茂源链霉菌原生质体的制备与再生

Preparation and regeneration of protoplast from Streptoverticillium mobaraensis

【目的】探索制备高纯度茂源链霉菌原生质悬液的条件,为原生质体融合提供支持。【方法】在茂源链霉菌菌丝体一级培养不同时间（4,8,12,16,20,24,28,32,36,40,44,48h）后,称取菌丝体干质量,确定一级培养最佳时间;将一级培养的菌丝体转接培养二级菌丝体,在不同培养时间（13,14.5,16,17.5,19,20.5h）根据原生质体制备率、再生率的综合情况及菌丝体形态显微镜观察结果,确定二级菌丝体的培养时间及培养基中添加甘氨酸的最佳质量浓度（0,2,5,10,20g/L）,根据原生质体制备率和再生率确定使用溶菌酶的质量浓度（2,5,8,10,20,50mg/mL）和酶解时间（1,2,3,4,5,6,7,8h）,使用不同方法（500r/min离心法与双层18μm和0.45μm滤膜过滤法）分离原生质体,对原生质体分离后的损耗情况进行比较,确定分离条件。【结果】茂源链霉菌一级菌丝体的最佳培养时间为28h;二级菌丝体培养的最佳条件为：在培养基中添加5g/L甘氨酸,培养16h,溶菌酶最适质量浓度为8mg/mL,酶解3~4h;以500r/min离心分离原生质体可以获得较纯净的原生质体悬液;茂源链霉菌原生质体制备率最高达97.82%,再生率最高达9.04%,纯度最高达87.53%。【结论】得到了制备高纯度的茂源链霉菌原生质体悬液的条件。

【Objective】The optimal preparation conditions of protoplast of Streptoverticillium mobaraensis were studied to provide information for fusion of protoplast.【Method】After being cultured for different times（4,8,12,16,20,24,28,32,36,40,44,and 48h）,the optimal time for primary culturing was determined by weighing the dry mass of mycelium.Then the optimal secondary culturing time and concentration of glycine in medium were determined by culturing the mycelium for different times（13,14.5,16,17.5,19 and 20.5h）and different glycine concentrations（0,2,5,10 and 20g/L）as well as considering the preparation ratio,regeneration ratio,and morphology.The concentrations of protoplast suspension（2,5,8,10,20 and 50 mg/mL）,the hydrolysis times（1,2,3,4,5,6,7and 8h）and different separation methods（500r/min centrifugation and 18μm and 0.45μm membrane filtration）were also tested and compared to determine optimal conditions.【Result】The culture time of primary strain body was 28 h.The optimal cul-turing conditions for secondary mycelium were：5g/L glycine,culture time 16 h,lysozyme concentration 8mg/mL,enzyme degradation time 3-4h,and using 500r/min centrifugation.With the optimal conditions,the preparation ratio,regeneration ratio and purity of protoplast were 97.82%,9.04%,and 87.53%,respectively.【Conclusion】The optimal conditions for preparation of protoplast with high purity fromStreptoverticillium mobaraensis were obtained.