State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China.
Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China.
Plant Methods. 2011 Sep 30;7(1):30. doi: 10.1186/1746-4811-7-30.
Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue.
Here, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts.
We show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.
植物原生质体是一种经过验证的生理和多功能细胞系统，广泛应用于高通量分析和基因功能表征。绿色原生质体已成功应用于与激素、代谢物和环境挑战相关的植物信号转导途径的研究。在水稻中，原生质体通常是从悬浮培养细胞或黄化幼苗中制备的，但只有少数研究探索了利用水稻绿色组织的原生质体。
在这里，我们报道了一种从正常培养的年轻水稻绿色组织中分离原生质体的简化方法，无需使用不必要的化学物质和真空设备。用广泛大小（4.5-13kb）的质粒对生成的原生质体进行转染，以及与多个质粒的共转染，实现了令人印象深刻的高效率，并允许通过 1）蛋白质免疫印迹分析、2）亚细胞定位测定、和 3）双分子荧光互补（BiFC）和萤火虫荧光素酶互补（FLC）的蛋白质-蛋白质相互作用分析进行评估。重要的是，水稻绿色组织原生质体具有光合作用活性，对逆行质体信号诱导剂 norflurazon（NF）敏感。GFP 标记的光相关转录因子 OsGLK1 的瞬时表达显著上调了内源性光合作用基因 OsLhcb1、OsLhcp、GADPH 和 RbcS 的转录水平，NF 处理在一定程度上降低了水稻绿色组织原生质体中的这些基因的转录水平。
我们在这里展示了一种使用具有光合作用活性的水稻绿色组织原生质体的简化和高效的瞬时基因表达系统，及其在蛋白质免疫印迹、定位和蛋白质-蛋白质相互作用测定中的广泛应用。这些水稻绿色组织原生质体将特别有助于研究与光/叶绿体相关的过程。
