Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids

文献类型： 外文期刊

第一作者： Ren, Rui

作者： Ren, Rui;Gao, Jie;Lu, Chuqiao;Wei, Yonglu;Jin, Jianpeng;Zhu, Genfa;Yang, Fengxi;Wong, Sek-Man;Wong, Sek-Man;Wong, Sek-Man

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关键词： Cymbidium orchids; protoplast isolation; protoplast-based transient expression system (PTES); subcellular location; bimolecular fluorescence complementation (BiFC) assay; gene regulation

期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：5.923； 五年影响因子：6.132 ）

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年卷期： 2020 年 21 卷 7 期

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收录情况： SCI

摘要： 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 x 10(7)/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.

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