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