Lanzhou University, School of Life Sciences, Lanzhou, China.
Biotechniques. 2010 Feb;48(2):125-33. doi: 10.2144/000113345.
Fluorescence imaging in plants is unusually challenging because of the large amounts of photosynthetic pigments contained in green plant tissues. For example, chlorophyll can obstruct the penetration of light and has high levels of autofluorescence at wavelengths that are often used for fluorescence imaging. Until now, mostly confocal laser scanning microscopy or the use of non-green parts of the plants, typically roots, have been used to overcome these limitations. We constructed tobacco (Nicotiana attenuata) plants expressing GFP-sporamin fusion polypeptide in their vascular tissues. As expected, it was not possible to visualize GFP fluorescence in tobacco leaves or stems using a stereomicroscope and filters specific for GFP detection; however, GFP fluorescence was readily detectable when virus-induced gene silencing (VIGS) was used to transiently silence the phytoene desaturase (PDS) gene in order to bleach chlorophyll-containing tissues. This method is an inexpensive alternative to confocal laser scanning microscopy for the detection of GFP fusion proteins or promoter-GFP reporter fusions in plant leaves.
在植物中进行荧光成像非常具有挑战性，因为绿色植物组织中含有大量的光合作用色素。例如，叶绿素会阻碍光的穿透，并且在荧光成像常用的波长处具有高水平的自发荧光。到目前为止，主要使用共聚焦激光扫描显微镜或植物的非绿色部分（通常是根）来克服这些限制。我们构建了在其维管束组织中表达 GFP-伴孢晶体蛋白融合多肽的烟草（Nicotiana attenuata）植株。正如预期的那样，使用立体显微镜和特定于 GFP 检测的滤光片无法在烟草叶片或茎部中观察到 GFP 荧光；然而，当使用病毒诱导的基因沉默（VIGS）来暂时沉默类胡萝卜素脱饱和酶（PDS）基因以漂白含有叶绿素的组织时，GFP 荧光很容易被检测到。这种方法是一种替代共聚焦激光扫描显微镜的廉价方法，用于检测植物叶片中的 GFP 融合蛋白或启动子-GFP 报告融合蛋白。
