Protoplast isolation and transient gene expression in the single-cell C<sub>4</sub> species, Bienertia sinuspersici

Shiu-Cheung Lung; Makoto Yanagisawa; Simon D. X. Chuong

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Protoplast isolation and transient gene expression in the single-cell C₄ species, Bienertia sinuspersici

机译：单细胞C _{4 物种Bienertia sinuspersici的原生质体分离和瞬时基因表达}

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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 C₄ species, despite their potential of serving as model systems for their extraordinary C₄ 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 C₄ 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 C₄ photosynthetic mechanism are discussed.

机译：尽管使用报道基因例如绿色荧光蛋白进行瞬时基因表达是检查基因功能和细胞内蛋白运输的通用工具，但高效基因操作方法的建立仍然是许多植物物种面临的挑战。尽管三个单细胞C _{4 物种具有潜在的作为其非常规C _{4 光合代谢模型系统的潜力，但尚未建立可靠的转化方案。我们报告了第一个优化的协议，用于从单细胞C _{4 物种Bienertia sinuspersici的绿藻细胞中分离出大规模且同质的原生质体。细胞化学染色证实，细胞壁消化后，在氯霉素细胞中细胞器的异常亚细胞区系得以保留。按照我们优化的聚乙二醇介导的转染程序，大约84％的分离的原生质体表达了报告荧光蛋白。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 光合机制中的进一步应用。}}}}

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《Plant Cell Reports》 |2011年第4期|p.473-484|共12页
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Shiu-Cheung Lung; Makoto Yanagisawa; Simon D. X. Chuong;
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1. Protoplast isolation and transient gene expression in the single-cell C species, Bienertia sinuspersici [J] . Lung Shiu-Cheung, Yanagisawa Makoto, Chuong Simon D. X. Plant Cell Reports . 2011,第4期

机译：单细胞C物种中的原生质体分离和瞬态基因表达，Bienertia Sinuspersici
2. Isolation of dimorphic chloroplasts from the single-cell C₄species Bienertia sinuspersici [J] . Shiu-Cheung Lung, Makoto Yanagisawa, Simon DX Chuong Plant methods . 2012,第10期

机译：从单细胞C _{4 物种Bienertia sinuspersici中分离出双形叶绿体}
3. Leaf development in the single-cell C-4 system in Bienertia sinuspersici: Expression of genes and peptide levels for C-4 metabolism in relation to chlorenchyma structure under different light conditions [J] . Lara MV, Offermann S, Smith M, Plant physiology . 2008,第1期

机译：Bienertia sinuspersici单细胞C-4系统中的叶片发育：在不同光照条件下与绿藻结构相关的C-4代谢基因和肽水平的表达
4. TRANSIENT EXPRESSION OF GENES WITH ALTERED METHYLATION LEVELS IN TOBACCO PROTOPLASTS [C] . N. BUCHERNA, L.E. HESZKY, I. NAGY Induced mutations and molecular techniques for crop improvement . 1995

机译：甲基化水平改变的基因在烟草原生质体中的瞬时表达
5. Spatial development of the cytoplasmic compartments for single cell C4 photosynthesis, and mechanisms of tolerance to salinity in Bienertia sinuspersici. [D] . Park, Joonho. 2008

机译：单细胞C4光合作用的细胞质区室的空间发育，以及对Bienertia sinuspersici盐度的耐受机制。
6. Leaf Development in the Single-Cell C4 System in Bienertia sinuspersici: Expression of Genes and Peptide Levels for C4 Metabolism in Relation to Chlorenchyma Structure under Different Light Conditions [O] . María Valeria Lara, Sascha Offermann, Monica Smith, 2008

机译：单能生物中的单细胞C4系统中的叶片发育：在不同光照条件下C4代谢基因和肽水平与小鳞茎结构相关的表达
7. Isolation of dimorphic chloroplasts from the single-cell C₄species Bienertia sinuspersici [O] . Lung Shiu-Cheung, Yanagisawa Makoto, Chuong Simon DX 2012

机译：从单细胞C _{4 物种 Bienertia sinuspersici 分离双态叶绿体}

Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici

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Protoplast isolation and transient gene expression in the single-cell C₄ species, Bienertia sinuspersici