产紫杉醇内生真菌Fusarium maire的原生质体制备和再生

Formation and Regeneration of Protoplasts of Taxol-Producing Endophytic Fungus Fusarium maire

通过正交设计等实验方法,详细的研究了培养基成分、酶系组成、菌龄、渗透压缓冲剂、pH、酶解温度、时间和再生培养方式等多种因素对Fusarium maire原生质体制备和再生的影响。实验结果表明:将Fusarium maire通过先静止2 d再以180 r/min震摇2 d后,以0.6 mol/L的KCl配制的pH为5的复合酶(3 mg/dL溶壁酶、2.5 mg/dL蜗牛酶1、mg/dL溶菌酶和0.5 mg/dL纤维素酶)中,32℃恒温酶解4 h,原生质体制备产量达3.2×107个/mL;纯化后的原生质体在25℃,以0.6 mol/L的蔗糖配制的YCM再生培养基上单层培养,其再生率达8.9%。为今后的通过原生质体融合和诱变研究,构建紫杉醇工程菌奠定了基础。

A fast and reliable method to obtain protoplasts from Fusarium maire which could produce taxol was reported. After 2 days of still culture and 2 days of shaking-culture at 25 ℃, the mycelials was treated with a combination of lywallzyme（3 mg/dL）, snailase（2. 5 mg/dL）, lysozyme（1 mg/dL） and cellulase（0.5 mg/dL） in pH 5,and 0. 6 mol/L KCl used as the osmotic stabilizer. The high protoplast yield of 3. 2 - 10^7/mL was obtained in 4 h at 32 ℃. The conditions of protoplast regeneration were also investigated. The protoplasts were purified and regenerated on the solid YCM at 25 ℃, using 0.6 mol/L sucrose as the osmotic stabilizer. A high regeneration rate was reached at 8.90%.