电压门控钾离子通道介导依枯草菌素A的抗真菌分子机制

真菌污染及其产生的真菌毒素广泛存在于食品生产和贮存过程中，严重威胁着食品安全和人类健康。由枯草芽孢杆菌分泌的环状脂肽依枯草菌素A（IA）具有广谱的抗真菌活性。然而，IA的抗真菌作用靶点及具体机制尚不清楚，限制了开发应用。申请人前期研究发现，IA可能通过激活电压门控钾离子通道（Kv），引起钾离子外流，导致真菌凋亡，但具体调控机制尚不清楚。为此，项目拟以炭黑曲霉为模式真菌，利用基因重组和X射线衍射技术解析Kv蛋白结构；通过中国仓鼠卵巢细胞（CHO）体外表达Kv蛋白和全细胞膜片钳技术分析IA对Kv电生理特性的影响；采用分子对接技术明确Kv蛋白与IA相互作用位点，并利用同源重组技术对真菌细胞内的可疑作用位点进行基因突变，以期揭示IA对Kv的调控作用及其与IA抗真菌作用之间的分子关联。本项目旨在为绿色、安全、高效抗真菌制剂的研制提供生物学途径，为IA的开发及推广应用奠定重要的前期理论基础。

Fungal contamination and mycotoxins produced by it are widely existed in the process of food production and storage, which seriously threaten food safety and human health. Iturin A (IA), a cyclic lipopeptide secreted by Bacillus subtilis, has broad-spectrum of antifungal activity. However, the antifungal target and specific mechanism of IA are still unknown, which limits the development and application of IA. According to the applicant's previous study, IA may cause the outflow of potassium ion through the activation of voltage-gated potassium ion channel (Kv), leading to fungal apoptosis, but the specific regulatory mechanism is still unclear. Therefore, the project plans to use Aspergillus carbonarius as the model fungus, use gene recombination and X-ray diffraction technology to analyze the structure of Kv protein. Use Chinese hamster ovary cells (CHO) to express Kv protein in vitro and use the whole cell patch clamp technology to analyze the influence of IA on the electrophysiological characteristics of Kv. Use molecular docking technology to identify the interaction site of Kv protein and IA, and use homologous recombination technology to carry out gene mutation on the suspected action sites in fungal cells, so as to reveal the regulatory role of IA on Kv and its molecular correlation with the antifungal effect of IA. This project aims to provide a biological pathway for the development of green, safe and effective antifungal agents and lay an important theoretical foundation for the development and application of IA.