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Optimization and Application of a Protoplast Preparation and Transformation System for Fusarium oxysporum
LIAOJiaoqun, ZHUWangjie, ZHANGHuawei
Chin Agric Sci Bull ›› 2026, Vol. 42 ›› Issue (11) : 50-56.
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Abbreviation (ISO4): Chin Agric Sci Bull
Editor in chief: Yulong YIN
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Optimization and Application of a Protoplast Preparation and Transformation System for Fusarium oxysporum
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.
Fusarium oxysporum / protoplast / preparation / transformation system / PEG-mediated transformation / virulence factor / gene knockout / genetic manipulation
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\n Fusarium\n, one of the most common fungal genera, has received considerable attention because of its biosynthetic exuberance, the result of many unique gene clusters involved in the production of secondary metabolites.\n
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Fungi have been assured to be one of the wealthiest pools of bio-metabolites with remarkable potential for discovering new drugs. The pathogenic fungi, Fusarium oxysporum affects many valuable trees and crops all over the world, producing wilt. This fungus is a source of different enzymes that have variable industrial and biotechnological applications. Additionally, it is widely employed for the synthesis of different types of metal nanoparticles with various biotechnological, pharmaceutical, industrial, and medicinal applications. Moreover, it possesses a mysterious capacity to produce a wide array of metabolites with a broad spectrum of bioactivities such as alkaloids, jasmonates, anthranilates, cyclic peptides, cyclic depsipeptides, xanthones, quinones, and terpenoids. Therefore, this review will cover the previously reported data on F. oxysporum, especially its metabolites and their bioactivities, as well as industrial relevance in biotechnology and nanotechnology in the period from 1967 to 2021. In this work, 180 metabolites have been listed and 203 references have been cited.
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