GFP基因在软枣猕猴桃愈伤组织原生质体中瞬间表达的初步研究  

The transient expression of GFP gene in callus protoplasts of Actinidia arguta

作　　者：朱道圩[1] 米银法[1] 陈延惠[1] 王静毅[1] 刘中杰[1] 

机构地区：[1]河南农业大学林学园艺学院,河南郑州450002

出　　处：《河南农业大学学报》2003年第2期145-148,共4页Journal of Henan Agricultural University

基　　金：河南省自然科学基金项目(0111011700)

摘　　要：用PEG介导法对游离出来的软枣猕猴桃原生质体进行遗传转化,成功地将绿色荧光蛋白(GFP)基因导入了原生质体,并获得了瞬间表达.结果表明,影响GFP基因转化的因素主要是材料的生理状态,PEG的种类和浓度;适宜的转化条件和方法为,将从白色、较为疏松愈伤组织中游离的原生质体,悬浮到质量分数为13%甘露醇的洗液(CPW13)中,置于45℃水浴中热激5min,再放在冰浴中迅速冷却;加入质粒,轻轻混匀后静止10min;逐滴加入质量分数40%PEG6000介导转化,终浓度为100g·L-1,在室温下放置20min;逐滴加入CPW13稀释转化后的原生质体,以避免PEG浓度剧烈变化造成细胞膜破裂.The green fluorescent protein(GFP) from the jellyfish Aequorea victoria has proven to be a useful tool in plant genetic transformation.The results showed that GFP gene was successfully transferred into A.arguta protoplasts by PEG method.The physiological condition of material,different PEG solution and the time of heat stimulus affected transformation.The protoplasts from white friable callus were suspended in CPW 13 solution,then incubated briefly with plasmid DNA for 10 min to allow sufficient contact between the DNA and the plasma membrane.A 40% PEG6000 solution was added to bring the final PEG concentration to 10%. PEG solution must be added slowly and dropwise,because the rapid addition of concentrated PEG can cause a sharp increase in osmotic potential and result in fusion or breakage of protoplasts.After a 15 min incubation,PEG was diluted with CPW13 solution,and after centrifuging the protoplasts were cultured in MS medium.Heat shock for 5 min in 45 ℃ water was included in this procedure.

关 键 词：软枣猕猴桃 愈伤组织 原生质体 瞬间表达 GFP基因 PEG介导法 遗传转化 绿色荧光蛋白 报告基因 

分 类 号：S663.4[农业科学—果树学]