BiFC imaging assay for plant protein-protein interactions.
BiFC imaging assay for plant protein-protein interactions.
复制标题
DOI：
10.1101/pdb.prot5380
复制
发表时间：
2010-02-01
影响因子：
--
通讯作者：
Spector, David L
中科院分区：
文献类型：
--
作者：
Fang, Yuda;Spector, David L
活细胞成像是研究植物在正常或胁迫生长条件下细胞结构、动态和功能的重要方法。微小的开花植物拟南芥为应用各种活体显微技术提供了理想的系统。它的小尺寸允许荧光穿透组织，并且它的小植株包含具有不同倍性水平和分化阶段的不同细胞类型。它的2C核只包含五对染色体，其中异染色质结构域被组织为染色中心，这些结构域在显微镜下很容易分辨。此外，强大的遗传工具的可用性促进了对各种细胞现象的分子机制的研究。在设计实时成像实验时，必须记住植物可以感知光、温度、渗透压、湿度、重力和营养。此外，植物还具有很强的生理行为和基因表达的昼夜节律。此外，植物组织通常是厚的（具有多个细胞层），并且可以具有强的自发荧光，特别是在绿色叶中。因此，优化的培养和成像条件对于成功的植物活细胞研究至关重要。该协议描述了一种双分子荧光互补（BiFC）成像检测蛋白质-蛋白质相互作用，通过其在烟草中的瞬时共表达。这里使用的例子检查了植物主要microRNA加工蛋白Dicer-like 1，Hyponastic Leaves 1和SERRATE之间的相互作用，但该过程可以很容易地修改以适应其他蛋白质或感兴趣的结构。
Live cell imaging is an essential approach for studying the structure, dynamics, and functions of cells in a living plant under normal or stressed growth conditions. The tiny flowering plant, Arabidopsis thaliana, provides an ideal system to apply various live microscopy techniques. Its small size allows fluorescent light to penetrate the tissues, and its plantlets contain different cell types with different ploidy levels and differentiation stages. Its 2C nucleus contains only five pairs of chromosomes in which heterochromatin domains are organized as chromocenters, and these domains are easily resolved under the microscope. In addition, the availability of powerful genetic tools facilitates the investigation of the molecular mechanisms underlying various cellular phenomena. In designing live imaging experiments, one must keep in mind that plants sense light, temperature, osmolarity, humidity, gravity, and nutrition. In addition, plants also have strong circadian rhythms of physiological behavior and gene expression. Moreover, plant tissues are normally thick (having multiple cell layers), and can have strong autofluorescence, especially in green leaves. Therefore, optimized culturing and imaging conditions are essential for successful live cell studies in plants. This protocol describes a bimolecular fluorescence complementation (BiFC) imaging assay for protein-protein interactions via their transient coexpression in tobacco. The example used here examines interactions among the plant primary microRNA processing proteins Dicer-like 1, Hyponastic Leaves 1, and SERRATE, but the procedure can be modified easily to accommodate other proteins or structures of interest.