Location: Crop Improvement and Genetics ResearchTitle: A bacterial gene codA encoding cytosine deaminase is an effective conditional negative selectable marker in Glycine max Author
|Shao, Min - University Of California|
|Michno, Jean-michel - University Of Minnesota|
|Thomson, James - Jim|
Submitted to: BioMed Central (BMC)Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2015
Publication Date: 6/17/2015
Citation: Shao, M., Michno, J., Blechl, A.E., Thomson, J.G. 2015. A bacterial gene codA encoding cytosine deaminase is an effective conditional negative selectable marker in Glycine max. BioMed Central (BMC)Biotechnology. doi: 10.1007/s00299-015-1818-5. Interpretive Summary: The use of site-specific recombination systems will be of great importance for precise modifications of plant chromosomes. Development of a gene targeting system for plants will require the use of appropriate negative selectable marker genes to identify plants that have lost such genes. In many plant species, the codA gene from E. coli bacteria, which encodes cytosine deaminase, has been successfully used as a negative selection marker. The gene product of codA converts the uracil analog 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), changing a harmless substrate to a product that is toxic to cells because it inhibits both RNA and DNA synthesis. Hence, plant cells that contain the codA gene are unable to thrive when grown in media containing 5-FC. This paper describes the use of 5-FC as a negative selection for offspring of transformed soybean plants that do not contain the codA gene. We show that 5-FC does not affect the growth of untransformed plants, but inhibits shoot and root growth of seedlings that express codA. The inhibited plantlets can be rescued by transplanting them to soil, where they recover and grow normally. We also show that leaf cuttings with the codA gene cannot root efficiently in 5-FC media. The protocols reported here will be useful in genetic engineering of soybean where they can be employed to remove selection marker genes from transgenic plants and to insert multiple genes into single chromosome locations.
Technical Abstract: Background Conditional negative selection is a powerful technique whereby the absence of a gene product allows survival in otherwise lethal conditions. In plants, the Escherichia coli gene codA has been employed as a negative selection marker. CodA is a conditionally lethal dominant gene encoding cytosine deaminase, which converts the nontoxic compound 5-fluorocytosine (5-FC) to the highly toxic compound 5-fluorouracil (5-FU). Results Here we show that codA can be used as a negative selection marker in soybean, Glycine max. Like most plants, soybean does not contain cytosine deaminase activity and we show here that wild type seedlings are not affected by inclusion of 5-FC in growth media. In contrast, transgenic G. max plants expressing codA and grown in the presence of 200 µg/mL 5-FC exhibit reductions in hypocotyl and branched taproot lengths, and severe suppression of lateral root development. Rescue of the 5-FC sensitive transgenic seedlings to soil results in restoration of normal growth. Leaf cuttings that express codA cannot form roots in media containing 5-FC. Conclusion These results demonstrate that codA can be used effectively in G. max for conditional negative selection. These protocols will be useful in deploying site-specific recombinases for the stacking of genes in specific locations of the soybean genome.