Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/26/2012
Publication Date: 4/22/2012
Citation: Blechl, A.E., Lin, J.W., Shao, M., Thilmony, R.L., Thomson, J.G. 2012. Bxb1 recombinase mediates site-specific deletions in transgenic wheat. Plant Molecular Biology Reporter. doi:10.1007/s11105-012-0454-2. Interpretive Summary: The research presented demonstrates the activity of the novel recombinase system Bxb1 within wheat cells. A transient assay system was developed that allows qualitative estimations of recombinase activity between various Bxb1 expression lines. Using the transient assay transgenic wheat lines expressing Bxb1 are identified. These findings are supported by more traditional molecular techniques such as PCR, northern blot and sequencing. The manuscript discusses the utility of the Bxb1 recombinase system for precise genomic engineering of wheat and benefits this can provide to the public.
Technical Abstract: The utility and commercial potential of genetically engineered (GE) plants would benefit from the ability to efficiently remove the marker genes used to identify transformants. Although wheat is one of the four most important food crops in the world, GE varieties have yet to be put into commercial production. To develop the Bxb1 site-specific recombination system (derived from the Mycobacterium smegmati bacteriophage Bxb1) for marker gene removal in transgenic wheat, we used biolistics to introduce into the wheat genome a codon optimized Bxb1 recombinase gene (BxbNom) under the control of the maize ubiquitin-1 promoter (Ubi1). Recombinase activity was monitored in wheat using a GUSPlus reporter gene activation assay. BxbNom recombinase-mediated excision of an att site-flanked stuffer DNA fragment activated B-glucuronidase reporter activity in callus, endosperm and leaves in transient assays. The system also detected activity in leaves and endosperm of multiple independent transgenic wheat lines stably expressing BxbNom over two generations. Bxb1-mediated excision was shown to be precise in wheat cells, yielding the predicted attL footprint. Our results demonstrate that the Bxb1 recombinase can be stably expressed in transgenic wheat plants and can potentially be a useful tool for generating marker-free GE plants. Establishing wheat lines capable of efficiently excising unneeded marker genes would remove one of the barriers to commercial deployment of GE wheat.