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Salicylic-Acid-Induced Self-excision of the Marker Gene nptII from Transgenic Tomato Using the Cre–loxP System

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Abstract

The presence of antibiotic-resistant genes in genetically engineered crops together with the target gene has generated a number of environmental and consumer concerns. In order to alleviate public concerns over the safety of food derived from transgenic crops, marker gene elimination is desirable. Marker-free transgenic tomato plants were obtained by using a salicylic-acid-regulated Cre–loxP-mediated site-specific DNA recombination system in which the selectable marker neomycin phosphotransferase nptII and cre genes were flanked by two directly oriented loxP sites. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding nptII and cre genes, sandwiched by two loxP sites from the tomato genome. Regenerant plants with the Cre–loxP system were obtained by selection on kanamycin media and polymerase chain reaction (PCR) screening. Transgenic plants were screened for excision by PCR using nptII, cre, and PR-1a promoter primers following treatment with salicylic acid. The footprint of the excision was determined by sequencing the T-DNA borders after a perfect recombination event. The excision efficiency was 38.7%. A new plant transformation vector, pBLNSC (Genbank accession number EU327497), was developed, containing six cloning sites and the self-excision system. This provided an effective approach to eliminate the selectable marker gene from transgenic tomato, thus expediting public acceptance of genetically modified tomato.

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Abbreviations

35S:

cauliflower mosaic virus 35S

Cef:

cefotaxime

CTAB:

cetyltrimethyl ammonium bromide

IAA:

indole-3-acetic acid

IBA:

indole-3-butyric acid

Kan:

kanamycin

LB:

Luria–Bertani

nptII:

neomycin phosphotransferase

PR-1a:

pathogenesis-ralated protein 1a

SA:

salicylic acid

ZT:

zeatin

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Acknowledgements

We are grateful to Dr. J. H. Hegemann from Heinrich-Heine-Universität, Institut für Mikrobiologie, Universitätsstrasse 1, Geb. 26.12.01.64, 40225 Düsseldorf, Germany who provided the plasmid pUG6 containing the loxP sites and the plasmid pSH47 containing the cre recombinase gene. This research was supported by the National Natural Science Foundation of China (No. 30460081) and the Scientific Research Program of the Higher Education Institution of XinJiang, China (No. XJEDU2005S15).

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Authors and Affiliations

  1. Department of Horticulture, Agricultural College, Shihezi University, Shihezi, 832003, People’s Republic of China

    Binggang Ma, Xiaoyu Duan, Chao Ma, Jianxin Niu, Huping Zhang & Lizhong Pan

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  1. Binggang Ma
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  2. Xiaoyu Duan
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Correspondence to Binggang Ma.

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Ma, B., Duan, X., Ma, C. et al. Salicylic-Acid-Induced Self-excision of the Marker Gene nptII from Transgenic Tomato Using the Cre–loxP System. Plant Mol Biol Rep 26, 199–212 (2008). https://doi.org/10.1007/s11105-008-0039-2

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