小菇属真菌(Mycena spp。)遗传转化体系是研究其促进天麻种子萌发机制的重要基础，而其原生质体的制备和再生是该体系构建的必要条件。以小菇菌株MY004为实验材料，通过单因素分析和正交试验策略，探究其原生质体制备和再生的影响因素，构建小菇原生质体制备和再生体系。结果表明，在含10 mM MES和0。7 M甘露醇的酶解缓冲液(pH=6)中，小菇MY004菌丝被10 mg/mL真菌溶壁酶在24 ℃条件下酶解3 h，其原生质体的释放率较高;经液体再生培养基(pH=5)培养3天，其原生质体再生效率可达2％。利用该体系，高效地完成了含报告基因eGFP转基因小菇的菌株纯化。小菇原生质体制备和再生体系可用于小菇遗传转化体系的快速构建，也将为小菇与天麻互作分子机制的研究提供基础平台。
Protoplast preparation,regeneration,and their application of a Mycena strain for promoting the seed germination of Gastrodia elata
The genetic transformation system of Mycena species is crucial for investigating the mo-lecular mechanism for studying the molecular mechanism behind the fungi's promotion of seed ger-mination in Gastrodia elata.To establish this system in Mycena spp.,it is essential to prepare and regenerate protoplasts.Single-factor analysis and multifactor orthogonal test were employed to in-vestigate the effects of protoplast preparation and regeneration on developing an optimal protoplast preparation and regeneration system in the Mycena strain MY004.The results exhibited that trea-ting MY004 mycelia with 10 mg/mL lywallzyme in an enzymatic buffer(pH=6)containing 10 mM MES and 0.7 M mannitol at 24 ℃ for 3 hours resulted in a higher rate of protoplast release.The protoplasts were then cultured in liquid regeneration medium at a pH of 5 for three days,resulting in a remarkable regeneration efficiency of up to 2％.Furthermore,this system was utilized to efficiently purify a transgenic strain containing the reporter gene eGFP(enhanced green fluorescent protein)in the Mycena strain MY004.The protoplast preparation and regeneration system can be used to rapid-ly establish the genetic transformation system in the Mycena strain MY004,and it provides a basic platform for studying the molecular mechanism of the interaction between Mycena and G.elata.
Mycenaprotoplast preparationprotoplast regenerationpurification of trans-genic strain
万方数据
维普
