Genetic transformation and mutagenesis of the entomopathogenic fungus paecilomyces fumosoroseus
Genetic transformation and mutagenesis of the entomopathogenic fungus paecilomyces fumosoroseus
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DOI：
10.1006/jipa.1999.4885
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发表时间：
1999-11
影响因子：
3.4
通讯作者：
F. Cantone;J. Vandenberg
中科院分区：
文献类型：
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作者：
F. Cantone;J. Vandenberg
我们开发了一种基于对除草剂草铵膦抗性的昆虫病原真菌烟熏拟青霉的dna介导转化系统。这种抗性是由吸湿链霉菌的bar基因提供的，并受中性曲霉trpC启动子和终止子序列的控制。在加入核酸酶抑制剂aurintricarboxylic acid （ATA）的辅助下，线性化质粒DNA的频率高达110个/g。在选择性和非选择性条件下连续5次植物移植，转化体的耐药性稳定。Southern分析显示，转化DNA以单拷贝和多拷贝的形式整合到烟状假人基因组中。通过限制性内切酶介导的整合（REMI）产生的单拷贝整合事件效率更高，尽管转化率通常不如含有ATA的处理高。利用这种方法恢复了两株对俄罗斯小麦蚜虫产孢能力和毒力发生改变的突变株。这种基于转化的操作将有助于插入诱变和各种基因的功能分析。
We have developed a DNA-mediated transformation system for the entomopathogenic fungus Paecilomyces fumosoroseus based on resistance to the herbicide glufosinate ammonium. The resistance is provided by the bar gene from Streptomyces hygroscopicus and is under the control of the Aspergillus nidulans trpC promoter and terminator sequences. Frequencies of up to 110 transformants/g of linearized plasmid DNA were obtained aided by the addition of the nuclease inhibitor, aurintricarboxylic acid (ATA). The transformants were stable for drug resistance for five consecutive vegetative transfers under selective and nonselective conditions. Southern analyses revealed that the transforming DNA integrated into the P. fumosoroseus genome in single and multiple copies. Single-copy integration events were generated with higher efficiency by restriction enzyme-mediated integration (REMI), although rates of transformation were generally not as high as those treatments containing ATA. Two mutant strains altered in sporulation capacity and virulence toward the Russian wheat aphid were recovered using this approach. This transformation-based manipulation of P. fumosoroseus will facilitate insertional mutagenesis and the functional analysis of various genes.