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Agrobacterium-mediated insertional mutagenesis of the ochratoxigenic fungus Aspergillus westerdijkiae

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Abstract:

Aspergillus westerdijkiae is a potent ochratoxin A (OTA) producer that has been found in coffee beans. OTA is known to have nephrotoxic effects and carcinogenic potential in animal species. Here we report for the first time the Agrobacterium-mediated transformation for Aspergillus westerdijkiae and the generation of ochratoxin-defective mutants. Conidia were transformed to hygromycin B resistance using strain AGL-1 of Agrobacterium tumefaciens. The obtained transformation frequency was up to 47 transformants per 106 target conidia. Among 600 transformants, approximately 5% showed morphological variations. Eight transformants with consistently reduced OTA production were obtained. Two of these transformants did not produce OTA (detection limit: 0.1 g/kg); the other six mutants produced lower amounts of OTA (1%–32%) compared with the wild-type strain. By using thermal asymmetric interlaced polymerase chain reaction, we successfully identified a putative flavin adenine dinucleotide monooxygenase gene.Key words: Aspergillus ochraceus, Aspergillus westerdijkiae, Agrobacterium-mediated transformation, Agrobacterium-mediated insertional mutagenesis, ochratoxin A.

Aspergillus westerdijkiae est un producteur important d'ochratoxine A (OTA) qui a été trouvé dans les fèves de café. L'OTA est reconnue pour ses effets néphrotoxiques et son potentiel cancérigène chez les espèces animales. Nous rapportons ici pour la première fois la transformation d'Aspergillus westerdijkiae par l'intermédiaire d'Agrobacterium et la génération de mutants défectifs en ochratoxine. Les conidies ont été transformées avec la souche AGL-1 d'Agrobacterium tumefaciens conférant la résistance à l'hygromycine B. La fréquence de transformation obtenue allait jusqu'à 47 transformants par million de conidies cibles. Parmi les 600 transformants obtenus, environ 5 % présentaient des variations morphologiques. Huit transformants dont la production d'OTA était régulièrement réduite ont été obtenus. Deux d'entre eux ne produisaient pas d'OTA (limite de détection : 0,1 µg/kg) tandis que les six autres mutants produisaient de plus faibles quantités d'OTA (1 % – 32 %) comparativement à la souche sauvage. En utilisant un PCR à asymétrique thermique entrelacée, nous avons pu identifier un gène potentiel d'une FAD monooxygénase.Mots clés : Aspergillus ochraceus, Aspergillus westerdijkiae, transformation via Agrobacterium, mutagenèse d'insertion via Agrobacterium, ochratoxine A.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: January 1, 2007

More about this publication?
  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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