Abstract
Dual-conditional positive/negative selection markers are versatile genetic tools for manipulating genomes. Plastid genomes are relatively small and conserved DNA molecules that can be manipulated precisely by homologous recombination. High-yield expression of recombinant products and maternal inheritance of plastid-encoded traits make plastids attractive sites for modification. Here, we describe the cloning and expression of a dao gene encoding D-amino acid oxidase from Schizosaccharomyces pombe in tobacco (Nicotiana tabacum) plastids. The results provide genetic evidence for the uptake of D-amino acids into plastids, which contain a target that is inhibited by D-alanine. Importantly, this nonantibiotic-based selection system allows the use of cheap and widely available D-amino acids, which are relatively nontoxic to animals and microbes, to either select against (D-valine) or for (D-alanine) cells containing transgenic plastids. Positive/negative selection with d-amino acids was effective in vitro and against transplastomic seedlings grown in soil. The dual functionality of dao is highly suited to the polyploid plastid compartment, where it can be used to provide tolerance against potential D-alanine-based herbicides, control the timing of recombination events such as marker excision, influence the segregation of transgenic plastid genomes, identify loci affecting dao function in mutant screens, and develop D-valine-based methods to manage the spread of transgenic plastids tagged with dao.
Highlights
Selectable marker genes provide powerful genetic tools for manipulating plant genomes (Miki and McHugh, 2004; Day and Goldschmidt-Clermont, 2011)
The dual functionality of dao is highly suited to the polyploid plastid compartment where it can be used to: provide tolerance against potential D-alanine based herbicides, control the timing of recombination events such as marker excision, influence the segregation of transgenic plastid genomes, identify loci affecting dao function in mutant screens, and develop D-valine based methods to manage the spread of transgenic plastids tagged with dao
Positive conditional selectable marker genes allow transgenic cells to divide in the presence of chemicals such as antibiotics or herbicides that inhibit wild type untransformed cells and underpin the development of herbicide resistant transplastomics crops (Daniell et al, 1998; Iamtham and Day, 2000; Lutz et al, 2001; Ye et al, 2001; Dufourmantel et al, 2007; Shimizu et al, 2008; Daniell et al, 2009)
Summary
Summary Dual conditional positive/negative selection markers are versatile genetic tools for manipulating genomes. The results provide genetic evidence for the uptake of D-amino acids into plastids, which contain a target that is inhibited by D-alanine. This non-antibiotic based selection system allows the use of cheap and widely available D-amino acids, which are relatively non-toxic to animals and microbes, to either select against (D-valine) or for (Dalanine) cells containing transgenic plastids. The dual functionality of dao is highly suited to the polyploid plastid compartment where it can be used to: provide tolerance against potential D-alanine based herbicides, control the timing of recombination events such as marker excision, influence the segregation of transgenic plastid genomes, identify loci affecting dao function in mutant screens, and develop D-valine based methods to manage the spread of transgenic plastids tagged with dao
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