Expression of a gfp gene in Penicillium nordicum under control of the promoter of the ochratoxin A polyketide synthase gene.
A gfp reporter gene strain of Penicillium nordicum was constructed in order to be able to study the influence of environmental parameters on ochratoxin A biosynthesis. To introduce the gfp gene an Agrobacterium tumefaciens mediated transformation system (ATMT) for P. nordicum was established resulting in a transformation efficiency of about 60 transformants per microg of DNA. The selection principle was based on the hygromycin B resistance gene located on the TI-DNA fragment of the binary vector system. PCR and Southern blot hybridization revealed that the TI-DNA was integrated into the chromosome of P. nordicum. To show that the GFP protein can be used as a reporter gene in P. nordicum, this species was subsequently transformed with a vector, carrying a gfp gene under the control of the strong constitutive gpd promoter of A. nidulans. Moreover in this vector construction the gfp gene contained a stuA nuclear localization domain. Successful transformed strains showed a strong GFP activity located in the nuclei after light stimulation in contrast to the wild type which showed only very weak unspecific auto fluorescence under these conditions. Based on this proof of principle a vector was constructed in which the promoter of the otapksPN gene, the gene of the ochratoxin A polyketide synthase of P. nordicum was placed in front of the gfp gene. This construct was transformed into P. nordicum by ATMT and the resulting transformants were analysed by fluorescence microscopy. In these transformants the whole mycelial cells showed GFP activity after light stimulation, whereas the wild type strain did not. When the transformed strains were grown on medium which suppressed ochratoxin A biosynthesis, a very low level of fluorescence could be detected, whereas a high level of fluorescence was seen after growth on medium supportive for ochratoxin A biosynthesis.
Schmidt-Heydt
Markus
M
Max Rubner-Institute, Karlsruhe, Germany.
Schunck
Tobias
T
Geisen
Rolf
R
D004271:DNA, Fungal; D005453:Fluorescence; D005506:Food Contamination / Q000032:analysis*; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006921:Hygromycin B; D009714:Nucleus Accumbens; D009793:Ochratoxins / Q000096:biosynthesis*; D010407:Penicillium / Q000235:genetics* / Q000378:metabolism; D036741:Plant Tumor-Inducing Plasmids; D048630:Polyketide Synthases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012231:Rhizobium / Q000235:genetics; D014170:Transformation, Genetic
D004271:DNA, Fungal; D009793:Ochratoxins; D049452:Green Fluorescent Proteins; C025589:ochratoxin A; D006921:Hygromycin B; D048630:Polyketide Synthases