Objective  The objective of this study is to examine the function of Zn(Ⅱ)₂Cys₆ transcription factor B8N6S4 in Aspergillus flavus, and to analyze the role of B8N6S4 in the growth, development, and Aflatoxin B₁ (AFB₁) biosynthesis of A. flavus.

Methods  Polyethylene glycol-mediated homologous recombination was used to construct deletion mutants of B8N6S4 gene, and the differences between the deletion mutants and the wild-type strain in terms of growth, development, AFB₁ biosynthesis and infection ability of A. flavus were compared.

Results  Bioinformatics analysis showed that B8N6S4 contained a typical GAL4-like Zn(Ⅱ)₂Cys₆ DNA-binding domain and a fungal-specific transcription factor domain. Phylogenetic analysis demonstrated that B8N6S4 exhibits the highest sequence similarity (97.99%) with the homologous protein KAB8271990.1 from the high aflatoxin-producing strain A. minisclerotigenes. Using homologous recombination, two A. flavus knockout strains, Δ B8N6S4-1 and Δ B8N6S4-2, were successfully constructed, and the successful deletion of the B8N6S4 gene and correct integration of selection marker genes were verified. Compared with the wild type, there was no significant change in the hyphal growth rate of two Δ B8N6S4 mutant on PDA medium, but the conidia yield was significantly reduced to7.5×10⁶, 8.0×10⁶ cells/mL, which was about 5.6 times lower than that of WT (4.4×10⁷ cells/mL). Notably, the sclerotia-forming capacity of the mutants was significantly improved, with the number of sclerotia reaching 767 and 836, which was about twice that of the WT strain (409 ± 39). The results of thin layer chromatography showed that the toxin synthesis ability of Δ B8N6S4-1 and Δ B8N6S4-2 mutants was enhanced, which increased by about 78% and 104%, respectively. In addition, the grain infection test of maize showed that the infection ability of two Δ B8N6S4 mutant was not significantly different from that of wild type. Spore counts following infection revealed comparable sporulation capacity between mutant and wild-type strains. However, AFB₁ production of Δ B8N6S4-1 and Δ B8N6S4-2 mutants on maize substrate was significantly increased, which was about 5.2 times and 3.9 times that of WT, respectively.

Conclusion  B8N6S4 is a Zn(Ⅱ)₂Cys₆ transcription factor of A. flavus, which positively regulates the asexual sporulation of A. flavus, negatively regulates the formation of sclerotia, and inhibits the biosynthesis of AFB₁. Δ B8N6S4 does not directly affect the pathogenicity of A. flavus but significantly enhances its AFB₁ production capacity. This study revealed for the first time the regulatory role of B8N6S4 in the aflatoxin biosynthesis network, providing a novel target for the prevention and control strategy to block aflatoxin biosynthesis.