Excision of selectable marker gene from transgenic tobacco using the GM-gene-deletor system regulated by a heat-inducible promoter
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To excise a selectable marker gene from transgenic plants, a new binary expression vector based on the ‘genetically modified (GM)-gene-deletor’ system was constructed. In this vector, the gene coding for FLP site-specific recombinase under the control of a heat shock-inducible promoter HSP18.2 from Arabidopsis thaliana and isopentenyltransferase gene (ipt), as a selectable marker gene under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter, were flanked by two loxP/FRT fusion sequences as recombination sites in direct orientation. Histochemical staining for GUS activity showed that, upon induction by heat shock, all exogenous DNA, including the selectable marker gene ipt, β-glucuronidase (gusA) gene and the FLP recombinase gene, between two loxP/FRT sites was eliminated efficiently from primary transgenic tobacco plants. Molecular analysis further confirmed that excision of the marker gene (ipt) was heritable and stable. Our approach provides a reliable strategy for auto-excising a selectable marker gene from calli, shoots or other tissues of transgenic plants after transformation and producing marker-free transgenic plants.
KeywordsGM-gene-deletor Heat shock ipt gene Marker-free Site-specific recombination
We would like to thank Miss Lindsey Tuominen for critically reading the manuscript. This work was supported by the Key Laboratory of Eco-environments in Three Gorges Reservoir Region, Ministry of Education of China, Southwest University and the Natural Science Foundation Project of CQ CSTC (to K. Luo).
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