在对红豆杉内生真菌(Ozoniumsp.)适生碳源、氮源和生长情况研究的基础上,通过正交试验筛选了其发酵培养基和原生质体制备的酶系统;用L9(34)安排了四因素三水平并考虑交互作用的正交试验,对实验结果进行了分析.结果表明,最优的发酵培养基为果糖1%、蔗糖1%、蛋白胨0.2%、酵母粉0.5%、KH2PO40.5%、MgSO4·7H2O0.3%、VB10.001%;分离原生质体的最优酶系统为1.5%溶壁酶+0.5%蜗牛酶+1.5%纤维素酶+1.0%溶菌酶;用此酶系统在30℃条件下酶解3h,原生质体的产量达6.55×107个/mL酶液;经荧光素二醋酸酯(FDA)染色评估原生质体活力,表明该条件下分离的原生质体活力较高,原生质体的再生率为2.56%.该研究为利用生物技术手段改良紫杉醇生产菌奠定了基础.图6表4参32 Based on the studies on the suitable carbon source, nitrogen source and growth of endophytic fungi (Ozonium sp.), The enzyme system of the fermentation medium and protoplast was screened by orthogonal test. The L9 (34) Four factors and three levels and considering the interaction of the orthogonal experiment, the experimental results were analyzed.The results showed that the optimal fermentation medium for fructose 1%, sucrose 1%, peptone 0.2%, 0.5% yeast extract, KH2PO40.5 %, MgSO4 · 7H2O0.3% and VB10.001%. The optimal enzyme system for protoplast isolation was 1.5% lyticase + 0.5% snail enzyme + 1.5% cellulase + 1.0% lysozyme. The proteolysis was carried out at 30 ℃ for 3h and the protoplast yield reached 6.55 × 107 / mL. The viability of protoplasts was evaluated by the staining of fluorescein diacetate (FDA), indicating that the protoplasts isolated under this condition had higher viability, Body regeneration rate of 2.56% .This study for the use of biotechnology means to improve paclitaxel-producing bacteria laid the foundation .Figure 6 Table 4 参 32