柄蓝状菌原生质体的制备与再生条件优化

Optimized Conditions for Preparation and Regeneration of Protoplasts from Talaromyces Stipitatus

为了建立以聚乙二醇/氯化钙(PEG/CaCl2)原生质体介导的柄蓝状菌(Talaromyces Stipitatus)高效的遗传转化系统,分别从材料选择,真菌菌龄,不同的渗透压稳定剂,酶的不同种类配比,酶的浓度,酶解时间及不同再生方式对柄蓝状菌EMM原生质的制备与再生条件进行优化。结果表明:以柄蓝状菌EMM接种生长48 h的菌丝为制备材料,1 mol/L MgSO4作为渗透压稳定剂,菌丝在温度为30℃,浓度为50 mg/mL的裂解酶溶液中酶解3 h,得到的原生质体先用再生培养基孵化培养12 h,然后再与PDA培养基混合铺板,以上组合条件下原生质体的释放量可达8.73×108/mL,再生率可达17.7%。

To establish a high efficient genetic transformation system mediated by the polyethylene glycol/calcium chloride (PEG/CaCl2) protoplast for Talaromyces Stipitatus (T. Stipitatus),the preparation andregeneration conditions of EMM protoplasts from T. Stipitatus were optimized such as materials,fungal age,different osmotic pressure stabilizers,different enzyme type ratios,enzyme concentration,enzymolysis timeand the regeneration mode. The results show that when the protoplasts are prepared with age of 48 h myceliafrom T. Stipitatus EMM as preparation materials,1 mol/L MgSO4 as the osmotic stabilizer in mass concentrationof 50 mg/mL lyase digested for 3 h at 30 ℃,the protoplasts are cultured in the regeneration medium for 12 hand finally plated with PDA medium,the release amount of protoplasts is 8.73 × 108/mL with 17.7% of the regeneration rate.