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

Content Provider	Springer Nature Link
Author	Han, Xuezhi Han, Fengying Ren, Xuesong Si, Jun Li, Chengqiong Song, Hongyuan
Copyright Year	2013
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.
Starting Page	529
Ending Page	542
Page Count	14
File Format	PDF
ISSN	01676857
Journal	Plant Cell, Tissue and Organ Culture (PCTOC)
Volume Number	113
Issue Number	3
e-ISSN	15735044
Language	English
Publisher	Springer Netherlands
Publisher Date	2013-04-09
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Split-Cre gene Ssp DnaE intein Protein trans-splicing Site-specific recombination Tobacco Plant Sciences Plant Physiology Plant Genetics & Genomics Plant Pathology
Content Type	Text
Resource Type	Article
Subject	Horticulture

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