Location: Crop Improvement and Genetics ResearchTitle: Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA
|SHAO, MIN - University Of California|
|Thomson, James - Jim|
Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/24/2017
Publication Date: 4/1/2017
Citation: Shao, M., Blechl, A.E., Thomson, J.G. 2017. Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA. Plant Biotechnology Journal. doi: 10.1111/pbi.12740.
Interpretive Summary: To help facilitate the freedom to operate and to stimulate development of this technology, our lab has developed and tested a number of novel site-specific recombinases for their abilities to perform engineering of plant genomes. In this report, we present evidence that the CinH and ParA recombinases can precisely remove DNA from the genomes of tobacco plastids, organelles that become chloroplasts in plant leaf cells. The deletion events can be inherited by the offspring in the absence of the recombinase genes, and the integrity of the target gene is maintained. These results demonstrate that the CinH and ParA recombinases are capable of removing unwanted antibiotic or herbicide genes used as selectable markers in genetic engineering. The use of these unique molecular tools will mitigate public concerns about the unwanted foreign DNA in genetically engineered crops and allow biotechnologists to add multiple genes to the same locations in plastid genomes.
Technical Abstract: Selectable marker genes (SMGs) are necessary for selection of transgenic plants. However, once stable transformants have been identified, the marker gene is no longer needed. In this study, we demonstrate the use of the small serine recombination systems, ParA-MRS and CinH-RS2, to precisely excise a marker gene from the plastid genome of tobacco. Transplastomic plants transformed with the pTCH-MRS and pTCH-RS2 vectors, containing the visual reporter gene DsRed flanked by directly oriented MRS and RS2 recognition sites, respectively, were crossed with nuclear-genome transformed tobacco plants expressing plastid-targeted ParA and CinH recombinases, respectively. One hundred percent of both types of F1 hybrids exhibited excision of the DsRed marker gene. PCR and Southern blot analyses of DNA from F2 plants showed that approximately 30% (CinH-RS2) or 40% (ParA-MRS) had lost the recombinase genes by segregation. The post-excision transformed plastid genomes were stable and the excision events heritable. The ParA-MRS and CinH-RS2 recombination systems will be useful tools for site-specific manipulation of the plastid genome and for generating marker-free plants, an essential step for reuse of SMG for gene stacking techniques and addressing concerns about the presence of antibiotic resistance genes in transgenic plants.