Generation of Aspergillus carbonarius mutants by Agrobacterium tumefaciens-mediated transformation method
DOI:
https://doi.org/10.5433/1679-0367.2006v27n2p95Keywords:
Aspergillus carbonarius, Ochratoxin A, Agrobacterium tumefaciens, Genetic transformation, Insertional mutants.Abstract
Aspergillus carbonarius is a potent ochratoxin A producer, a mycotoxin that has nephrotoxic and carcinogenic effects. The knowledge of genes involved in biosynthesis of this toxin may be useful for the development of detection and control methods. The Agrobacterium tumefaciens-mediated transformation method has been demonstrated as a powerful tool to obtain insertional mutants for the characterization of new genes. The aim of this work was to adapt the A. tumefaciens-mediated transformation method for ITAL187 strain of A. carbonarius and to obtain transformants with alteration in ochratoxin A production. Conidia were transformed to hygromicin B resistance using AGL-1 strain of A. tumefaciens. The transformation frequency was 20,04 transformants per 105 target conidia. The transformation evidence was obtained by PCR and Southern Blot analysis. The last one showed us that the hph gene integration was randomly in the genome fungi. Among 238 transformants, a total of 12 (5,042%) showed morphological variations. Three transformants (T44, T47 and T188) with consistently reduced and two transformants (T238 and T162) with increased ochratoxin A production were obtained. The identification of nocauted genes will contribute to ochratoxin A biosynthesis clarify.
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