|Shao, Min -|
|Kumar, Shahsi -|
Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 4, 2013
Publication Date: April 1, 2014
Repository URL: http://dx.doi.org/10.1111/pbi.12139
Citation: Shao, M., Kumar, S., Thomson, J.G. 2014. Precise excision of plastid DNA by the large serine recombinase Bxb1. Plant Biotechnology Journal. 12:322-329 DOI: 10.1111/pbi.12139. Interpretive Summary: To help facilitate the freedom to operate and stimulate development of this technology the USDA's Western Regional Research Center 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 tobacco plastid 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.
Technical Abstract: To elucidate the precise excision of transgene, tobacco plastid genome was transformed with a vector (pTCH-BxbPB) that contains a stuffer DNA fragment flanked by directly oriented attB and attP recognition sites for the Bxb1 recombinase. The transformed plastid genomes containing the recognition sites were shown to be stable in the absence of the Bxb1 enzyme. Further, separate nuclear transgenic tobacco lines were generated with Bxb1 recombinase gene, modified to include a stroma targeting peptide (STD) and driven by a CaMV 35S promoter (35S) and nos3’ terminator cassette. Crossing the two lines led to an efficient transgene excision from the plastid genome. The expression of Bxb1 recombinase has resulted in complete excision of the target sequence in a conservative manner from all samples. The recombination events in transplastomic plants are stable and capable of heritable transmission to subsequent generations in the absence of the recombinase transgene.