A NOVEL CIRCADIAN PHENOTYPE BASED ON FIREFLY LUCIFERASE EXPRESSION IN TRANSGENIC PLANTS
A NOVEL CIRCADIAN PHENOTYPE BASED ON FIREFLY LUCIFERASE EXPRESSION IN TRANSGENIC PLANTS
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DOI：
10.1105/tpc.4.9.1075
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发表时间：
1992-09-01
期刊：
影响因子：
11.6
通讯作者：
KAY, SA
中科院分区：
文献类型：
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作者：
MILLAR, AJ;SHORT, SR;KAY, SA
拟南芥cab2启动子的320 bp片段足以介导光敏色素和生物钟的转录调控。我们将该启动子片段与萤火虫荧光素酶（Luc）基因融合，以获得完整植物中调控基因表达的实时报告基因。Cab2：：Luc转录物以预期的模式积累，并且在黄化和绿色幼苗中荧光素酶活性与Cab2：：Luc mRNA丰度密切相关。大量荧光素酶蛋白的浓度没有反映这些模式，但保持相对恒定的水平，这意味着翻译后机制导致荧光素酶活性的高幅度调节。我们使用了低光视频成像系统，以建立荧光素酶生物发光在体内准确报告的时间和空间调节cab2转录在单个幼苗。萤火虫荧光素酶系统的独特性质使我们能够在单个多细胞生物中真实的时间内监测受调控的基因表达。这种在分子水平上用于时间调节的非侵入性标记物构成了昼夜节律表型，其可用于分离昼夜节律钟中的突变体。
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 c ate 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.