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首页> 外文期刊>Plant molecular biology reporter >A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants
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A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants

机译：用于对转基因植物中的mGFP5-ER和sGFP（S65T）进行荧光定量的协议

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The Green Fluorescent Protein (GFP) from Aequorea victoriahas begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP i,n transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant ill sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor~(TM) Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 +- 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 #mu#g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 #mu#g and 2.11 #mu#g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfp5-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 #mu#g mGFP5-ER per mg extractable protein.
机译：维多利亚水母（Aequorea victoria）的绿色荧光蛋白（GFP）已开始用作植物中的报告蛋白。由于可以以非破坏性方式检测GFP荧光，因此它特别有用，而基于酶的报道分子的检测通常需要破坏植物组织。使用GFP作为报道基因使得在任何生长阶段都可以在体内筛选转基因植物组织。 GFP i，n转基因植物提取物的定量将增加GFP作为报告蛋白的效用。我们在此报告了使用BioRad VersaFluor〜（TM）荧光计系统或Labsystems Fluoroskan Ascent通过紫外光激发对烟草叶提取物中的mGFP5-ER变体进行定量分析，以及通过蓝光激发对sGFP（S65T）生病的甘蔗叶和愈伤组织提取物进行了定量分析FL配备了窄带发射滤光片（510±5 nm）。从在未转化的植物提取物中制备的GFP标准系列中测定转基因植物提取物中的GFP浓度，其浓度范围为0.1至4＃μg/ ml纯化的rGFP。玉米遍在蛋白启动子驱动下，甘蔗愈伤组织和叶片中sgfp（S65T）的表达水平分别为每毫克可提取蛋白0.525＃mu＃g和2.11＃mu＃g sGFP（S65T）。在菜叶中，由花椰菜花叶病毒（CaMV）35S启动子驱动的mgfp5-ER的表达范围为每mg可提取蛋白高达7.05＃mu＃g mGFP5-ER。

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