Aspergillus flavus infection is a major issue for safe food storage. In this study, we constructed an efficient prokaryotic expression system for puroindoline B (PINB) protein to detect its antifungal activity. The Puroindoline b gene was cloned into pET-28a (+) vector and expressed in Escherichia coli. Treatment with fusion PINB revealed that it inhibits mycelial growth of A. flavus, a common grain mold. Moreover, fusion PINB-treated A. flavus mycelium withered and exhibited a sunken spore head. As fusion PINB concentration increased, electrical conductivity in mycelium also increased, indicative of cell membrane damage. Furthermore, intracellular malate dehydrogenase and succinate dehydrogenase activity decreased, revealing a disruption in the tricarboxylic acid cycle. Moreover, the dampened activity of the ion pump Na⁺K⁺-ATPase negatively affected the intracellular regulation of both ions. Catalase and superoxide dismutase activity decreased, thus reducing antioxidant capacity, a result confirmed with an increase in malondialdehyde content. Changes to the GSH/GSSG ratio indicated a shift to an intracellular oxidative state. At the same time, laser scanning confocal microscopy assay showed the accumulation of reactive oxygen species and nuclear damage. Therefore, the PINB fusion protein may have the potential to control A. flavus in grain storage and food preservation.

黄曲霉感染是安全保存食物的主要问题。在这项研究中，我们构建了一个有效的puroindoline B（PINB）蛋白原核表达系统，以检测其抗真菌活性。将Puroindoline b基因克隆到pET-28a（+）载体中并在大肠杆菌中表达。融合PINB的处理表明，它可以抑制常见谷物霉菌黄曲霉的菌丝体生长。此外，融合PINB处理的黄曲霉菌丝体枯萎，孢子头凹陷。随着融合PINB浓度的增加，菌丝体中的电导率也增加，表明细胞膜受损。此外，细胞内的苹果酸脱氢酶和琥珀酸脱氢酶活性降低，揭示了三羧酸循环的破坏。此外，离子泵Na ⁺ K ⁺的阻尼活性-ATPase对两种离子的细胞内调节均产生负面影响。过氧化氢酶和超氧化物歧化酶的活性降低，从而降低了抗氧化能力，结果证实丙二醛含量增加。GSH / GSSG比率的变化表明已转变为细胞内氧化态。同时，激光扫描共聚焦显微镜分析显示了活性氧的积累和核损伤。因此，PINB融合蛋白可能具有控制谷物存储和食品保存中黄曲霉的潜力。