根癌农杆菌介导的球毛壳菌遗传转化及T-DNA插入突变体的获得

Agrobacterium tumefaciens-mediated transformation of Chaetomium globosum and its T-DNA insertional mutagenesis

根癌农杆菌介导的遗传转化系统是植物基因工程常用方法 ,目前已将这一转化系统应用到酵母、丝状真菌以及人类细胞的转化。利用这一转化系统 ,成功地实现了丝状真菌球毛壳菌 (Chaetomiumglobosum)的遗传转化 ,转化率约为 6 0～ 180个转化子 10 7个孢子。通过对转化子的PCR检测和Southern杂交分析表明 ,T DNA已整合进毛壳菌基因组中 ,而且在所检测的转化子中都是以单拷贝的形式整合 ,转化子都能够稳定遗传。根癌农杆菌介导的遗传转化具有转化率高、低拷贝、遗传稳定、操作简便等优点 。

Agrobacterium tumefacines-mediated transformation is a routine method in the genetic transformation of a wide range of plant species, and so far, its application has been extended to the transformation of yeasts, fungi and even human cells. By using this transformation system, filamentous fungus Chaetomium globosum was successfully transformaed with an efficiency of 60～180 transformants per 10 7 spores. PCR and Southern analysis showed that the T-DNA was integrated into the genome. Among of the examined transformants contained a single copy, and was stable through mitotic cell division. The tranformation system mediated by Agrobacterium tumefaciens may prove to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.