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Bacteriophage 5′ untranslated regions for control of plastid transgene expression

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Abstract

Expression of foreign proteins from transgenes incorporated into plastid genomes requires regulatory sequences that can be recognized by the plastid transcription and translation machinery. Translation signals harbored by the 5′ untranslated region (UTR) of plastid transcripts can profoundly affect the level of accumulation of proteins expressed from chimeric transgenes. Both endogenous 5′ UTRs and the bacteriophage T7 gene 10 (T7g10) 5′ UTR have been found to be effective in combination with particular coding regions to mediate high-level expression of foreign proteins. We investigated whether two other bacteriophage 5′ UTRs could be utilized in plastid transgenes by fusing them to the aadA (aminoglycoside-3′-adenyltransferase) coding region that is commonly used as a selectable marker in plastid transformation. Transplastomic plants containing either the T7g1.3 or T4g23 5′ UTRs fused to Myc-epitope-tagged aadA were successfully obtained, demonstrating the ability of these 5′ UTRs to regulate gene expression in plastids. Placing the Thermobifida fusca cel6A gene under the control of the T7g1.3 or T4g23 5′ UTRs, along with a tetC downstream box, resulted in poor expression of the cellulase in contrast with high-level accumulation while using the T7g10 5′ UTR. However, transplastomic plants with the bacteriophage 5′ UTRs controlling the aadA coding region exhibited fewer undesired recombinant species than plants containing the same marker gene regulated by the Nicotiana tabacum psbA 5′ UTR. Furthermore, expression of the T7g1.3 and T4g23 5′ UTR::aadA fusions downstream of the cel6A gene provided sufficient spectinomycin resistance to allow selection of homoplasmic transgenic plants and had no effect on Cel6A accumulation.

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Abbreviations

5′ UTR:

5′ Untranslated region

AadA:

Aminoglycoside-3′-adenyltransferase

DB:

Downstream box

EPSPS:

5-Enolpyruvylshikimate-3-phosphate synthase

GUS:

β-Glucuronidase

IEE:

Intercistronic expression element

PPR:

Pentatricopeptide repeat

RPS1:

Ribosomal protein S1

SD:

Shine-Dalgarno

SSC:

Saline-sodium citrate

T4g23:

T4 gene 23

T7g10:

T7 gene 10

T7g1.3:

T7 gene 1.3

TetC:

Fragment C of tetanus toxin

TSP:

Total soluble protein

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Acknowledgments

We thank Prof. David Wilson (Cornell University) for the anti-Cel6A antibody. BNG was the recipient of a National Science Foundation Graduate Research Fellowship. This work was supported by a USDA Grant (USDA NRI 2007-02133) to BAA and MRH. This research was also supported in part by the Cornell University Agricultural Experiment Station federal formula funds, Project No. NYC-165425 received from Cooperative State Research, Education and Extension Service, US Department of Agriculture. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the US Department of Agriculture.

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Author notes

  1. Huijun Yang

    Present address: Biology Department, Brookhaven National Laboratory, Building 463, Upton, NY, 11973, USA

  2. Benjamin N. Gray

    Present address: Agrivida, Inc., 200 Boston Ave., Ste. 3100, Medford, MA, 02155, USA

Authors and Affiliations

  1. Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY, 14853, USA

    Huijun Yang, Benjamin N. Gray & Beth A. Ahner

  2. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, 14853, USA

    Huijun Yang & Maureen R. Hanson

Authors
  1. Huijun Yang
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  2. Benjamin N. Gray
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  3. Beth A. Ahner
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  4. Maureen R. Hanson
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Correspondence to Beth A. Ahner.

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A contribution to the Special Issue on Evolution and Biogenesis of Chloroplasts and Mitochondria.

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Yang, H., Gray, B.N., Ahner, B.A. et al. Bacteriophage 5′ untranslated regions for control of plastid transgene expression. Planta 237, 517–527 (2013). https://doi.org/10.1007/s00425-012-1770-3

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