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Ssp DnaE split-intein mediated split-Cre reconstitution in tobacco

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Abstract

The Cre/loxP system is increasingly exploited for spatial and temporal gene activation or inactivation. In this study, a novel approach for gene activation using a Cre/loxp system in tobacco is described. As the DnaE intein in Synechocystis sp. strain PCC6803 is capable of catalyzing a protein trans-splicing reaction to assemble a mature protein from two separate precursors, the N- and C-terminal ends of the Cre enzyme, split between Gly190 and Gly191, were fused to N- and C-terminals of the Ssp DnaE split intein,respectively. Subsequently, in-frame fusions of NCre/NInt and CInt/CCre are assembled into the pCAMBIA1300 cloning vector, and used for co-expression, along with the BAR selectable marker gene for BASTA herbicide resistance in tobacco. A Cre-dependent excision recombination event is monitored when tobacco leaf explants are screened for resistance to Basta, but along with absence of beta-glucuronidase activity. Based on herbicide resistance, an efficient recombination event is observed, in vivo Bar activation following co-expression of NCre/NInt and CInt/CCre fusion genes in pCAGUS/BAR transgenic lines. Moreover, the recombination efficiency is comparable to that of intact Cre gene expression. However, no Cre recombination event is observed when only the NCre and CCre genes or the NCre/NInt fusion gene and the CCre genes are co-expressed. Thus, the Ssp DnaE split intein-mediated Cre activity reconstitution observed in this study provides an alternative approach for the traditional Cre/loxP system, and this may aid in achieving dynamic regulation of gene expression in transgenic plants.

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Abbreviations

YEB:

Yeast extract broth

6-BA:

6-Benzyladenine

PPT:

Phosphinothricin

MES:

2-(N-Morpholino)ethanesulfonic acid

HYG:

Hygromycin

Kan:

Kanamycin

GUS:

β-Glucuronidase

MS:

Murashige and Skoog

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Acknowledgments

This work was supported by the Natural Science Foundation Project of CQ CSTC (No. CSTC2011jjA80014), the National Natural Science Foundation of China (No. 30771462), and the Fundamental Research Funds for the Central Universities (No. XDJK2009B024).

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Authors and Affiliations

  1. College of Horticulture and Landscape Architecture/Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education, Southwest University, Chongqing, 400715, China

    Xuezhi Han, Fengying Han, Xuesong Ren, Jun Si, Chengqiong Li & Hongyuan Song

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  1. Xuezhi Han
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  2. Fengying Han
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  3. Xuesong Ren
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  4. Jun Si
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Correspondence to Hongyuan Song.

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Han, X., Han, F., Ren, X. et al. Ssp DnaE split-intein mediated split-Cre reconstitution in tobacco. Plant Cell Tiss Organ Cult 113, 529–542 (2013). https://doi.org/10.1007/s11240-013-0294-2

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