PEG-mediated and Agrobacterium-mediated transformation in the mycopathogen Verticillium fungicola
PEG-mediated and Agrobacterium-mediated transformation in the mycopathogen Verticillium fungicola
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DOI：
10.1017/s0953756201005251
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发表时间：
2002-01-01
影响因子：
--
通讯作者：
Foster, GD
中科院分区：
文献类型：
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作者：
Amey, RC;Athey-Pollard, A;Foster, GD
利用聚乙二醇介导和农杆菌介导两种技术，成功地转化了双孢蘑菇中的一种严重的支原真菌黄萎病菌。peg介导的湿霉素B抗性（hph）、β -葡萄糖醛酸酶GUS （uidA）和绿色荧光蛋白（GFP）基因共转化成功。农杆菌介导的hph基因转化成功。peg介导和农杆菌介导的转化频率分别高达每杯DNA 102个和每105个分生孢子4068个转化子。在非选择性培养基上连续继代培养18个月后，整合基因在共转化体中的表达稳定，并在-80℃的甘油中保存。peg介导的转化体的分子分析显示转化基因整合到目标基因组中。农杆菌介导的转化体的分子分析显示，转化DNA在目标基因组内以单拷贝的形式整合。共转化菌在接种实验中表现出疾病症状，其毒性至少与野生型真菌一样强。在体内观察到GFP和GUS的表达，GFP标记的菌株在流行病学和宿主-病原体相互作用研究中具有很大的潜力。真菌弧菌转化系统的开发将使该生物与双孢霉相互作用的分子研究更加深入。
Verticillium funigicola, a severe mycopathogen of the cultivated mushroom Agaricus bisporus, was successfully transformed using both PEG-mediated and Agrobacterium-mediated techniques. PEG-mediated co-transformation was successful with hygromycin B resistance (hph), uidA (beta-glucuronidase GUS), and green fluorescent protein (GFP) genes. Agrobacterium-mediated transformation was successful with the hph gene. Transformation frequencies of up to 102 transformants per mug DNA and 4068 transformants per 105 conidia were obtained for PEG-mediated and Agrobacterium-mediated transformation respectively. Expression of integrated genes in co-transformants was stable after 18 months of successive sub-culturing on non-selective medium, and following storage at -80 degreesC in glycerol. Molecular analysis of PEG-mediated transformants showed integration of the transforming genes into the target genome. Molecular analysis of Agrobacterium-mediated transformants showed integration of transforming DNA as single copies within the target genome. Co-transformants exhibited symptoms of disease in inoculation experiments and were at least as virulent as the wild-type fungus. GFP and GUS expression were observed in-vivo with the GFP-tagged strain showing great potential as a tool in epidemiological and host-pathogen interaction studies. The development of transformation systems for V.fungicola will allow in-depth molecular studies of the interaction of this organism with A. bisporus.