Using STJs to measure the expression and interactions of Arabidopsis fluorescently-labelled proteins involved in pollen development

花粉发育是植物生命周期的关键步骤，在作物育种和种子生产中具有重要的商业意义。我们一直在研究对花粉形成至关重要的MS1蛋白在这一过程中所起的作用。我们已经确定了一些MS1似乎与之相互作用的蛋白质，但需要在植物中验证这些相互作用。要做到这一点，我们需要显示这些蛋白质表达的时间和地点，并确认它们与MS1相互作用。一种方法是制造融合蛋白，将荧光标记物与感兴趣的蛋白质结合，并寻找荧光，以及与用不同荧光标记物标记的MS1蛋白相互作用是否会导致荧光变化。荧光标记蛋白的检测通常通过共聚焦显微镜进行，然而，这是非常困难的，因为蛋白质在组织内有限数量的细胞中表达，叶绿素自身荧光部分掩盖了融合蛋白的荧光，并且检测荧光的微小变化是非常困难的。虽然这些问题适用于我们的工作，但它们也是其他植物科学研究人员遇到的常见限制。一种可能缓解这些问题的方法是使用一种新型的探测器（STJ）来探测单个光子的能量。欧洲空间局开发了用于光学波长的stj，并将其应用于莱斯特大学空间研究中心的生物荧光探测。stj以高效率和极低的内部背景测量单个光子的能量。探测器提供了一个优势，超过其他光子计数系统，因为整个荧光光谱是在一个事件一个事件的基础上进行检测。这意味着，不同的荧光标签的贡献可以区分，而不需要过滤器和自身荧光的问题可以消除。我们希望将这种技术应用于高等植物花药中荧光标记蛋白的检测和相互作用。这将验证该技术在植物中蛋白的体外表达和相互作用的研究。在植物生物学中使用基于stj的检测系统可以克服目前在植物荧光分子可视化中遇到的许多限制。这项工作也将为花粉发育过程提供有价值的信息。

Pollen development is a critical step in the plant life cycle with commercial importance in crop breeding and seed production. We have been investigating the role that the MS1 protein, which is vital for pollen formation, plays in this process. We have identified a number of proteins that MS1 appears to interact with, but need to verify these interactions in planta. To do this we need to show when and where these proteins are expressed and confirm that they interact with MS1. One way to do this is to make fusion proteins combining a fluorescent marker to the protein of interest and to look for fluorescence and whether there is a change in the fluorescence as a consequence of interacting with the MS1 protein labelled with a different fluorescent marker. Detection of fluorescently labelled proteins are usually conducted by confocal microscopy, however, this is very difficult as the proteins are expressed in a limited number of cells within a tissue, chlorophyll autofluorescence partly masks the fusion protein fluorescence, and detecting small changes in fluorescence is extremely difficult. Although these issues apply to our work they are also common limitations encountered by other Plant Science researchers. One approach that may alleviate many of these problems is to use a novel form of detector (STJ) that detects the energy of individual photons. STJs for optical wavelengths have been developed by the European Space Agency and applied to the detection of biological fluorescence at the University of Leicester Space Research Centre. STJs measure the energies of individual optical photons with high efficiency and extremely low internal background. The detector offers an advantage over other photon counting systems because the entire fluorescent spectrum is detected on an event-by-event basis. This means that the contributions of different fluorescent labels can be distinguished without the need for filters and problems of autofluorescence can be eliminated. We wish to apply this technique to the detection and interaction of fluorescently labelled proteins in the anthers of a higher plant. This will validate this technique for the study of in vitro expression and interaction of proteins in plants. The use of STJ-based detection systems in plant biology may overcome many of the limitations that are currently encountered in the visualisation of fluorescent molecules in planta. This work will also provide valuable information on the process of pollen development.