Arabidopsis 2010: Bioluminescence Resonance Energy Transfer (BRET)- A Tool to Explore Protein-Protein Interactions in Arabidopsis

该项目的目标是开发一种新的光学技术，称为生物发光共振能量转移(BRET)，作为植物功能基因组学社区的资源，特别是在模式物种拟南芥中。Bret已经展示了作为一种工具的前景，它可以在活组织中实时绘制特定细胞蛋白质之间的物理接触图，以及它们之间的时间分辨相互作用。虽然这种相互作用的重要性在生命科学中早已得到承认，但相应的实验工具在很大程度上仅限于体外条件或异种、非植物生物。因此，Bret技术将为描绘拟南芥基因组中被认为包含的25,000个基因的功能提供独特的贡献。BRET是一种无辐射的能量转移形式，当两个兼容的光学探针进入分子附近时，就可以发生这种转移。具体来说，为了探索两个给定的配对蛋白之间的蛋白质-蛋白质相互作用，这两个蛋白质在基因上融合到一个发出蓝光的荧光素酶和一个吸收蓝色光的黄色荧光蛋白。如果两个杂交蛋白相互作用，荧光素酶的激发能量可能会转移到荧光蛋白上，导致发光光谱容易检测到黄移。该项目的第一个目标是构建分子遗传学工具，如克隆载体，用于表达Bret杂交蛋白。其次，仪器和实验方案将针对时间分辨Bret数据采集进行优化。此外，作为Bret在植物研究中有效性的原型例子，Bret将被应用于阐明通过光和生物钟调节核基因表达的遗传网络。在本项目中开发的资源应广泛传播，以鼓励它们在整个植物科学界(http://chicory.bio.vanderbilt.edu/chj/arabidopsisbret中的应用

The goal of this project is to develop a novel optical technique, termed Bioluminescence Resonance Energy Transfer (BRET), as a resource for the plant functional genomics community, specifically in the model species Arabidopsis thaliana. BRET has shown promise as a tool to chart the physical contacts between specific cellular proteins, and their time-resolved interactions, in living tissue and in real time. While the importance of such interactions has long been recognized in the life sciences, the corresponding experimental tools have largely been limited to in vitro conditions or to heterologous, non-plant organisms. Therefore, the BRET technique will provide a unique contribution toward delineating the functions of the 25,000 genes thought to be contained within the Arabidopsis genome. BRET is a form of radiation-free energy transfer that can occur when two compatible optical probes are brought into molecular proximity. In detail, to probe for a protein-protein interaction between two given partner proteins, the two proteins are genetically fused to a blue light emitting luciferase and to a blue light absorbing yellow fluorescent protein. If the two hybrid proteins interact, the excitation energy of the luciferase may be transferred to the fluorescent protein, resulting in an easily detected yellow-shift in the luminescence spectrum. The first goal of this project is to construct molecular genetic tools, such as cloning vectors, for the expression of BRET hybrid proteins. Second, instrumentation and experimental protocols will be optimized for time-resolved BRET data acquisition. Moreover, as a prototype example of the efficacy of BRET for research in plants, BRET will be applied to illuminate the genetic networks that mediate the control of nuclear gene expression by light and the circadian clock. Resources developed in this project shall be widely disseminated to encourage their application within the entire plant science community (http://chicory.bio.vanderbilt.edu/chj/arabidopsisbret