Optimization of Agrobacterium-mediated Transformation of Verticillium dahliae
Optimization of Agrobacterium-mediated Transformation of Verticillium dahliae
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发表时间：
2011
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通讯作者：
Zhang Bao-long
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作者：
Zhang Bao-long
为了研究黄萎病的分子基础和黄萎病菌致病的分子机制，利用携带潮霉素抗性基因和绿色荧光蛋白(GFP)基因的双元载体对农杆菌介导的黄萎病菌转化进行了优化，使黄萎病菌菌株V991和BP2的每1×106个分生孢子产生100~550个GFP转化子。转化条件：将农杆菌溶液调至OD600=0.2，在液体IM培养基中预培养4h，然后加入等量的分生孢子悬浮液(每毫升1×106个分生孢子)，在查氏培养基中混合将该混合培养基(每皿200μL)接种于硝酸纤维素膜上，置于共培养的IM培养基上，25℃培养60h，转移到含有500 mg·L-1头孢菌素和50 mg·L-1潮霉素B的PDA型培养基上。农杆菌菌株LBA4404和AGL-1对黄萎病病原菌V991和BP2的转化效率高于农杆菌SK1044和EHA105。
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.