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当前位置: 首页 > 文章 > 农杆菌介导转化大丽轮枝菌的体系优化 棉花学报 2011,23 (6) 29-36
Position: Home > Articles > Optimization of Agrobacterium-mediated Transformation of Verticillium dahliae Cotton Science 2011,23 (6) 29-36

农杆菌介导转化大丽轮枝菌的体系优化

作  者：
陈天子;袁洪波;杨郁文;刘蔼民;张保龙
单  位：
江苏省农业科学院江苏省农业生物学重点实验室
关键词：
农杆菌;大丽轮枝菌;潮霉素;GFP;遗传转化;遗传稳定性;表型变异
摘  要：
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=0.2并预培养4 h,然后与等体积的浓度为106个.mL-1的大丽轮枝菌孢子混匀,每皿取200μL混合液涂于固体IM共培养基的纤维素膜上,25℃共培养60 h,转移至固体PDA培养基上以头孢噻肟钠500mg.L-1+潮霉素B 50 mg.L-1进行筛选。研究发现农杆菌LBA4404和AGL-1比农杆菌SK1044和EHA105更适用于大丽轮枝菌转化。对转化子观察和鉴定发现:从表型上,70.8%转化子保持野生型形态,29.2%转化子发生了形态变化;从致病力上,81%转化子的致病力相对于野生型没有发生显著变化,19%转化子的致病力或增强或减弱。
译  名：
Optimization of Agrobacterium-mediated Transformation of Verticillium dahliae
作  者：
CHEN Tian-zi,YUAN Hong-bo,YANG Yu-wen,LIU Ai-min,ZHANG Bao-long(Provincial Key Laboratory of Agrobiology,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)
关键词：
Agrobacterium;Verticillium dahliae;hygromycin;GFP;genetic transformation;genetic stability;phenotypic variation
摘  要：
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.

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