根癌农杆菌介导的哈茨木霉菌遗传转化的研究

T-DNA Insertional Mutation of Trichoderma harzianum Mediated by Agrobacterium tumefaciens and Characteristic Analysis of the Transformants

利用根癌农杆菌EHA105进行了哈茨木霉Th-33的转化,在优化的转化条件下,EHA105对木霉菌的转化效率约45-100个转化子/106个孢子。利用该方法已建立了含4000多个转化子的木霉T-DNA插入突变体库。随机挑选24个转化子进行遗传稳定性分析,结果显示转化子经过5代无选择压力连续转接后都能在选择平板上正常生长;潮霉素抗性基因的PCR扩增和Southern blot分析表明,木霉转化子含有该基因,Southern blot进一步表明转化子多为单拷贝随机插入。该转化体系的改进将有利于木霉菌生防功能基因的克隆和作用机制的研究。

Filamentous fungus Trichoderma harzianum Th-33 was successfully transformed with Agrobacterium tumefaciens EHA105 for random integration of transforming DNA（T-DNA）. Co-cultivation of T. harzianum Th-33 conidia with A. tumefaciens EHA105 in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency（ about 45 -100 transformants per 10^6 conidia）. T-DNA insertional library of T. harzianum Th-33 mediated by A. consisted of more than 4000 transformants. Twenty-four randomly selected resistant clones were proved to be stable through five generations mitotic cell division. The integration of the hph gene into harzianum genome was determined by PCR and Southern blot analysis. The results showed that T-DNA was inserted into all the transformants with random and single copy mainly. These suggest that A. potentially powerful tool towards ns mediated transformation is a mutagenesis and gene clone for T. harzianum.