雷公藤悬浮细胞原生质体的制备及瞬时转化体系的建立

为探索药用植物雷公藤(Tripterygium wilfordii)悬浮细胞原生质体提取的最优条件,并建立雷公藤原生质体瞬时转化体系,以雷公藤悬浮细胞为材料,对酶解液配比、酶解时间、甘露醇浓度及处理转速进行考察。用PEG介导的瞬时转化法将外源基因转化到雷公藤原生质体中。结果表明,以雷公藤悬浮细胞为材料提取原生质体的最佳条件是酶液配比为2.0%纤维素酶+0.5%果胶酶+0.5%离析酶,甘露醇浓度为0.6 mol·L–1,酶解10小时,处理转速为67×g;用PEG介导法将含有编码GFP的植物表达载体转化雷公藤悬浮细胞原生质体,激光共聚焦扫描显微镜下细胞显示绿色荧光。通过实验筛选得到雷公藤悬浮细胞原生质体的最佳提取条件,建立了雷公藤悬浮细胞原生质体的瞬时转化体系,为进一步开展雷公藤功能基因及合成生物学研究奠定了基础。

Protoplast Isolation and Establishment of Transient Expression System of Tripterygium wilfordii Suspension Culture Cells

We aimed to find the best protoplast extraction conditions with Tripterygium wilfordii suspension culture cells and establish an efficient transient expression system. Key factors in protoplast isolation, such as concentration of enzymes, time of enzymolysis, osmotic pressure and centrifugation speed, were studied. PEG mediation was used to transfer the GFP gene into protoplasts. The optimal extraction system was with cellulose R-10 2.0%, pectinase Y-23 0.5%, macerozyme R-10 0.5%, 0.6 mol?L^-1 mannitol. The suitable enzymolysis time was 10 h, and the optimal centrifugation speed was 67 ×g. Under LSCM, the protoplast emitted green fluorescence after transforming the plasmid encoding green fluorescent protein into it. We revealed the optional conditions to isolate protoplast of T. wilfordii suspension cells and established the transient transformation system; it laid a foundation for further study on the functional genes and syn- thetic biology of T. wilfordii.