Millar A J, Short S R, Chua N H, Kay S A
Laboratory of Plant Molecular Biology, Rockefeller University, New York, New York 10021-6399.
Plant Cell. 1992 Sep;4(9):1075-87. doi: 10.1105/tpc.4.9.1075.
A 320-bp fragment of the Arabidopsis cab2 promoter is sufficient to mediate transcriptional regulation by both phytochrome and the circadian clock. We fused this promoter fragment to the firefly luciferase (Luc) gene to create a real-time reporter for regulated gene expression in intact plants. Cab2::Luc transcript accumulated in the expected patterns and luciferase activity was closely correlated to cab2::Luc mRNA abundance in both etiolated and green seedlings. The concentration of the bulk of luciferase protein did not reflect these patterns but maintained a relatively constant level, implying that a post-translational mechanism(s) leads to the high-amplitude regulation of luciferase activity. We used a low-light video imaging system to establish that luciferase bioluminescence in vivo accurately reports the temporal and spatial regulation of cab2 transcription in single seedlings. The unique qualities of the firefly luciferase system allowed us to monitor regulated gene expression in real time in individual multicellular organisms. This noninvasive marker for temporal regulation at the molecular level constitutes a circadian phenotype, which may be used to isolate mutants in the circadian clock.
拟南芥cab2启动子的一个320碱基对的片段足以介导光敏色素和生物钟对转录的调控。我们将这个启动子片段与萤火虫荧光素酶（Luc）基因融合，以创建一个用于完整植物中受调控基因表达的实时报告基因。Cab2::Luc转录本以预期模式积累，并且在黄化苗和绿色幼苗中，荧光素酶活性都与cab2::Luc mRNA丰度密切相关。大部分荧光素酶蛋白的浓度并未反映这些模式，而是维持在相对恒定的水平，这意味着一种翻译后机制导致了荧光素酶活性的高幅度调控。我们使用了一个弱光视频成像系统来确定体内荧光素酶生物发光能够准确报告单个幼苗中cab2转录的时空调控。萤火虫荧光素酶系统的独特特性使我们能够在单个多细胞生物体中实时监测受调控的基因表达。这种在分子水平上用于时间调控的非侵入性标记构成了一种昼夜节律表型，可用于分离生物钟突变体。
