Chromosomal location plays a role in regulation of aflatoxin gene expression in Aspergillus parasiticus.
The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-beta-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.
Chiou
Ching-Hsun
CH
Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824, USA.
Miller
Michael
M
Wilson
David L
DL
Trail
Frances
F
Linz
John E
JE
D000348:Aflatoxins / Q000096:biosynthesis*; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015825:Chromosomes, Fungal / Q000235:genetics*; D005656:Fungal Proteins; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014170:Transformation, Genetic
D000348:Aflatoxins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; C062609:norsolorinic acid ketoreductase; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D005966:Glucuronidase