农杆菌介导转化大丽轮枝菌的体系优化  

Optimization of Agrobacterium-mediated Transformation of Verticillium dahliae

作　　者：陈天子[1] 袁洪波[1] 杨郁文[1] 刘蔼民[1] 张保龙[1] 

机构地区：[1]江苏省农业科学院,江苏省农业生物学重点实验室,江苏南京210014

出　　处：《棉花学报》2011年第6期507-514,共8页Cotton Science

基　　金：国家重大专项(2009ZX08005-003B);2010年江苏省农业科技自主创新资金(CX(10)434)

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。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 × 10^6 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 × 10^6 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.

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 

分 类 号：S432.4[农业科学—植物病理学]