Abstract
Marker-free transgenic tomato plants harboring a synthetic Bacillus thuringiensis endotoxin gene, cryIAc, were obtained by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination system, in which the selectable marker neomycin phosphotransferase gene flanked by two directly oriented loxP sites was located between the cauliflower mosaic virus 35S promoter and a promoterless cryIAc. Upon induction by 2 μM β-estradiol, sequences encoding the selectable marker and cre sandwiched by two loxP sites were excised from the tomato genome, leading to activation of the downstream endotoxin gene cryIAc with high expression levels as shown by Northern blot and ELISA assay (250–790 ng g−1 fresh wt) in T1 generation. For transgenic line with single transgenic loci, 15% of T1 progenies were revealed marker-free. This autoexcision strategy provides an effective approach to eliminate a selectable marker gene from transgenic tomato, thus expediting the public acceptance of genetically modified crop.
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Acknowledgements
The authors thank Dr Chua Nam-Hai (Laboratory of Plant Molecular Biology, Rockefeller University, USA), Dr Zhang Qi-Fa (National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, China) for the gift of pX6-GFP vector and pUBC vector respectively, and Dr Guo Wen-Wu (National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, China) for reading this manuscript. This work was supported by the Hi-Tech Research and Development (863) Program of China (No. 2001AA212221) and the National Natural Science Foundation of China (No. 30270914).
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Zhang, Y., Li, H., Ouyang, B. et al. Chemical-induced autoexcision of selectable markers in elite tomato plants transformed with a gene conferring resistance to lepidopteran insects. Biotechnol Lett 28, 1247–1253 (2006). https://doi.org/10.1007/s10529-006-9081-z
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DOI: https://doi.org/10.1007/s10529-006-9081-z