Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids
Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids
复制标题
高效原生质体分离和瞬时表达系统用于兰花开花相关基因的功能表征
DOI：
10.3390/ijms21072264
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发表时间：
2020-04-01
影响因子：
5.6
通讯作者：
Yang, Fengxi
中科院分区：
文献类型：
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作者：
Ren, Rui;Gao, Jie;Yang, Fengxi
原生质体系统已被证明是现代植物生物学的有力工具。然而，成功制备丰富的原生质体仍然是大蕙兰面临的挑战。本研究通过优化酶解条件，建立了一种高效的兰花花瓣原生质体分离方法。最佳条件为d -甘露醇浓度（0.5 M）、酶浓度（1.2% （w/v）纤维素和0.6% （w/v）宏观酶）和酶解时间（6 h）。在该方案下，蕙兰花瓣原生质体产量最高（3.50 × 107/g鲜重），活力最高（94.21%）。此外，我们通过优化影响聚乙二醇（PEG）介导原生质体转染的因素，包括孵育时间、最终PEG4000浓度和质粒DNA数量，获得了较高的转染效率（80%）。该高效原生质体瞬时表达系统（PTES）进一步应用于兰花蛋白亚细胞定位、双分子荧光互补（BiFC）检测和开花相关基因的基因调控研究。总之，我们的原生质体分离和转染方案是高效、稳定和节省时间的。它可用于兰花和其他经济上重要的单子叶作物的基因功能和分子分析。
Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for Cymbidium orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 107/g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of Cymbidium. In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in Cymbidium orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops.