title: Agrobacterium-mediated insertional mutagenesis of the ochratoxigenic fungus Aspergillus westerdijkiae.
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 10(6) 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 microg/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.
authors.affiliation: Centro de Ciências Biológicas, Universidade Estadual de Londrina, Paraná, Brazil.
authors.forename: Marcia M
authors.initials: MM
authors.lastname: Mata
authors.forename: Marta H
authors.initials: MH
authors.lastname: Taniwaki
authors.forename: Beatriz T
authors.initials: BT
authors.lastname: Iamanaka
authors.forename: Daniele
authors.initials: D
authors.lastname: Sartori
authors.forename: André L M
authors.initials: AL
authors.lastname: Oliveira
authors.forename: Márcia C
authors.initials: MC
authors.lastname: Furlaneto
authors.forename: Maria Helena P
authors.initials: MH
authors.lastname: Fungaro
mesh_terms: D001230:Aspergillus / Q000235:genetics* / Q000378:metabolism*; D003069:Coffee / Q000382:microbiology; D004271:DNA, Fungal / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000502:physiology*; D009793:Ochratoxins / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D012231:Rhizobium / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D003069:Coffee; D004271:DNA, Fungal; D009793:Ochratoxins; C025589:ochratoxin A