中国生物工程杂志

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 106 conidia). T-DNA insertional library of T.harzianum Th-33 mediated by A. tumefaciens was constructed, which 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 T. 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 .tumefaciens mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene clone for T. harzianum .