモンにより同種を認識し付着・変態 (着生)することで群居する. この特性を利用したフェロモントラップを用いることで, 環境負荷の少ない新たな付着対策技術の開発が可能と考えられる. そこで各種カラムクロマトグラフィーを用いてタテジマフジツボ成体抽出液から32 kDaのタンパク質性着生フェロモンを精製した. 次にフェロモンのcDNAクローニングを行い, この情報を基に大腸菌を用いた着生フェロモンの発現系を構築した. さらに親水性DNゲルを担体とし, 組換え体フェロモンを用いた着生フェロモン含有ゲルを試作した. シャーレを用いた試験の結果, フジツボ幼生はフェロモンにより付着が誘導され, 80%以上がフェロモン含有ゲル周辺に付着した. 以上から, 着生フェロモンを用いることでフェロモントラップの開発が可能であると考えられた.

概要 （英文）

Novel larval settlement-inducing protein was purified from extracts of adult barnacle, Balanus amphitrite. The settlement of the larvae was significantly induced with the purified protein. Results of the assay using agarose gels indicated that this protein is released into seawater and attracts to settle cypris larvae. This result suggests that the purified protein is a waterborne type pheromone which induces settlement of larvae of B. amphitrite. Then a pair of degenerate primers was designed on the basis of the respective partial amino acid sequences, and complete cDNA of the pheromone protein was cloned by reverse transcription polymerase chain reaction using degenerate primers and 3'- and 5'-rapid amplification of cDNA ends. cDNA of this pheromone consisted of 907 bp nucleotides and included an open reading frame of 756 bp nucleotides. Deduced protein was 201-residues (28,716.53 Da), and included 15 residues signal peptide. The pheromone was heterologously expressed in Escherichia coli cell with a N-terminal hexa His-tag without 15 residues signal peptide. Purified recombinant pheromone protein had larval settlement-inducing activity similar to those of wild-type isolated from the extract of B amphitrite. Double-network hydrogel that combined rigid poly(2-acrylamido-2-methylpropanesulfonic acid) gels with flexible poly(acrylamide) gels was used for a carrier, pheromone trap gel for barnacle larvae was developed using the recombinant pheromone protein. Pheromone trap gel attracted cypris larvae and induced larval settlement in the laboratory experiments. The results suggest that this pheromone is available as antifouling techniques as pheromone traps for the barnacles.

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