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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。
In order to explore the optimal conditions for protoplast extraction from the suspension cell of Tripterygium wilfordii and to establish the transient transformation system of the protoplast of Tripterygium wilfordii, the suspension cells of Tripterygium wilfordii were used as materials, the ratio of the hydrolyzate, the enzymolysis time, Mannitol concentration and processing speed were investigated. The exogenous gene was transformed into tripterygium wilfordii protoplasts by PEG-mediated transient transformation. The results showed that the optimal conditions for extracting protoplasts from the suspension cells of Tripterygium wilfordii were as follows: 2.0% cellulase + 0.5% pectinase + 0.5% isozyme, mannitol concentration 0.6 mol·L-1 , Hydrolysis for 10 hours, the processing speed of 67 × g; using PEG-mediated method will contain plant expression vector encoding GFP transformed tripterygium wilfordii suspension cell protoplasts, laser scanning confocal microscope cells showed green fluorescence. Through the experimental screening, the optimal extraction conditions of the protoplasts of the suspension cells of Tripterygium wilfordii were established, and the instantaneous transformation system of the protoplasts of the suspension cells of Tripterygium wilfordii was established, which lays the foundation for the further study on the functional genes and the synthetic biology of Tripterygium wilfordii.