链霉菌702原生质体的制备和再生条件研究

Studies on Condition of Formation and Regeneration of Protoplasts from Streptomyces 702

为了确定链霉菌702菌株原生质体制备和再生较优组合条件,以链霉菌702菌株为试验材料,试验设计以单因素和多因素多水平的正交试验。在单因素试验结果中探索该菌的对数生长期为35 h^50 h,在菌丝体培养基中添加1.0%甘氨酸有利于该菌原生质体制备和再生;正交试验分别以影响该菌原生质体制备和再生的菌丝体培养时间、溶菌酶使用浓度、酶解温度和酶解时间的四因素三水平的L9(34)正交试验,试验表明:链霉菌702菌株原生质制备和再生较优组合为A2B2C3D1,即菌丝体培养时间为43 h,溶菌酶浓度为2.0 mg/mL,酶解温度为37℃,酶解时间60 m in,原生质体的制备率和再生率分别达到96.5%和27.8%。本试验为该菌进一步进行原生质体诱变打下良好的基础。

The better composition condition of the factors that affected the formation and regeneration of protoplasts from Streptomyces 702 were investigated. The experimental material was Streptomyces 702. And single factor and multifactor orthogonal experiment were used in this experiment. From single factor experiment the result proved that the logarithmic phase of this Streptomyces was 35 h - 50 h. Furthermore, it was propitious to form and regenerate the protoplasts of Streptomyces 702 that 1.0% of glycin was added in mycelium substrate. The L9 （3^4） orthogonal experiment was mycelium cultured time, lysozyme concentration, enzymolysis temperature and time. By using range analysis, varimace analysis and aggregative score, we analyse the outcome of the test. The results indicated that the better assembly condition of formation and regeneration of protoplasts from Streptomyces 702 was A2B2C3D1 （ mycelium cultured for 43 h, 2 mg/mL of lysozyme concentration, enzymolysis at 37℃ for 60 min）. The protoplasts rate of forming and regenerating was 96.5% and 27.8%. It laid good foundation for further mutagenesis of the protoplasts.