South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China.
Fungal Biol. 2011 Mar;115(3):265-74. doi: 10.1016/j.funbio.2010.12.011. Epub 2010 Dec 31.
Cordyceps militaris is an insect-born fungus with various biological and pharmacological activities. The mutant library of C. militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation (ATMT), for the ultimate identification of genes involved in isolate degeneration during fruiting body production. Successful transformation of C. militaris JM4 by A. tumefaciens AGL-1 carrying vector pATMT1 was performed, with efficiency in the range of 30-600 transformants per 1×10(5) conidia. Acetosyringone (AS) supplement in C. militaris ATMT was not necessary during either precultivation or cocultivation. The transformation procedure was optimised based on the ratios between donor A. tumefaciens and recipient conidia, and pH value of cocultivation media. The integration of the hyg gene into C. militaris genome was determined by PCR and Southern blot analysis, suggesting that 67-88% resulting transformants in cultivation conditions with or without AS were inserted by T-DNA and 55-80% were single-copy. Special mutants with altered phenotypes and growth potentials were characterised. The efficient TAIL-PCR approach was established for identifying T-DNA flanking sequences from C. militaris mutants. The successful construction of the mutant library indicated the usefulness of this approach for functional genetic analysis in this important fungus.
蛹虫草是一种昆虫源真菌，具有多种生物学和药理学活性。通过改良的根癌农杆菌介导转化（ATMT）构建了蛹虫草的突变体文库，用于最终鉴定在子实体生产过程中分离物退化相关的基因。成功地将携带载体 pATMT1 的根癌农杆菌 AGL-1 转化蛹虫草 JM4，转化效率范围为每 1×10(5)个分生孢子 30-600 个转化子。在预培养或共培养过程中，蛹虫草 ATMT 中不需要乙酰丁香酮（AS）的补充。基于供体根癌农杆菌和受体分生孢子的比例以及共培养培养基的 pH 值，优化了转化程序。通过 PCR 和 Southern blot 分析确定了 hyg 基因整合到蛹虫草基因组中，表明在有或没有 AS 的培养条件下，67-88%的转化子是通过 T-DNA 插入的，55-80%是单拷贝的。对具有改变表型和生长潜力的特殊突变体进行了表征。建立了高效的 TAIL-PCR 方法，用于从蛹虫草突变体中鉴定 T-DNA 侧翼序列。突变体文库的成功构建表明，该方法在这种重要真菌的功能遗传分析中是有用的。
