Agrobacterium tumefaciens-mediated transformation of the dermatophyte, Trichophyton mentagrophytes: an efficient tool for gene transfer

作者：Yamada Tsuyoshi; Makimura Koichi^*; Satoh Kazuo; Umeda Yoshiko; Ishihara Yumiko; Abe Shigeru

来源：Medical Mycology, 2009, 47(5): 485-494.

DOI：10.1080/13693780802322240

• dermatophyte
• Agrobacterium tumefaciens
• genetic transformation
• targeted gene disruption

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to facilitate gene transfer into the clinically important dermatophyte, Trichophyton mentagrophytes (teleomorph: Arthroderma vanbreuseghemii). A binary vector containing a hygromycin B resistance cassette was introduced into A. tumefaciens, and the resultant strain was co-cultivated with fungal small conidia. Transformation yielded a large number of hygromycin B-resistant transformants. Hybridization analysis showed that most of the transformants harboured a single copy of T-DNA randomly integrated into the genome. Transformation frequency was increased to more than 200 per 10 7 conidia by optimizing the co-cultivation time and temperature. ATMT was then used for targeted gene disruption mediated by homologous recombination. Using a PCR-based strategy, we isolated the areA/nit2-like nitrogen regulatory gene (tnr) Trichophyton nitrogen regulator) from T. mentagrophytes. A binary vector containing two regions of the tnr locus flanking the hygromycin B resistance cassette was constructed and introduced into T. mentagrophytes via ATMT. Transformants with disruption of the areA/nit-2-like gene (tnr) were obtained in three of four independent disruption experiments, most of which showed homologous recombination via double crossover without additional ectopic integration of the disruption construct.

• 出版日期2009
• 单位河北医科大学