Salicylic-Acid-Induced Self-excision of the Marker Gene <Emphasis Type="Italic">npt</Emphasis>II from Transgenic Tomato Using the Cre–<Emphasis Type="Italic">loxP</Emphasis> System |
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Authors: | Binggang Ma Xiaoyu Duan Chao Ma Jianxin Niu Huping Zhang Lizhong Pan |
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Institution: | (1) Department of Horticulture, Agricultural College, Shihezi University, Shihezi, 832003, People’s Republic of China |
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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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Keywords: | Cre– loxP recombination system Marker-free Self-excision Solanum lycopersicum Transgenic tomato |
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