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Specific objectives are to utilize the basic information obtained from aflatoxin biosynthesis (1) to identify the defects in the Aspergillus sojae gene homologs and (2) to determine the molecular basis of why A. sojae does not produce aflatoxins.
(1) The gene sequences of aflR and aflJ, pivotal to the regulation of aflatoxin biosynthesis in A. parasiticus, will be determined from several A. sojae strains to see what nucleotide variations are present. (2) The ability of the predicted A. sojae AFLR regulatory protein to activate gene expression will be examined by a reporter gene system. (3) The interaction of A. sojae AFLR with A. sojae AFLJ will be investigated by a yeast two hybrid system, which studies protein-protein interaction. (4) Regions and/or amino acid residues, crucial for interaction, in A. sojae as well as A. parasiticus AFLR and AFLJ, will be localized by deletion and site directed mutagenesis analyses. (5) The function of AFLJ in the activation of gene expression will be elucidated in a yeast system by a combination of gene co-expression, reporter gene, real-time PCR, and enzyme activity assays.