Lin Hsiang-Yin, Chen Jhun-Chen, Fang Su-Chiung
Biotechnology Center in Southern Taiwan, Academia Sinica, Tainan, Taiwan.
Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan.
Front Plant Sci. 2018 Jun 22;9:843. doi: 10.3389/fpls.2018.00843. eCollection 2018.
The enigmatic nature of the specialized developmental programs of orchids has fascinated plant biologists for centuries. The recent releases of orchid genomes indicate that orchids possess new gene families and family expansions and contractions to regulate a diverse suite of developmental processes. However, the extremely long orchid life cycle and lack of molecular toolkit have hampered the advancement of orchid biology research. To overcome the technical difficulties and establish a platform for rapid gene regulation studies, in this study, we developed an efficient protoplast isolation and transient expression system for . This protocol was successfully applied to protein subcellular localization and protein-protein interaction studies. Moreover, it was confirmed to be useful in delineating the PaE2F/PaDPdependent cell cycle pathway and studying auxin response. In summary, the established orchid protoplast transient expression system provides a means to functionally characterize orchid genes at the molecular level allowing assessment of transcriptome responses to transgene expression and widening the scope of molecular studies in orchids.
几个世纪以来，兰花特殊发育程序的神秘本质一直吸引着植物生物学家。最近公布的兰花基因组表明，兰花拥有新的基因家族以及家族的扩增和收缩，以调控一系列不同的发育过程。然而，兰花极长的生命周期以及缺乏分子工具阻碍了兰花生物学研究的进展。为了克服技术难题并建立一个快速基因调控研究平台，在本研究中，我们开发了一种高效的原生质体分离和瞬时表达系统。该方案已成功应用于蛋白质亚细胞定位和蛋白质-蛋白质相互作用研究。此外，已证实其在描绘依赖于PaE2F/PaDP的细胞周期途径和研究生长素反应方面很有用。总之，所建立的兰花原生质体瞬时表达系统提供了一种在分子水平上对兰花基因进行功能表征的方法，能够评估转录组对转基因表达的反应，并拓宽了兰花分子研究的范围。
