刺糖多孢菌原生质体制备与再生条件优化

Protoplast formation and regeneration for Saccharopolyspora spinosa

目的研究多杀菌素产生菌刺糖多孢菌(Saccharopolyspora spinosa)原生质体制备与再生的最佳条件。方法利用数理统计的方法研究了不同制备培养基、菌龄、甘氨酸浓度、溶菌酶处理条件以及再生培养基对原生质体制备和再生的影响,并考察了原生质体的适宜保藏温度。结果菌体在添加0.3%甘氨酸的EHC培养基中培养72h,用2mg/mL溶菌酶32℃酶解40min后,涂布在再生培养基R6上再生,原生质体制备率超过99%,再生数可达到107cfu/mL。刺糖多孢菌原生质体可置于4℃短期保存72h,长期保存需要放置于-80℃条件下。结论优化的结果为刺糖多孢菌原生质体融合育种和遗传转化体系建立奠定了基础。

Objective The optimal conditions for the formation, regeneration and preservation of protoplasts were established in Saccharopolyspora spinosa N-15-50-4B, a spinosad-producing strain. Methods Several crucial factors were tested on protoplast formation and regeneration including the preparation medium, spawn age, glycine concentration, regeneration medium and the treatment conditions of lysozyme. Results The results presented that the protoplast could be efficiently formed and regenerated under a certain condition. When the collected mycelia from S. spinosa N-15-50-4B grown in EHC medium with 0.3% glycine for 72h and were treated by 2mg/mL lysozyme at 32℃for 40min, then plated on the R6 medium, the rate ofprotoplasts formation could reach more than 99% and the number of regeneration protoplast was up to 107cfu/mL. We also studied the effects of temperature on protoplast preservation. Short-term storage for protoplasts was 4℃ for 72h, and long-term storage needed -80℃. Conclusion These studies enable to improve spinosad-producing strains though protoplast fusion and to establish genetic transformation system ofS. spinosa.