Objective

To investigate the protoplast preparation and transformation system of endophytic fungus Falciphora oryzae.

Results

F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9 M KCl solution and 10 mg mL⁻¹ glucanase at 30 °C with shaking at 80 rpm for 2–3 h. When the protoplasts were plated on a regenerations-agar medium containing 1 M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired green fluorescent protein-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots.

Conclusions

This study provided a protoplast transformation system of F. oryzae, creating opportunities for future genetic research in other endophytic fungi.

中文翻译：

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一种有效的暗中隔内生菌 Falciphora oryzae 基因操作方案

客观的

研究内生真菌Falciphora oryzae原生质体制备及转化体系。

结果

用含有 0.9 M KCl 溶液和 10 mg mL ^-1葡聚糖酶的酶消化液在 30 °C 下以 80 rpm 振荡 2-3 h处理米曲霉菌株获得更高的原生质体产量和有效转化。当原生质体接种在含有 1 M 蔗糖的再生琼脂培养基上时，原生质体的再生长率最高。我们通过将 pKD6-GFP 载体转化为原生质体，成功获得了表达绿色荧光蛋白的转化体。此外，真菌菌丝中的 GFP 表达在水稻根系共生过程中具有良好的稳定性和强度。

结论

该研究提供了一种稻瘟病菌的原生质体转化系统，为未来其他内生真菌的遗传研究创造了机会。