农杆菌介导菜豆几丁质酶基因转化洋桔梗

摘要

An antifungal chitinase gene was transferred into the leaf segments of lisianthus [ Eustoma grandiflorum ( Raf. ) Shinn. ] Double Mariachi Pink to enhance the resistance to fungal diseases by the Agrobacterium-mediated method. Thirteen kanamycin-resistant lines were obtained and analyzed by PCR, among which, three transgenic lines were con-firmed nptII gene positive by RT-PCR. The chitinase activities of the three transgenic lines were 0. 215 U, 0. 225 U and 0. 286 U, significantly higher than the non-transformed line (0. 133 U). The inhibitory effects (in terms of the diameter of inhibition zone) on three fungi ( Streptomyce 5A, 5E and boytrytis cinerea) were significantly greater than the non-trans-formed line;The strongest inhibitory transgenic line showed larger diameters of inhibition zone by 106. 00%, 90. 91% and 71. 53%.%通过农杆菌介导法将抗真菌的几丁质酶基因转入洋桔梗，以提高其对真菌病害的抗性。结果表明，成功克隆了抗真菌活性较强的菜豆几丁质酶基因，构建了植物表达载体，并对洋桔梗Double Mariachi Pink叶块进行转化，获得了卡那霉素抗性植株，对抗性植株进行了2次PCR检测( nptⅡ基因)，第1次PCR检测获得了13个阳性株系，培养60 d后再进行第2次PCR检测，获得11个阳性株系，再对生长良好的5个转基因株系进行RT-PCR检测，获得了3个阳性株系。3个阳性株系的几丁质酶平均活性(0.215 U、0.225 U和0.286 U)均显著高于未转化植株(0.133 U)，转基因株系几丁质酶粗提液对大豆链霉菌5A、5E和灰葡萄孢菌3种指示真菌的抑菌圈直径均显著大于未转化植株，其中抑菌效果最好的株系对上述3种指示真菌的抑菌圈直径与对照相比，分别增大了106.00％、90.91％和71.53％。