Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus
Open Access
- 26 November 2007
- journal article
- research article
- Published by Springer Science and Business Media LLC in BMC Microbiology
- Vol. 7 (1), 104
- https://doi.org/10.1186/1471-2180-7-104
Abstract
Background: An available whole genome sequence for Aspergillus flavus provides the opportunity to characterize factors involved in pathogenicity and to elucidate the regulatory networks involved in aflatoxin biosynthesis. Functional analysis of genes within the genome is greatly facilitated by the ability to disrupt or mis-express target genes and then evaluate their result on the phenotype of the fungus. Large-scale functional analysis requires an efficient genetic transformation system and the ability to readily select transformants with altered expression, and usually requires generation of double (or multi) gene deletion strains or the use of prototrophic strains. However, dominant selectable markers, an efficient transformation system and an efficient screening system for transformants in A. flavus are absent. Results: The efficiency of the genetic transformation system for A. flavus based on uracil auxotrophy was improved. In addition, A. flavus was shown to be sensitive to the antibiotic, phleomycin. Transformation of A. flavus with the ble gene for resistance to phleomycin resulted in stable transformants when selected on 100 μg/ml phleomycin. We also compared the phleomycin system with one based on complementation for uracil auxotrophy which was confirmed by uracil and 5-fluoroorotic acid selection and via transformation with the pyr4 gene from Neurospora crassa and pyrG gene from A. nidulans in A. flavus NRRL 3357. A transformation protocol using pyr4 as a selectable marker resulted in site specific disruption of a target gene. A rapid and convenient colony PCR method for screening genetically altered transformants was also developed in this study. Conclusion: We employed phleomycin resistance as a new positive selectable marker for genetic transformation of A. flavus. The experiments outlined herein constitute the first report of the use of the antibiotic phleomycin for transformation of A. flavus. Further, we demonstrated that this transformation protocol could be used for directed gene disruption in A. flavus. The significance of this is twofold. First, it allows strains to be transformed without having to generate an auxotrophic mutation, which is time consuming and may result in undesirable mutations. Second, this protocol allows for double gene knockouts when used in conjunction with existing strains with auxotrophic mutations. To further facilitate functional analysis in this strain we developed a colony PCR-based method that is a rapid and convenient method for screening genetically altered transformants. This work will be of interest to those working on molecular biology of aflatoxin metabolism in A. flavus, especially for functional analysis using gene deletion and gene expression.Keywords
This publication has 25 references indexed in Scilit:
- Tuber borchiimycelial protoplasts isolation, characterization and functional delivery of liposome content, a new step towards truffles biotechnologyFEMS Microbiology Letters, 2005
- Clustered Pathway Genes in Aflatoxin BiosynthesisApplied and Environmental Microbiology, 2004
- Template preparation for rapid PCR in Colletotrichum lindemuthianumBrazilian Journal of Microbiology, 2003
- Current Research on Reducing Pre‐ and Post‐harvest Aflatoxin Contamination of U.S. Almond, Pistachio, and WalnutJournal of Toxicology: Toxin Reviews, 2003
- Practice Guidelines for Diseases Caused by AspergillusClinical Infectious Diseases, 2000
- An improved colony-PCR method for filamentous fungi for amplification of PCR-fragments of several kilobasesJournal of Biotechnology, 1998
- Design and testing of a plant‐specific PCR primer for ecological and evolutionary studiesMolecular Ecology, 1992
- Optimized vectors and selection for transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and phleomycin resistanceGene, 1990
- Bleomycin resistance: a new dominant selectable marker for plant cell transformationPlant Molecular Biology, 1986
- Development of a high-frequency transforming vector for Aspergillus nidulansGene, 1985