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Applied and Environmental Microbiology, January 2006, p. 484-490, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.484-490.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Analysis of an Inactive Aflatoxin Biosynthesis Gene Cluster in Aspergillus oryzae RIB Strains

National Research Institute of Brewing (NRIB), 3-7-1 Kagamiyama, Higashi-Hiroshima 739-0046, Japan,¹ Department of Molecular Biotechnology, Graduate School of Advanced Science of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan,² Bio’c Co., Ltd., 111-1 Murocho-Uchida, Toyohashi 441-8087, Japan³

Received 2 March 2005/ Accepted 12 October 2005

To help assess the potential for aflatoxin production by Aspergillus oryzae, the structure of an aflatoxin biosynthesis gene homolog cluster in A. oryzae RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99% similarity to those of Aspergillus flavus, three genes shared 93% similarity or less. A 257-bp deletion in the aflT region, a frameshift mutation in norA, and a base pair substitution in verA were found in A. oryzae RIB 40. In the aflR promoter, two substitutions were found in one of the three putative AreA binding sites and in the FacB binding site. PCR primers were designed to amplify homologs of aflT, nor-1, aflR, norA, avnA, verB, and vbs and were used to detect these genes in 210 A. oryzae strains. Based on the PCR results, the A. oryzae RIB strains were classified into three groups, although most of them fell into two of the groups. Group 1, in which amplification of all seven genes was confirmed, contained 122 RIB strains (58.1% of examined strains), including RIB 40. Seventy-seven strains (36.7%) belonged to group 2, characterized by having only vbs, verB, and avnA in half of the cluster. Although slight expression of aflR was detected by reverse transcription-PCR in some group 1 strains, including RIB 40, other genes (avnA, vbs, verB, and omtA) related to aflatoxin production were not detected. aflR was not detected in group 2 strains by Southern analysis.

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