Transformation of Tobacco Cultured Cell by Particle Bombardment : Expression of Phenylalanine Ammonia-lyase Gene in Transgenic Tobacco Cells.
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The plasmid DNA containing a NPTⅡ as a selectable marker gene and a tobacco PAL cDNA combined with the CaMV 35S promoter and NOS terminator was transformed into tobacco cells by a particle bombardment device based on flowing helium. The most effcient transformation was achieved when the amount of DNA-coated tungsten particle was 0.2㎎ per projectile, the amount of plasmid DNA was 4μg/㎎ of tungsten particles, the helium pressure accelerated was 3㎏/c㎡, and the distance between the sample and the projectile was 15cm. Genetic analysis of kanamycin-resistant transformants obtained showed that the PAL cDNA construct integrated into the genome of tobacco cells. PAL activity of the transformant increased almost 4-fold and scopoletin content increased more than 2-fold as compared to nontrasformed cells.ヘリウムガス圧式パーティクルガンにより，タバコ培養細胞のPALcDNAを同細胞へ再導入し，形質転換体を得た．タバコ培養細胞のPALcDNAをカリフラワーモザイクウイルス(CaMV)の35Sプロモーターとノパリン合成酵素(NOS)遺伝子のターミネーターに接続し，ネオマイシンホスホトランスフェラーゼⅡ(NPTⅡ)遺伝子とともにタバコ培養細胞へ導入し，形質転換体をカナマイシン培地で選抜した．また，パーティクルガンによる遺伝子導入の最適条件としては，加速圧力3㎏/c㎡，0.2㎎タングステン粒子/プロジェクタイル，4μg/㎎タングステン粒子，プロジェクタイルからサンプルまでの距離は15㎝であった．そして得られた形質転換体の中には，非形質転換体と比較して，PAL酵素活性で4倍，スコポレチン生成量で2倍以上のものが確認された．
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- Transformation of Tobacco Cultured Cell by Particle Bombardment : Expression of Phenylalanine Ammonia-lyase Gene in Transgenic Tobacco Cells.