Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the fungus Penicillium marneffei
| Autor: | Shuangxi Ren, Shen Huan, Zhen Qian, Li Yunqian, Bin Xu, Shengrong Tang, Piyan Zhang, Yuezhu Wang |
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| Rok vydání: | 2008 |
| Předmět: |
Genetic Vectors
Mutant Mutagenesis (molecular biology technique) Plant Science Biology Polymerase Chain Reaction law.invention Microbiology Insertional mutagenesis Transformation Genetic law Genetics Cloning Molecular Ecology Evolution Behavior and Systematics Polymerase chain reaction Southern blot Penicillium Agrobacterium tumefaciens biology.organism_classification Mutagenesis Insertional Transformation (genetics) Phenotype Penicillium marneffei Biotechnology |
| Zdroj: | Mycological Research. 112:943-949 |
| ISSN: | 0953-7562 |
| DOI: | 10.1016/j.mycres.2008.01.017 |
| Popis: | Penicillium marneffei is an opportunistic fungal pathogen of humans, causing respiratory, skin, and systemic mycosis in south-east Asia. Here we describe the transformation of P. marneffei with Agrobacterium tumefaciens, and the optimization of the transformation procedure. Transformations in different combinations between A. tumefaciens stains (LBA4404 and EHA105) and binary vectors (pCB309A, pBI129A, and pCaMBIA1312A) showed that EHA105/pBI129A were the most efficient partners. Southern blot analysis suggested that 87.5 % of transformants obtained with this protocol displayed single hybridization bands, indicating a single insert of T-DNA in each of the transformants. Unique hybridization patterns, along with thermal asymmetric interlaced PCR (TAIL-PCR) analysis of T-DNA insertion sites, suggested that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in P. marneffei. Several mutants with altered phenotypes were obtained during the construction of the mutant library, indicating the usefulness of the approach for functional genetic analysis in this important fungal pathogen. |
| Databáze: | OpenAIRE |
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