拮抗木霉gz-2菌株原生质体制备及转化条件优化

摘要

[目的]优化拮抗木霉gz-2菌株原生质体制备及转化条件,快速、高效获得绿色荧光标记菌株,为哈茨木霉及同类真菌的GFP标记提供参考,并为下一步研究其在不同环境中的定殖动态、分布规律及生防特性等打下基础.[方法]采用PEG-CaCl2介导的原生质体转化体系,从木霉菌株的菌龄、酶解时间、转化体系中质粒的含量及再生培养基中潮霉素B(HmB)的浓度等方面对哈茨木霉gz-2菌株原生质体制备及转化条件进行优化,获得表达稳定的转化子,并与出发菌株的生长特性进行比较,评价转化效果.[结果]gz-2菌株培养36 h菌丝所形成的原生质体稳定性最好,菌丝体酶解3.5 h获得的原生质体数量最多,达11.67×106个/mL;每240μL转化体系中质粒DNA含量为40μL,得到的转化子数量最多,为8.67个/皿;再生培养基中HmB含量为600μg/mL时可获得稳定的强转化子.[结论]筛选获得1株表达稳定的转化子GFP-gz-2菌株,该菌株在菌落形态、菌丝生长速率及产孢量上与出发菌株gz-2无显著差异,且具有较好的遗传稳定性.%[Objective]In this study,protoplast preparation and transformation conditions of antagonistic Trichoder-ma strain gz-2 were optimized to obtain strains marked by green fluorescent tag rapidly and effectively,provide reference for GFP tags of T. harzianum and other fungi of the same kind,and lay a foundation for the study of colonization dynamics, distribution regulation and biocontrol features.[Method]The conditions of protoplast preparation and transformation of T. harzianum strain gz-2 was optimized with the protoplast transformation system mediated by PEG-CaCl2. The condi-tions were optimized from following aspects: age and digestion time of T. harzianum strain,content of plasmid in the transformation system and the concentration of hygromycin B(HmB) in regeneration medium. Transformants whose expressions were stable were obtained ,and the growth features of them were compared with the original strains to evaluate transfon effects.[Result]Protoplast prepared by hypha of strain gz-2 cultured for 36 h was the most stable. The number of protoplast(11.67 × 106 protoplast/mL) was the largest when mycelium enzymolysis time was 3.5 h. When plasmid DNA content was 40μL in every 240μL transformation system,the obtained transformant number was the larg-est,reaching 8.67 transformant/plate. When HmB content in regeneration medium plate was 600μg/mL,stable strong transformants could be obtained.[Conclusion]A stable transformant GFP-gz-2 strain is obtained which has sound genetic stability. There is no significant difference in colony morphology,hypha growth rate and spore quantity between it and original strain gz-2.