农杆菌介导转化大丽轮枝茵的体系优化

为了揭示大丽轮枝菌的遗传变异和致病机理,以含潮霉素为抗性筛选标记、绿色荧光蛋白GFP为报告基因载体,优化了农杆菌介导转化大丽轮枝菌的遗传转化体系,获得了大丽轮枝菌菌株V991和BP2含绿色荧光蛋白GFP的转化子,转化率达到100×10-6～550×10-6.优化的转化条件为:农杆菌以IM液体培养基调节浓度至OD600=...

Optimization of Agrobacterium-mediated Transformation of Verticillium dahliae

To facilitate the study on the molecular basis of Verticillium dahliae and the molecular mechanisms that Verticillium spp. employ to cause disease, a binary vector carrying hygromycin resistant gene and green fluorescent protein(GFP) gene was used to optimize the Agrobacterium-mediated transformation of Verticillium dahliae, leading to the production of 100~550 GFP-carrying transformants per 1×10⁶ conidia of Verticillium dahliae strain V991 and strain BP2. The transformation conditions: Agrobacterium solution was adjusted to OD₆₀₀=0.2 and precultured in liquid IM medium for 4 h, then was mixed with an equal volume of a conidial suspension(1×10⁶ conidia per mL) in Czapek’s medium, this mixed medium(200 μL per plate) was plated on a nitrocellulose filter and placed on co-cultivation IM medium at 25℃ for 60 h, the filters were transferred to PDA medium containing 500 mg·L^-1 cefotaxime and 50 mg·L^-1 hygromycin B as selection agents. Agrobacterium strains LBA4404 and AGL-1 were more efficient in transformation of Verticillium dahliae strain V991 and strain BP2 than Agrobacterium strains SK1044 and EHA105. In the aspect of morphology, 70.8% transformants remained wild-type phenotype, while 29.2% transformants performed mutation in phenotype, in contrast to 19% transformants with changes in pathogenicity.