PEG-Mediated Genetic Transformation of Fusarium oxysporum f. sp conglutinans to Study Pathogenesis in Cabbage

文献类型： 外文期刊

第一作者： Zhang, Wei

作者： Zhang, Wei;Feng, Jianhai;Yan, Hong;Li, Xinghong;Yan, Jiye;Zhao, Wensheng;Huang, Jinguang;Feng, Jianhai;Yang, Laying

作者机构：

关键词： Fusarium oxysporum f. sp conglutinans;genetic transformation;restriction enzyme mediated integration;pathogenesis

期刊名称：CHIANG MAI JOURNAL OF SCIENCE （ 影响因子：0.523； 五年影响因子：0.505 ）

ISSN： 0125-2526

年卷期： 2014 年 41 卷 4 期

页码：

收录情况： SCI

摘要： Establishment of an efficient and stable genetic transformation system in cabbage Fusarium wilt pathogen (Fusarium oxysporum f. sp. conglutinans) is a key step for studying gene function and the specific phenotypes of mutants. The optimal conditions for protoplast preparation and generation are as follows: A mycelia growth time of 32 h in CM medium, Driselase as the cell wall-degrading enzyme, 1.5 ml of enzyme solution (20 mg/ml Driselase) to 1 gram of mycelia, a treatment time of 4 h at 28 degrees C, and 0.7 M NaCl buffer. With the above conditions, the pKNTG vector containing green fluorescent protein gene was transformed into the protoplasts of F. oxysporum strain A6 by polyethylene glycol (PEG), and 20 positive transformants were obtained and used for the pathogenicity test. The linearized pUCATPH plasmid containing the hygromycin resistance gene was also transformed into the protoplasts of A6 strain, and HindIII and EcoRI were used in this procedure. Finally, a library with 1506 transformants was constructed by the restriction enzyme mediated integration (REMI) method. Southern blot analysis was used to prove the feasibility-of the transformation system, and different phenotypic mutants were evaluated. The results show that efficient PEG-mediated genetic transformation and the REMI transformation system of the Fusarium oxysporum f. sp. conglutinans were established successfully.

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