Molecular Tools to Minimize Risk in Genetically Engineered Crops
Location: Crop Improvement & Utilization Research
Title: The Bxb1 recombination system demonstrates heritable transmission of site-specific excision in Arabidopsis
Submitted to: BioMed Central (BMC)Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 21, 2012
Publication Date: March 1, 2012
Citation: Thomson, J.G., Chan, R., Smith, J.D., Thilmony, R.L., Yau, Y., Wang, Y., Ow, D.W. 2012. The Bxb1 recombination system demonstrates heritable transmission of site-specific excision in Arabidopsis. BioMed Central (BMC)Biotechnology. 12:9 doi:10.1186/1472-6750-12-9.
Interpretive Summary: To help facilitate the freedom to operate and stimulate development of this technology our lab has developed and tested a number of novel recombinases to better control genomic engineering in plants. We present evidence demonstrating that the Bxb1 recombinase can precisely remove DNA from the Arabidopsis genome and the deletion event can be inherited in progeny plants independent of the Bxb1 gene. The conservative nature of the recombinase enzymes does not add or delete nucleotides thereby maintaining gene integrity. Thus they are unique moelcular tools capable of removing unwanted antibiotic or herbicide genes used as selectable markers during genetic engineering prior to public release.
Background: The mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has catalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and mammalian cells. Results: In this work, the Bxb1 recombinase gene was transformed and constitutively expressed in Arabidopsis thaliana plants harboring a chromosomally integrated attP and attB-flanked target sequence. The Bxb1 recombinase successfully excised the target sequence in a conservative manner and the resulting recombination event was heritably transmitted to subsequent generations in the absence of the recombinase transgene. In addition, we also show that Bxb1 recombinase expressing plants can be manually crossed with att-flanked target transgenic plants to generate excised progeny. Conclusion: The Bxb1 large serine recombinase performs site-specific recombination in Arabidopsis thaliana germinal tissue, producing stable lines free of unwanted DNA. The precise site-specific deletion produced by Bxb1 in planta demonstrates that this enzyme can be a useful tool for the genetic engineering of plants without selectable marker transgenes or other undesirable exogenous sequences.