Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici
Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici
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DOI：
10.1007/s00299-010-0953-2
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发表时间：
2011-04-01
影响因子：
6.2
通讯作者：
Chuong, Simon D. X.
中科院分区：
文献类型：
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作者：
Lung, Shiu-Cheung;Yanagisawa, Makoto;Chuong, Simon D. X.
尽管使用绿色荧光蛋白等报告基因进行瞬时基因表达是检查基因功能和细胞内蛋白质运输的通用工具，但建立高效的基因操作方法在许多植物物种中仍然是一个挑战。尽管这三种单细胞 C-4 物种有可能作为其非凡的 C-4 光合代谢的模型系统，但尚未建立可靠的转化方案。我们报告了第一个优化的方案，用于从单细胞 C-4 物种 Bienertia sinospersici 的绿藻细胞中分离大规模且同质的原生质体群体。细胞化学染色证实了细胞壁消化后，绿藻细胞中不寻常的细胞器亚细胞区室的保存。经过我们优化的聚乙二醇介导的转染程序后，大约 84% 的分离原生质体表达报告荧光蛋白。标记有各种细胞内分选信号的荧光融合蛋白证明了瞬时基因表达系统在亚细胞蛋白定位和细胞器动力学研究中的潜在用途。讨论了当前原生质体分离和转染技术在理解新型单细胞 C-4 光合作用机制中的进一步应用。
Although transient gene expression using reporters such as green fluorescent protein is a versatile tool for examining gene functions and intracellular protein trafficking, the establishment of a highly efficient gene manipulation method remains a challenge in many plant species. A reliable transformation protocol has not yet been established for the three single-cell C-4 species, despite their potential of serving as model systems for their extraordinary C-4 photosynthetic metabolism. We report the first protocol optimized for isolating a large-scale and homogenous population of protoplasts from chlorenchyma cells of the single-cell C-4 species Bienertia sinuspersici. Cytochemical staining confirmed the preservation of the unusual subcellular compartmentation of organelles in chlorenchyma cells after cell wall digestion. Approximately 84% of isolated protoplasts expressed the reporter fluorescent protein following our optimized polyethylene glycol-mediated transfection procedures. Fluorescent fusion protein tagged with various intracellular sorting signals demonstrated potential use of the transient gene expression system in subcellular protein localization and organelle dynamics studies. Further applications of the current protoplast isolation and transfection techniques in understanding the novel single-cell C-4 photosynthetic mechanism are discussed.