农杆菌介导的曼地亚红豆杉细胞遗传转化体系的建立

The Establishment of Genetic Transformation System of Taxus Mediated by Agrobacterium

建立了根癌农杆菌介导的红豆杉细胞遗传转化体系,为红豆杉细胞的遗传改良打下基础。最优转化条件是:菌液光密度A600=0.6,侵染时间25 min,共培养最适温度21℃,共培养时间48 h;最适头孢霉素(Cephamycin,Cef)抑菌浓度为300 mg/L;最优的除菌及筛选方式为:用含100 mg/L头孢霉素的无菌水冲洗共培养后的细胞10 s,然后将细胞转至含300 mg/L头孢霉素的抑菌培养基上,两周后转至含150 mg/L卡那霉素(Kanamycin,Kan)或8 mg/L潮霉素(Hygromycin,Hyg)的筛选培养基上筛选。采用小愈伤团法筛选转基因纯系,GUS染色显示,筛选后转化细胞株的阳性细胞占80%以上。

In order to facilitate the genetic modification of Taxus cells, the genetic transformation system of Tax- us media cell mediated by Agrobacterium tumefaciens EHA 105 strain was established in the current work. The optimal bacterial concentration, infection time, temperature and co-culture time were A600 =0.6, 25 min, 21℃ and 48 h, respectively. The optimal cephalosporins chloramphenicol （Cef） bacteriostatic concentration was 300 mg/L. The most optimum methods for bacteria removing and Taxus cells screening were as follows： the co-cul- ture cells were washed for 10 s with distilled water containing 100 mg/L Cef, and then transferred to medium containing 300 mg/L Cef, and finally transferred to medium containing 150 mg/L kanamycin （Kan） or 8 mg/L hygromycin （Hyg） for further selection. A method of small callus culture was used to select the trans- genic pure cells. The results of GUS dyeing showed the ratio of the positive transformed cells in selected cell lines was above 80%.