JELLYFISH GREEN FLUORESCENT PROTEIN AS A REPORTER FOR VIRUS-INFECTIONS
JELLYFISH GREEN FLUORESCENT PROTEIN AS A REPORTER FOR VIRUS-INFECTIONS
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DOI：
10.1046/j.1365-313x.1995.07061045.x
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发表时间：
1995-06-01
期刊：
影响因子：
7.2
通讯作者：
CRUZ, SS
中科院分区：
文献类型：
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作者：
BAULCOMBE, DC;CHAPMAN, S;CRUZ, SS
将编码绿色荧光蛋白(GFP)的基因导入以马铃薯X病毒(PVX)为载体的病毒载体中。接种PVX.GFP的PVX寄主植株发生系统侵染。这些植物在接种后1至2天内最初检测到GFP的产生，方法是在接种的叶片上存在在紫外光下发出明亮绿色荧光的区域。随后，这种绿色荧光在系统感染的组织中很明显。荧光可以用多种方法检测。其中最简单的方法是在黑暗的房间里观察紫外线照射的植物。对感染PVX.GFP的组织进行了荧光或共聚焦激光扫描显微镜分析。这些显微镜方法使病毒的存在定位于单个受感染的细胞。也可以通过光谱或受感染植物提取物的电泳法来检测绿色荧光。为了说明该报告基因在病毒学研究中的潜在应用，构建了PVX.GFP的衍生物，其中PVX的外壳蛋白基因被GFP取代。对接种组织的激光共聚焦扫描显微镜显示，病毒仅限于接种的细胞，从而证实了早先的猜测，即PVX外壳蛋白对于细胞间的移动是必不可少的。GFP很可能在转基因植物和病毒感染组织中用作报告基因。
The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.