Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants
Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants
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DOI：
10.1007/s00299-003-0638-1
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发表时间：
2003-09-01
影响因子：
6.2
通讯作者：
Stewart, CN
中科院分区：
文献类型：
--
作者：
Richards, HA;Halfhill, MD;Stewart, CN
绿色荧光蛋白（GFP）在生物学研究中的应用是显而易见的。开发了一种基于荧光的方法来定量转基因植物和蛋白质提取物中的GFP水平。荧光强度与GFP水平的增加呈线性关系，其范围包括花椰菜花叶病毒35S启动子在植物中的转基因表达。使用标准曲线来估计植物和蛋白质提取物中的GFP浓度。这些值与来自转基因植物的蛋白质提取物中GFP的ELISA测量值一致，表明该技术是重组GFP表达的可靠测量。然后估计纯合和半合植物中的植物内GFP表达水平。纯合转基因植物表达的GFP量是半合植物的两倍，表明加性转基因表达。该方法可用于简化植物中转基因表达的表征。
The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.