Positive Fluorescent Selection Permits Precise, Rapid, and In-Depth Overexpression Analysis in Plant Protoplasts
Positive Fluorescent Selection Permits Precise, Rapid, and In-Depth Overexpression Analysis in Plant Protoplasts
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DOI：
10.1104/pp.108.133975
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发表时间：
2009-03-01
期刊：
影响因子：
7.4
通讯作者：
Birnbaum, Kenneth D.
中科院分区：
文献类型：
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作者：
Bargmann, Bastiaan O. R.;Birnbaum, Kenneth D.
植物原生质体的瞬时遗传修饰是一种简单而快速的技术，可用于研究植物生物学的许多方面。最近对后生动物系统的研究已经利用基于细胞的测定法来通过高通量方法询问信号转导途径。植物生物学家可以从新工具中受益，这些新工具扩大了细胞培养在大规模基因功能分析中的应用。我们开发了一种系统，采用荧光阳性选择结合流式细胞术分析和荧光激活细胞分选来专门分离转化原生质体中的反应。该系统克服了转染的原生质体悬浮液通常是已成功转化和尚未成功转化的细胞的异质混合物的缺点。该网关兼容系统能够利用过表达对遗传电路进行高通量筛选。红色荧光蛋白选择标记的结合可以与广泛使用的绿色荧光蛋白 (GFP) 工具结合使用。例如，这种双标记方法允许对转化细胞中的 GFP 报告基因激活进行细胞计数分析，或对特定 GFP 标记细胞群中的荧光激活细胞分选介导的分离和下游过表达效应进行检查。在这里，作为一个例子，该系统的新用途被应用于生长素信号传导的研究，利用红色荧光蛋白/GFP双重标记能力。为了响应通过显性负生长素信号成分的过度表达来操纵生长素响应网络，我们量化了对生长素响应 DR5::GFP 报告基因激活的影响，并分析了表达根表皮标记的细胞中的全基因组转录变化。
Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the study of numerous aspects of plant biology. Recent studies in metazoan systems have utilized cell-based assays to interrogate signal transduction pathways using high-throughput methods. Plant biologists could benefit from new tools that expand the use of cell culture for large-scale analysis of gene function. We have developed a system that employs fluorescent positive selection in combination with flow cytometric analysis and fluorescence-activated cell sorting to isolate responses in the transformed protoplasts exclusively. The system overcomes the drawback that transfected protoplast suspensions are often a heterogeneous mix of cells that have and have not been successfully transformed. This Gateway-compatible system enables high-throughput screening of genetic circuitry using overexpression. The incorporation of a red fluorescent protein selection marker enables combined utilization with widely available green fluorescent protein (GFP) tools. For instance, such a dual labeling approach allows cytometric analysis of GFP reporter gene activation expressly in the transformed cells or fluorescence-activated cell sorting-mediated isolation and downstream examination of overexpression effects in a specific GFP-marked cell population. Here, as an example, novel uses of this system are applied to the study of auxin signaling, exploiting the red fluorescent protein/GFP dual labeling capability. In response to manipulation of the auxin response network through overexpression of dominant negative auxin signaling components, we quantify effects on auxin-responsive DR5::GFP reporter gene activation as well as profile genome-wide transcriptional changes specifically in cells expressing a root epidermal marker.