摘要
以耐药标记的鸭大肠杆菌ZBO78(Eryr,Chls)为亲本菌株,在DNaseⅠ始终存在的条件下,采用溶菌酶-EDTA法制备原生质体,然后在高渗琼脂平板上再生。通过优化溶菌酶-EDTA的工作浓度与作用时间、原生质体不同的处理方式和再生培养基中蔗糖浓度等条件,摸索出ZBO78以0.2g/L溶菌酶作用20min后补加EDTA至终浓度为0.01mmol/L继续作用16min为最佳试验条件,并成功获得了91.94%的原生质体制备率和37.29%的再生率,为下一步进行融合试验提供了依据。
With drug resistance tag on Esherichia coli 078 （Ery^r,Chl^s） strain as parental strain,protoplasts were prepared with lysozyme and EDTA while DNase I always existed ,then protoplasts can regenerate on medium with high osmotic pressure. Lots of conditions such as concentration and action time of lysozyme and EDTA,different preparation methods and concentration of＇sucrose in regeneration medium were systematic optimized,Esherichia coli 078 added with lysozyme 0.2 g/L acted for 20 minutes,EDTA was then added slowly to a final concentration of 0.01 mmol/L for another 16 minutes as the best experimental condition,91.94% protoplast formation frequency and 37.29% regeneration frequency were obtained,the results provide a way for the next fusion experiment.
出处
《中国家禽》
北大核心
2009年第7期22-26,共5页
China Poultry
基金
浙江省重点科技攻关项目(2005E60014)
浙江省农科院创新能力提升工程项目
关键词
大肠杆菌
原生质体
制备
再生
Eshe richia coli
protoplast
formation
regeneration
