| Autor: |
Uriel Urquiza-García, Andrew J. Millar |
| Jazyk: |
angličtina |
| Rok vydání: |
2019 |
| Předmět: |
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| Zdroj: |
Plant Methods, Vol 15, Iss 1, Pp 1-11 (2019) |
| Druh dokumentu: |
article |
| ISSN: |
1746-4811 |
| DOI: |
10.1186/s13007-019-0454-4 |
| Popis: |
Abstract Background Protein data over circadian time scale is scarce for clock transcription factors. Further work in this direction is required for refining quantitative clock models. However, gathering highly resolved dynamics of low-abundance transcription factors has been a major challenge in the field. In this work we provide a new tool that could help this major issue. Bioluminescence is an important tool for gathering data on circadian gene expression. It allows data collection over extended time periods for low signal levels, thanks to a large signal-to-noise ratio. However, the main reporter so far used, firefly luciferase (FLUC), presents some disadvantages for reporting total protein levels. For example, the rapid, post-translational inactivation of this luciferase will result in underestimation of protein numbers. A more stable reporter protein could in principle tackle this issue. We noticed that NanoLUC might fill this gap, given its reported brightness and the stability of both enzyme and substrate. However, no data in plant systems on the circadian time scale had been reported. Results We tested NanoLUC activity under different scenarios that will be important for generating highly quantitative data. These include enzyme purification for calibration curves, expression in transient plant systems, stable transgenic plants and in planta time series over circadian time scales. Furthermore, we show that the difference in substrate use between firefly luciferase and NanoLUC allows tracking of two different reporters from the same samples. We show this by exploring the impact of a BOAp:BOA-NanoLUC construct transformed into a Col-0 CCA1p:FLUC background. Conclusions We concluded that NanoLUC reporters are compatible with established instrumentation and protocols for firefly luciferase. Overall, our results provide guidelines for researchers gathering dynamic protein data over different time scales and experimental setups. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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