Aspergillus oryzae, although closely related to Aspergillus flavus, does not produce aflatoxin (AF). A. oryzae RIB strains can be classified into three groups (group 1-3) based on the structure of the AF biosynthesis gene homolog cluster (AFHC). In group 1 strains, where AFHC is present, the expression level of the aflR gene is extremely low and there is no expression of the other four AF homologue genes (avnA, verB, omtA and vbs). We conducted a detailed structural comparison of AFLR ORF and AFLJ ORF from A. oryzae and A. flavus and identified several amino-acid substitutions. If these substitutions induce inactivation of AFLR and AFLJ, AF biosynthesis of A. oryzae will be doubly inhibited at the transcriptional and translational level. In this study, we transferred aflR and aflJ to A. oryzae RIB67, a group 2 strain where more than half of AFHC is missing. Under control of the pgkA promoter, aflR and aflJ was expressed and avnA, verB, omtA and vbs gene expression were monitored by RT-PCR. We prepared six types of forced-expression vectors, including aflR (from A. oryzae RIB40 or its three mutants) or aflJ (from A. oryzae RIB40 or A. flavus RIB4011). RT-PCR analysis showed that transformants containing aflJ from A. oryzae displayed no expression of AF biosynthetic homologue genes, whereas aflR substitutions had no such effect. These results strongly suggest that the amino-acid substitutions in AFLJ of A. oryzae induce inactivation at the protein level.
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