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Transgene excision in pollen using a codon optimized serine resolvase CinH-RS2 site-specific recombination system

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Abstract

Transgene escape, a major environmental and regulatory concern in transgenic crop cultivation, could be alleviated by removing transgenes from pollen, the most frequent vector for transgene flow. A transgene excision vector containing a codon optimized serine resolvase CinH recombinase (CinH) and its recognition sites RS2 were constructed and transformed into tobacco (Nicotiana tabacum cv. Xanthi). CinH recombinase recognized 119 bp of nucleic acid sequences, RS2, in pollen and excised the transgene flanked by the RS2 sites. In this system, the pollen-specific LAT52 promoter from tomato was employed to control the expression of CinH recombinase. Loss of expression of a green fluorescent protein (GFP) gene under the control of the LAT59 promoter from tomato was used as an indicator of transgene excision. Efficiency of transgene excision from pollen was determined by flow cytometry (FCM)-based pollen screening. While a transgenic event in the absence of CinH recombinase contained about 70% of GFP-synthesizing pollen, three single-copy transgene events contained less than 1% of GFP-synthesizing pollen based on 30,000 pollen grains analyzed per event. This suggests that CinH-RS2 recombination system could be effectively utilized for transgene biocontainment.

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Acknowledgments

The authors would like to thank Murali R. Rao for helping on statistical analysis and Spencer Wei for his help with vector construction. We thank Patrick Gallois for the gift of pPK100 that was included in our vectors. This research was funded by USDA Biotechnology Risk Assessment Grants to C. N. Stewart, Jr. and D. W. Ow. We especially wish to express our gratitude to collaborator Yi Li, also a co-PI on the above grant, for the extensive help in this line of research and for the many conversations and innovations he and his lab has accomplished.

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Author notes

  1. Robert Blanvillain

    Present address: Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS UMR 5004, 2 Place Viala, 34060, Montpellier Cedex 1, France

  2. David W. Ow

    Present address: Gene Engineering Center, South China Botanical Garden, 723 Xingke Road, 510650, Tianhe, Guangzhou, China

Authors and Affiliations

  1. Department of Plant Sciences, University of Tennessee, Knoxville, TN, 37996, USA

    Hong S. Moon, Laura L. Abercrombie & C. N. Stewart Jr.

  2. Center for Wildlife Health, Department of Forestry, Wildlife and Fisheries, University of Tennessee, Knoxville, TN, 37996, USA

    Shigetoshi Eda

  3. Plant Gene Expression Center, 800 Buchanan Street, Albany, CA, 94710, USA

    Robert Blanvillain & David W. Ow

  4. Crop Improvement and Utilization Research Unit, Western Regional Research Center, USDA-ARS, 800 Buchanan Street, Albany, CA, 94710, USA

    James G. Thomson

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  1. Hong S. Moon
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Correspondence to C. N. Stewart Jr..

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Moon, H.S., Abercrombie, L.L., Eda, S. et al. Transgene excision in pollen using a codon optimized serine resolvase CinH-RS2 site-specific recombination system. Plant Mol Biol 75, 621–631 (2011). https://doi.org/10.1007/s11103-011-9756-2

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  • DOI: https://doi.org/10.1007/s11103-011-9756-2

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