Abstract
A transgene-expression system for wheat that relies on the complementation of inactive precursor protein fragments through a split-intein system is described. The N- and C-terminal fragments of a barnase gene from Bacillus amyloliquifaciens were fused to intein sequences from Synechocystis sp. and transformed into wheat plants. Upon translation, both barnase fragments are assembled by an autocatalytic intein-mediated trans-splicing reaction, thus forming a cytotoxic enzyme. This chapter focuses on the use of introns and flexible polypeptide linkers to foster the expression of a split-barnase expression system in plants. The methods and protocols that were employed with the objective to test the effects of such genetic elements on transgene expression and to find the optimal design of expression vectors for use in wheat are provided. Split-inteins can be used to form an agriculturally important trait (male sterility) in wheat plants. The use of this principle for the production of hybrid wheat seed is described. The suggested toolbox will hopefully be a valuable contribution to future optimization strategies in this commercially important crop.
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Acknowledgements
The critical comments of Angelika Gils and Dr. Heike Gnad (Saaten-Union Biotec GmbH) on the manuscript are gratefully acknowledged. The author thanks Dr. Volker Lein (Saaten-Union Recherche, France) for providing the purple-colored spring wheat variety that was used in the hybrid cross.
The majority of experiments described here were performed within a joint project between the Nordsaat GmbH and the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben and between Nordsaat GmbH and Saaten-Union Biotec GmbH under funding of the Bundesministerium für Bildung und Forschung (Grants FZK0315889 and 031B0030; “WEIZEN 2.0”).
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Gils, M. (2017). A Recessive Pollination Control System for Wheat Based on Intein-Mediated Protein Splicing. In: Mootz, H. (eds) Split Inteins. Methods in Molecular Biology, vol 1495. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6451-2_12
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DOI: https://doi.org/10.1007/978-1-4939-6451-2_12
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