title: Agrobacterium-mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis.
abstract: Filamentous fungus Trichoderma reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell division. The integration of the hph gene into T. reesei genome was determined by PCR and dot blot analysis. Transgenic T. reesei strains were analyzed using TAIL-PCR for their T-DNA contents. The results showed that T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination, which suggests that Agrobacterium-mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene transfer technology for T. reesei.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, Shandong, People's Republic of China.
authors.forename: Yao Hua
authors.initials: YH
authors.lastname: Zhong
authors.forename: Xiao Li
authors.initials: XL
authors.lastname: Wang
authors.forename: Tian Hong
authors.initials: TH
authors.lastname: Wang
authors.forename: Qiao
authors.initials: Q
authors.lastname: Jiang
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001483:Base Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D016681:Genome, Fungal; D006921:Hygromycin B / Q000494:pharmacology; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic / Q000235:genetics*; D014242:Trichoderma / Q000187:drug effects / Q000235:genetics*
chemical_list: D004271:DNA, Fungal; D006921:Hygromycin B