| 摘要: |
| 为探索“红颜”草莓悬浮细胞系原生质体提取的最优条件，并建立“红颜”草莓原生质体瞬时转化体系，以“红颜”草莓悬浮细胞为材料，对酶液组成、酶解温度、酶解方式进行研究。用PEG介导的瞬时转化法将标记基因GFP转化到“红颜”草莓原生质体中。结果显示：以“红颜”草莓悬浮细胞系作为分离材料，酶液组合为CPW中含有0.5%PVP+0.1%MES+1%纤维素酶+0.5%离析酶+0.01%半纤维素酶+0.9 mol/L甘露醇，在低速（50 r/min）恒温（31 ℃）震摇下进行酶解反应，酶解10 h时，达到“红颜”草莓原生质体最佳分离效果，每克鲜重产量可得原生质体6×108 个，活力值可达93.0%。PEG介导法成功将含有绿色荧光蛋白（green fluorescent protein, GFP）的植物表达载体转化“红颜”草莓悬浮细胞原生质体，转化效率达44%。通过实验筛选得到“红颜”草莓悬浮细胞原生质体的最佳制备条件，建立“红颜”草莓悬浮细胞原生质体的瞬时转化体系，为进一步开展“红颜”草莓功能基因及合成生物学研究奠定基础。 |
| 关键词: “ 红颜 ” 草莓 悬浮细胞系 原生质体制备 瞬时转化体系 |
| DOI：10.14188/j.ajsh.2019.06.008 |
| 分类号:Q7 |
| 基金项目:三峡大学高层次人才科研启动基金(2016GCRC02) |
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| Preparation of protoplasts and establishment of transient transformation system of Hongyan strawberry |
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DU Xiaoyun1,WANG Zhen1,WANG Cuihua1,ZENG Gongjian1,SHEN Xiangling1
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Key Laboratory of Three Gorges Regional Plant Genetics & Germplasm Enhancement, China Three Gorges University, Yichang 443000, Hubei, China
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| Abstract: |
| In order to explore the optimal conditions for protoplast extraction of Hongyan strawberry suspension cell line, the transient transformation system was established, and the suspension cell of Hongyan strawberry was used as material to study the composition of enzyme solution, enzymatic hydrolysis temperature and enzymatic hydrolysis method. The marker gene GFP was transformed into the protoplast of Hongyan strawberry by PEG-mediated transient transformation.When the Hongyan strawberry suspension cells were used as the separation material, and the composition of enzyme solution was 0.5% PVP+0.1% MES+1% cellulase+0.5% isolation enzyme+0.01% hemicellulase+0.9 mol/L mannitol in CPW, enzymatic hydrolysis was carried out under low speed (50 r/min), constant temperature (31 °C) shaking for 10 h, the best separation effect of protoplasts of Hongyan strawberry can be achieved. The protoplast yield was up to 6×108 and the vitality value was up to 93.0%. By PEG-mediated method, the plant expression vector containing GFP was transformed into the protoplast of the Hongyan strawberry suspension cell with a transformation efficiency of 44%. The optimal preparation conditions of protoplasts of Hongyan strawberry suspension cells were obtained, and transient transformation system of protoplasts of Hongyan strawberry suspension cells was established. These studies lay a foundation for further research on functional genes and synthetic biology of Hongyan strawberry. |
| Key words: Hongyan strawberry suspension cell line protoplast preparation transient transformation system |
