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Intein-mediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis

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Abstract

Key message

An approach for restoring recombination activity of complementation split-Cre was developed to excise the transgene in hybrid progeny of GM crops.

Abstract

Growing concerns about the biosafety of genetically modified (GM) crops has currently become a limited factor affecting the public acceptance. Several approaches have been developed to generate selectable-marker-gene-free GM crops. However, no strategy was reported to be broadly applicable to hybrid crops. Previous studies have demonstrated that complementation split-Cre recombinase restored recombination activity in transgenic plants. In this study, we found that split-Cre mediated by split-intein Synechocystis sp. DnaE had high recombination efficiency when Cre recombinase was split at Asp232/Asp233 (866 bp). Furthermore, we constructed two plant expression vectors, pCA-NCre-In and pCA-Ic-CCre, containing NCre866-In and Ic-CCre866 fragments, respectively. After transformation, parent lines of transgenic Arabidopsis with one single copy were generated and used for hybridization. The results of GUS staining demonstrated that the recombination activity of split-Cre could be reassembled in these hybrid progeny of transgenic plants through hybridization and the foreign genes flanked by two loxP sites were efficiently excised. Our strategy may provide an effective approach for generating the next generation of GM hybrid crops without biosafety concerns.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (31300990, 31370672 and 31171620), the National Key Project for Research on Transgenic Plants (2016ZX08010-003), the Natural Science Foundation Project of CQ CSTC (CSTC2013JJB8007) and the Fundamental Research Funds for the Central Universities (XDJK2013c147 and XDJK2014a005).

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    Authors and Affiliations

    1. Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing, 400715, China

      Jia Ge, Lijun Wang, Chen Yang, Lingyu Ran, Mengling Wen, Xianan Fu, Di Fan & Keming Luo

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    1. Jia Ge
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    2. Lijun Wang
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    3. Chen Yang
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    8. Keming Luo
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    Correspondence to Keming Luo.

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    Communicated by P. Lakshmanan.

    J. Ge and L. Wang contributed equally to this work.

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    Ge, J., Wang, L., Yang, C. et al. Intein-mediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis . Plant Cell Rep 35, 2045–2053 (2016). https://doi.org/10.1007/s00299-016-2015-x

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