In planta comparative analysis of improved green fluorescent proteins with reference to fluorescence intensity and bimolecular fluorescence complementation ability
In planta comparative analysis of improved green fluorescent proteins with reference to fluorescence intensity and bimolecular fluorescence complementation ability
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DOI：
10.5511/plantbiotechnology.15.0120a
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发表时间：
2015-03-01
影响因子：
1.6
通讯作者：
Kodama, Yutaka
中科院分区：
文献类型：
--
作者：
Fujii, Yuta;Kodama, Yutaka
绿色荧光蛋白（GFP）是从水母Aequorea维多利亚中发现的，并且已经进行了几项改进以改变其物理化学性质。所得到的改进的GFP变体已被用作报告蛋白，用于包括植物科学在内的各种研究领域中的生物成像技术。几乎所有的GFP变体都是使用大肠杆菌开发的，以改善哺乳动物细胞中的荧光特性，但在其他生物体（如植物细胞）中的影响仍有待确定。在这项研究中，我们进行了比较分析的四个改进的GFP变体，GFP-S65 T，eGFP，frGFP和sfGFP，在拟南芥原生质体中的荧光强度为参考，并发现sfGFP是最明亮的。使用来自GFP变体的非荧光片段，我们还进行了双分子荧光互补（BiFC）测定，以找到合适的基于GFP的BiFC片段对，用于活植物细胞中蛋白质-蛋白质相互作用的可视化。我们的观察显示，最亮的是基于sfGFP的BiFC。此外，作为基于sfGFP的BiFC的评价方法，首次在植物中成功完成了BiFC竞争测定。本研究为绿色荧光蛋白分子的筛选和改良及其在植物BiFC技术中的应用提供了有用的信息。
Green fluorescent protein (GFP) was discovered from the jellyfish Aequorea victoria, and several improvements have been carried out to change its physicochemical properties. The resulting improved GFP variants have been used as reporter proteins for bioimaging techniques in various research fields including plant science. Almost all GFP variants were developed using Escherichia coli to improve fluorescence properties in mammalian cells, but the impact in other organisms such as plant cells remains to be determined. In this study, we performed comparative analysis of four improved GFP variants, GFP-S65T, eGFP, frGFP and sfGFP, with reference to the fluorescence intensity in Arabidopsis protoplasts, and found that sfGFP is the brightest. Using non-fluorescent fragments from the GFP variants, we also conducted bimolecular fluorescence complementation (BiFC) assays to find appropriate fragment pairs of GFP-based BiFC for visualization of protein-protein interactions in living plant cells. Our observations revealed that the brightest is the sfGFP-based BiFC. Further, as an evaluation method for the sfGFP-based BiFC, a BiFC competition assay was successfully completed for the first time in planta. The present study provides useful information for selection and improvement of the GFP molecule and its application to BiFC technology in plants.