Generation of marker free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters |
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Authors: | Suchandra Deb Roy Mukesh Saxena Prasanna S Bhomkar Mikhail Pooggin Thomas Hohn Neera Bhalla-Sarin |
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Institution: | (1) School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India;(2) Institute of Botany, University of Basel, Schoenbeinstrasse 6, 4056 Basel, Switzerland |
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Abstract: | Despite the advances in transgenesis, transformation technologies still rely on the introduction of a selectable marker gene
to identify cells and tissues that have integrated the gene of interest in their genome. The continuous presence of the marker
genes in the transgenics is often controversial as it can potentially have multiple undesirable impacts. The present study
employed the self-excising Cre-loxP system to generate marker-free Arabidopsis thaliana expressing the agronomically important glyoxalase I (glyI) gene from Brassica juncea to confer salt stress tolerance. A binary vector was constructed wherein the salt-inducible rd29A promoter was used to drive
the expression of the glyI gene and the transformants of A. thaliana were recovered using kanamycin resistance as the selectable marker. The neomycin phosphotransferase II (nptII) gene was flanked by the loxP sites followed by the introduction of a heat-inducible Cre-recombinase in between the loxP sites. The kanamycin-resistant transgenic lines of A. thaliana using this vector showed an ability to withstand stress imposed by 150 mM NaCl. The exposure of the T2 transgenic lines to
a mild heat shock (37°C) resulted in the recovery of salt-tolerant, kanamycin-sensitive T3 progeny. Molecular analyses of
the T3 transgenic lines following the heat shock treatment confirmed the excision of the nptII gene and the completion of their life cycle in the presence of 150 mM NaCl-induced stress. |
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Keywords: | Arabidopsis thaliana hsp-cre npt-lox Bj glyoxalase I gene rd29A promoter Salt stress tolerance |
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