Atkins Simon D, Mauchline Tim H, Kerry Brian R, Hirsch Penny R
Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK.
Mycol Res. 2004 Jun;108(Pt 6):654-61. doi: 10.1017/s0953756204009906.
The nematophagous fungus Pochonia chlamydosporia is a potential biocontrol agent against root knot and cyst nematodes. Genetic transformation of the fungus to introduce visual marker genes, novel traits, or changes in expression levels of endogenous genes, would greatly enhance understanding of its behaviour on nematode-infested roots and of its interactions with other soil and rhizosphere microorganisms. A transformation system for the introduction of novel genes into P. chlamydosporia has been developed. Methods to generate protoplasts, introduce DNA and regenerate transformed viable fungal mycelium have been optimised, using plasmids carrying the green fluorescent protein marker gene gfp and the hygromycin resistance gene hph. Cultures of P. chlamydosporia were resistant to high levels of a range of fungal inhibitors, including hygromycin, that are commonly used with dominant selectable marker genes in the transformation of other fungi. However, regenerating protoplasts transformed with hph could be selected by their ability to grow through an agar overlay containing 1 mg ml(-1) hygromycin. Green fluorescence was observed in protoplasts and regenerating mycelium after transformation with gfp, but the GFP phenotype was lost on subculture. Maintenance of introduced genes was not stable, and during subculture, PCR assays indicated that the transformants lost both hph and gfp. When these genes were introduced on the same plasmid, segregation of hph and gfp was observed prior to their loss. It was unclear whether the introduced plasmids were able to replicate autonomously in P. chlamydosporia, or if they integrated transiently into the fungal genome. Possible reasons for the instability of the transformants are discussed.
食线虫真菌厚垣普可尼亚菌是一种潜在的防治根结线虫和孢囊线虫的生物防治剂。对该真菌进行遗传转化以引入视觉标记基因、新性状或改变内源基因的表达水平，将极大地增进我们对其在受线虫侵染的根上的行为以及与其他土壤和根际微生物相互作用的理解。已开发出一种将新基因导入厚垣普可尼亚菌的转化系统。利用携带绿色荧光蛋白标记基因gfp和潮霉素抗性基因hph的质粒，对原生质体的生成、DNA的导入以及转化后的活真菌菌丝体的再生方法进行了优化。厚垣普可尼亚菌培养物对一系列高水平的真菌抑制剂具有抗性，包括潮霉素，而潮霉素常用于其他真菌转化中的显性选择标记基因。然而，用hph转化的再生原生质体可以通过其在含有1 mg/ml潮霉素的琼脂覆盖层上生长的能力来筛选。用gfp转化后，在原生质体和再生菌丝体中观察到绿色荧光，但在继代培养时GFP表型消失。导入基因的维持不稳定，在继代培养期间，PCR分析表明转化体同时丢失了hph和gfp。当这些基因在同一质粒上导入时，在它们丢失之前观察到hph和gfp的分离。尚不清楚导入的质粒是否能够在厚垣普可尼亚菌中自主复制，或者它们是否暂时整合到真菌基因组中。讨论了转化体不稳定的可能原因。
