热研4号王草原生质体的制备  

A Effective Protocol for King Grass Reyan No.4 Protoplasts Preparation of King Grass Reyan No.4

作　　者：张继友[1] 景晓辉[2] 吴伦英[1] 刘国道[3] 吴琳[4] 

机构地区：[1]海南大学农学院,海口570228 [2]北京大学生命科学学院,北京100871 [3]中国热带农业科学院品种资源研究所,儋州571737 [4]中国热带农业科学院环境与植物保护研究所,海口571101

出　　处：《分子植物育种》2015年第4期915-918,共4页Molecular Plant Breeding

基　　金：国家自然科学基金项目(70963003);国家973计划项目(2007CB08903)共同资助

摘　　要：开展原生质体融合育种以及利用原生质体瞬时表达系统进行分子生物学实验的前提和基础是建立高效、有活力的原生质体制备体系。为建立热研4号王草原生质体制备体系，本研究以热研4号王草叶片为原生质体制备的外植体，将切成细丝的热研4号王草幼叶置于含2.0%纤维素酶＋0.5%果胶酶Y-23＋0.5%崩溃酶＋0.6 mol/L甘露醇＋40 mmol/L KCl＋6 mmol/L MES，pH 5.7＋20 mmol/L CaCl2＋0.15%BSA的酶液中，在避光条件下置于50 r/min的摇床酶解6~8 h，获得了大量有活力且均匀一致的原生质体。经过荧光素双醋酸酯（fluoresceindiacetate, FDA）染色和Western blot检测目的蛋白表达，结果发现采用酶解法制备的热研4号王草原生质体活力可达75%，且可用于表达拟南芥外源蛋白。本研究可为下一步利用热研4号王草开展分子生物学研究奠定基础。The prerequisites and foundations of the system for efficient plant regeneration from protoplast via somatic hybridization and transient expression in protoplast for molecular biology research is to build efficient and effective protoplast preparation system. Establishment of efficient isolation protoplasts from King Grass Reyan No.4 have failed thus far. In this paper, enzymatic method was used to isolate high-quality protoplasts from Pennisetumpur pureum＆#237;P. americanum cv. Reyan No.4. High-quality protoplast with a viability of 75 % were achieved by incubating the slimmed young leaves in a digestion enzymolic solution comprising 2%cellulase R-10＋0.5%pectolyase Y-23＋0.5%macerozyme R-10＋0.6 mol/L Mannitol＋40 mmol/L KCl＋6 mmol/L MES, pH 5.7＋20 mmol/L CaCl2＋0.15% BSA. Arabidopsis gene A TA F1 was further transfected into King Grass Reyan No.4 protoplasts to testify the efficiency of our system. SDS-PAGE electrophoresis and subsequent Western blot detection demonstrate that ATAF1 protein was accumulated in the transformed protoplasts. Together, these results showed that an efficient protoplast isolation system from young leaves was established for King Grass Reyan No.4.

关 键 词：热研4号王草 原生质体制备 酶解法 荧光素双醋酸酯染色 Western BLOT 

分 类 号：Q943[生物学—植物学]