Engineered GFP as a vital reporter in plants
Engineered GFP as a vital reporter in plants
复制标题
DOI：
10.1016/s0960-9822(02)00483-9
复制
发表时间：
1996-03-01
期刊：
影响因子：
9.2
通讯作者：
Sheen, J
中科院分区：
文献类型：
--
作者：
Chiu, WL;Niwa, Y;Sheen, J
背景资料：绿色荧光蛋白（GFP）的水母Aequorea维多利亚最近被用作一个通用的记者在广泛的异源活细胞和生物体。虽然在一些基于强启动子或高拷贝数病毒载体的植物瞬时表达测定中是成功的，但是GFP要用作完整植物中的标记物，需要进一步提高表达效率和荧光强度。在这里，我们报告说，一个广泛修改的GFP是一个通用的和敏感的记者在各种活的植物细胞和转基因plants.Results：我们表明，一个重新设计的GFP基因序列，与高度表达的人类蛋白质的青睐密码子，给出了20倍以上的GFP在玉米叶细胞中的表达比原来的水母GFP序列。当与发色团中的突变相结合时，用苏氨酸替换位置65处的丝氨酸，新的GFP序列在用490 nm（蓝色）光激发时给出超过100倍更亮的荧光信号，并且形成更大的发色团。我们还表明，这种修饰的GFP在各种瞬时表达系统中具有广泛的用途，并且允许容易地检测弱启动子活性，可视化的蛋白质靶向到细胞核和各种质体，并在活的单细胞和转基因plants.Conclusions的信号转导通路的分析：修改后的GFP是一个简单而经济的新工具，直接可视化的启动子活动的强度和细胞特异性的范围广泛。它可以用于测量信号转导途径的动态响应、转染效率和嵌合蛋白的亚细胞定位，并且应该适用于高等植物的遗传修饰活细胞和组织的许多其他应用。这些数据还表明，密码子使用效应可能是普遍的，允许设计在进化上遥远的物种如人类和玉米中具有高表达效率的重组蛋白。
Background: The green-fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently been used as a universal reporter in a broad range of heterologous living cells and organisms. Although successful in some plant transient expression assays based on strong promoters or high copy number viral vectors, further improvement of expression efficiency and fluorescent intensity are required for GFP to be useful as a marker in intact plants. Here, we report that an extensively modified GFP is a versatile and sensitive reporter in a variety of living plant cells and in transgenic plants.Results: We show that a re-engineered GFP gene sequence, with the favored codons of highly expressed human proteins, gives 20-fold higher GFP expression in maize leaf cells than the original jellyfish GFP sequence. When combined with a mutation in the chromophore, the replacement of the serine at position 65 with a threonine, the new GFP sequence gives more than 100-fold brighter fluorescent signals upon excitation with 490 nm (blue) light, and swifter chromophore formation, We also show that this modified GFP has a broad use in various transient expression systems, and allows the easy detection of weak promoter activity, visualization of protein targeting into the nucleus and various plastids, and analysis of signal transduction pathways in living single cells and in transgenic plants.Conclusions: The modified GFP is a simple and economical new tool for the direct visualization of promoter activities with a broad range of strength and cell specificity. It can be used to measure dynamic responses of signal transduction pathways, transfection efficiency, and subcellular localization of chimeric proteins, and should be suitable for many other applications in genetically modified living cells and tissues of higher plants. The data also suggest that the codon usage effect might be universal, allowing the design of recombinant proteins with high expression efficiency in evolutionarily distant species such as humans and maize.