苦荞叶肉细胞原生质体的分离纯化及瞬时转化

Isolation,Purification and Transient Expression of Mesophyll Protoplast in Tartary Buckwheat

高效分离原生质体是遗传转化、细胞融合和再生培养的基础性工作。该实验以苦荞（Fagopyrum tartaricum）品种‘榆6-21’的叶肉细胞为材料,研究了酶类组合、甘露醇浓度、酶解时间以及离心速度对苦荞叶肉细胞原生质体分离纯化的影响。结果表明：酶解液组成为1.5%纤维素酶R-10＋0.5%离析酶R-10＋0.5mol/L甘露醇＋20mmol/L MES＋20mmol/L KCl＋10mmol/L CaCl2＋0.1%牛血清白蛋白,以第5~7片真叶为材料,用胶带纸撕去叶片下表皮后,25℃黑暗酶解4h,以900r/min离心收集,可以获得高质量的原生质体,原生质体产量可达6×106个/g,活力达到90%以上;用双荧光素酶报告基因载体检测原生质体的转化效率,以分离纯化的苦荞叶肉细胞原生质体为受体,将双荧光素酶报告基因载体与其混合后,在终浓度为20%PEG4000介导下,黑暗转化20min,可以检测到较高活性的萤火虫荧光素酶和海肾荧光素酶,证明双荧光素酶报告载体可成功转化到原生质体中。研究结果为苦荞原生质体瞬时转化及遗传操作提供了技术基础。

The basic work for the system establishment about genetic transformation,cell fusion and regeneration is to formulate efficient protoplast isolation system.The influence of different factors such as enzyme liquid combination,mannitol concentration,enzymatic hydrolysis time and centrifugal speeds on the isolation and purification of protoplast from tartary buckwheat‘YU 6-21＇mesophyll cells were studied.The results showed that the optimal enzyme solution for protoplast isolation was 1.5% cellulase R-10＋0.5% macerozyme R-10＋0.5mol/L mannitol＋20mmol/L MES＋20mmol/L KCl＋10mmol/L CaCl2＋0.1% BSA.Fifth to sevevth true leaves,which were removed the lower epidermal layer with tape,were incubated in enzyme solution for 4hat 25℃in dark,and centrifugal speeds was 900r/min for protoplast collection.The protoplast yield amounted to 6×106/g fresh weight and the vitality was up to 90%;Dual-luciferase reporter vector was used as the reporter gene and protoplasts were regarded as the receptor to measure protoplast transformation efficiency,when the concentration of PEG4000 was 20% and transfection time was 20 min in dark,the high luciferase activities of Photinus pyralis and Renilla reniformis could be detected,indicating that dual-luciferase reporter vector can be successfully loaded into protoplasts.The study provide technical basis for tartray buckwheat protoplast transient expression system and genetic manipulation.