Abstract
Objectives
To exploit cold-inducible biochemical processes beneficial for foreign mRNA transcription, translation and storage, as well as protein product stability, during Agrobacterium-mediated transient expression.
Results
The efficiency of three different 5′-regulatory sequences to achieve transient expression of the GFP-based reporter gene under chilling conditions (6–8 °C since the 3rd day post inoculation) was compared. We studied the upstream sequences of a cold-inducible Arabidopsis thaliana cor15a gene, the core element of 35S CaMV promoter fused to the TMV omega 5′-UTR, and the synthetic promoter including the 35S core sequence and two binding sites for cold-inducible CBF transcription factors (P_DRE::35S). Cultivation of plants transiently expressing reporter gene under control of the synthetic P_DRE::35S promoter under chilling conditions since the 3rd dpi led to the reliably higher reporter accumulation as compared to the other tested regulatory sequences under chilling or greenhouse conditions. Reporter protein fluorescence under chilling conditions using P_DRE::35S reached 160% as compared to the transient expression in the greenhouse. Period of transient expression considerably extended if plants were cultivated at chilling temperature since the 3rd dpi: reporter protein fluorescence reached its maximum at the 20th dpi and was detected in leaves up to the 65th dpi. The enhanced protein accumulation at low temperature was accompanied by the prolonged period of corresponding mRNA accumulation.
Conclusion
Transient expression under chilling conditions using synthetic cold-inducible promoter enhances target protein accumulation and may decrease greenhouse heating expenses.
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Acknowledgement
The authors are grateful for financial support to National Academy of Science of Ukraine, Grant № UkrISTEI 0115U004171, and Alexander von Humboldt Foundation (Georg Forster Research Fellowship (HERMES) for experienced researchers 2015). We thank Dr. V. Dosenko (O. O. Bohomolets Institute of Physiology, Kyiv, Ukraine) for help with RT-qPCR measurements and Dr. Polina V. Lishko (University of California, Berkeley, USA) for linguistic advice.
Supporting information
Supplementary Figure 1—Scheme of transcriptional units for expression of the bifunctional reporter gene GFP::LicBM3 under control of different 5'-regulatory sequences.
Supplementary Figure 2—The comparison of GFP fluorescence in plants transiently expressing reporter gene driven with P_35S and P_DRE::35S under chilling and greenhouse conditions.
Supplementary Figure 3—Relative level of the reporter transcript compared to the actin mRNA and the mean Ct values of the reporter and actin mRNA.
Supplementary Table 1–21—The results of detailed t-test analysis for difference between fluorescence means for the three studied regulatory sequences for chilling and greenhouse conditions.
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Gerasymenko, I.M., Sheludko, Y.V. Synthetic cold-inducible promoter enhances recombinant protein accumulation during Agrobacterium-mediated transient expression in Nicotiana excelsior at chilling temperatures. Biotechnol Lett 39, 1059–1067 (2017). https://doi.org/10.1007/s10529-017-2336-z
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DOI: https://doi.org/10.1007/s10529-017-2336-z