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中国农学通报

中国农学通报 ›› 2026, Vol. 42 ›› Issue (11): 50-56.doi: 10.11924/j.issn.1000-6850.casb2025-1041

• 生物科学 • 上一篇    下一篇

尖孢镰刀菌原生质体制备与转化体系优化及其应用

廖姣群1,2(), 祝王婕1,2, 章华伟1,2()   

  1. 1 浙江工业大学药学院， 杭州 310014
    2 绿色化学合成与转化技术全国重点实验室， 杭州 310014
  • 收稿日期:2025-12-28 修回日期:2026-03-20 出版日期:2026-06-12 发布日期:2026-06-12
  • 通讯作者:
    章华伟，男，1978年出生，安徽绩溪人，教授，博士，主要从事海洋候选药物临床前研究。通信地址：310014 浙江杭州拱墅区潮王路18号 浙江工业大学药学院，Tel：0571-13968063069，E-mail：。
  • 作者简介:

    廖姣群，女，2000年出生，湖南郴州人，硕士，研究方向：农业致病菌的基因改造。通信地址：310014 浙江杭州拱墅区潮王路18号 浙江工业大学药学院，E-mail：。

  • 基金资助:
    国家重点研发计划课题“基于多尺度发酵过程优化的候选药物绿色生物制造关键技术研究”(2022YFC2804203)

Optimization and Application of a Protoplast Preparation and Transformation System for Fusarium oxysporum

LIAO Jiaoqun1,2(), ZHU Wangjie1,2, ZHANG Huawei1,2()   

  1. 1 School of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014
    2 National Key Laboratory of Green Chemical Synthesis and Transformation Technology, Hangzhou 310014
  • Received:2025-12-28 Revised:2026-03-20 Published:2026-06-12 Online:2026-06-12

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摘要：

尖孢镰刀菌（Fusarium oxysporum）是重要农业致病菌与活性次级代谢产物资源真菌，高效稳定的原生质体转化体系是其基因功能研究与代谢调控的关键技术前提。为优化尖孢镰刀菌原生质体制备与转化体系，提高质粒转化效率；本研究以土大黄内生尖孢镰刀菌R1为材料，采用PEG介导转化法，通过单因素试验优化原生质体制备与转化条件；尖孢镰刀菌原生质体最佳制备条件为：CMC-Na培养基中产孢72 h，YEPD培养基中培养菌丝24 h，复合酶（崩溃酶: Yatalase: 溶菌酶=3: 1: 1，20 mg/mL）于36℃、 180 r/min、0.7 mol/L NaCl中酶解4 h，原生质体产量达3.81×107个/mL；以TB3为转化培养基，转化效率达27,000/mg（以单位质量的DNA转化子数计算）。应用研究表明，该体系成功敲除了镰刀菌R1内毒力因子fusarubin生物合成核心基因T1PKS，效率达62.5%；本研究构建了一套高产、稳定、高效的尖孢镰刀菌R1原生质体制备与PEG介导遗传转化体系，为该真菌的基因编辑、毒力机制解析、次级代谢产物挖掘及农业致病菌防控研究提供可靠技术支撑。

关键词: 尖孢镰刀菌, 原生质体, 制备条件, 转化体系, PEG介导转化, 毒力因子, 基因敲除, 遗传操作

Abstract:

Fusarium oxysporum is an important agricultural pathogen and active secondary metabolite resource fungus. Efficient and stable protoplast transformation system is the key technical prerequisite for its gene function research and metabolic regulation. To improve plasmid transformation efficiency, this study optimized the protoplast preparation and transformation system in Fusarium oxysporum; Using F. oxysporum R1 as the research object, the PEG-mediated transformation and single-factor experiments were employed to optimize the conditions for protoplast preparation and transformation. The optimal conditions for protoplast preparation of F. oxysporum were as follows: sporulation in CMC-Na medium for 72 h, mycelial culture in YEPD medium for 24 h, and enzymatic digestion with a composite enzyme cocktail (drislase: yatalase: lysozyme = 3: 1: 1, 20 mg/mL) in 0.7 mol/L NaCl at 36 ℃ and 180 r/min for 4 h. Under these conditions, the protoplast yield reached 3.81×107 protoplasts/mL. Using TB3 as the transformation medium, the transformation efficiency reached 27,000/mg (calculated as the number of transformants per unit mass of DNA). Experiment demonstrated that this system successfully knocked out the core virulence factor gene T1PKS involved in fusarubin biosynthesis in F. oxysporum R1, achieving an efficiency of 62.5%. In this study, a high-yield, stable and efficient protoplast preparation and PEG-mediated genetic transformation system of F. oxysporum R1 was constructed, which provided reliable technical support for gene editing, virulence mechanism analysis, secondary metabolite mining and prevention and control of agricultural pathogenic bacteria.

Key words: Fusarium oxysporum, protoplast, preparation, transformation system, PEG-mediated transformation, virulence factor, gene knockout, genetic manipulation

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