Abstract
The development of tools which ensure the desired level of transgene expression in plastids is a prerequisite for the effective utilization of these plant organelles for the deployment of bioactive proteins. High-level accumulation of target proteins is considered as a positive feature of transplastomic plants, but excessive accumulation of foreign proteins may have deleterious effects on host plants. On the other hand, expression at low levels can result in ineffective phenotypes. We compared the effectiveness of different 5′-regulatory sequences in driving the expression of a reporter gene, β-glucuronidase (uidA), in tobacco chloroplasts. To achieve varying expression levels, we have chosen heterologous 5′-regulatory sequences which either differ significantly from their homologous counterparts or depend on specific nuclear encoded factors. The Medicago truncatula psbA promoter/5′-UTR supported the highest levels of protein accumulation, surpassing the other tested sequences by two to three orders of magnitude. The heterologous regulatory sequence of Phaseolus vulgaris rbcL gene was as efficient in tobacco chloroplasts as the corresponding homologous promoter/5′-UTR. The Arabidopsis thaliana ndhF promoter/5′-UTR supported as high reporter activity levels as the rbcL 5′-sequences, whereas the effectiveness of A. thaliana psbN promoter/5′-UTR was three fold lower. The characterized regulatory sequences can be utilized to establish transplastomic lines with desirable levels of target protein accumulation. The ability to control transgene expression should be useful for achieving appropriate levels of protein accumulation and thereby avoid their negative impacts on host plant physiology.
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Acknowledgments
Work was supported by the U.S. Department of Energy GIPP program through the Science and Technology Center in Ukraine; Grant # LBNL-0221-UA (P415). Authors are also grateful for financial support of DuPont Corporation (DuPont Young Professor Award 2012, the recipient was IG). YS is grateful for financial support to Alexander von Humboldt Foundation (Georg Forster Research Fellowship (HERMES) for experienced researchers 2015).
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Gerasymenko, I.M., Sheludko, Y.V., Klebanovych, A.A. et al. Comparison of effectiveness of 5′-regulatory sequences in transplastomic tobacco chloroplasts. Transgenic Res 26, 65–75 (2017). https://doi.org/10.1007/s11248-016-9980-2
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DOI: https://doi.org/10.1007/s11248-016-9980-2