杏鲍菇原生质体制备、再生及其遗传转化体系的建立  

Preparation and Regeneration of Protoplasts of Pleurotus eryngii and Establishment of Its Genetic Transformation System

作　　者：邹优花 连玲丹 钟武杰[1] 杜敏如 黄颖茵 王杰[1] ZOU Youhua;LIAN Lingdan;ZHONG Wujie;DU Minru;HUANG Yingyin;WANG Jie(College of Food Science,South China Agricultural University,Guangzhou 510642,Guangdong,China)

机构地区：[1]华南农业大学食品学院,广东广州510642

出　　处：《食品研究与开发》2023年第21期138-144,共7页Food Research and Development

基　　金：国家自然科学基金项目(31772020)。

摘　　要：为优化杏鲍菇GIM5.344原生质体的制备、再生条件及建立稳定有效的遗传转化体系,对裂解酶、酶解温度、酶解时间、再生培养基稳渗剂等原生质体制备、再生条件以及潮霉素浓度、聚乙二醇(polyethylene glycol,PEG)介导转化方法进行优化,探究不同原生质体制备、再生条件对原生质体产量和再生率的影响。结果表明:选用溶壁酶1进行酶解效果最佳,当酶解温度30℃、酶解时间2 h、再生培养基稳渗剂为甘露醇时,原生质体的产量可达到7×108CFU/mL,再生率达到0.40%。得到转化子最多的转化方法为固体预培养+双层培养法,即PEG介导转化后在无抗性再生培养基上28℃预培养48 h,然后再添加一层含有30μg/mL潮霉素的培养基进行拟转化子的筛选。所得转化子中的绿色荧光蛋白基因能稳定有效表达。In order to optimize the preparation and regeneration of the protoplasts of Pleurotus eryngii GIM5.344strain and to establish a stable and effective genetic transformation system,lytic enzymes,enzymatic hydrolysis temperature,enzymatic hydrolysis time,regeneration medium stabilizer in protoplast preparation conditions,as well as concentrations of hygromycin B and the polyethylene glycol(PEG)-mediated transformation method were optimized.The effects of different protoplast preparation and regeneration conditions on protoplast yield and regeneration rate were investigated.The results showed that the use of lysing enzyme 1 for enzymatic hydrolysis was the most effective.When enzymatic hydrolysis was conducted at 30 ℃ for 2 h using regeneration medium containing mannitol,the protoplast yield reached 7 × 108CFU/mL and the regeneration rate reached 0.40%.The transformation method that yielded the highest number of transformants was the solid pre-culture followed by a double-layer culture method.This method involved pre-culturing the protoplasts at 28 ℃ on non-selective regeneration medium for 48 h after PEG-mediated transformation.Subsequently,a layer of culture medium containing 30 μg/mL hygromycin was added for selection of putative transformants.The green fluorescent protein gene in the obtained transformants exhibited stable and effective expression.

关 键 词：杏鲍菇 原生质体 制备 再生 转化体系 

分 类 号：TS219[轻工技术与工程—粮食、油脂及植物蛋白工程]