Growth of transplastomic cells expressing D-amino acid oxidase in chloroplasts is tolerant to D-alanine and inhibited by D-valine

Martin F. Gisby, Elisabeth A. Mudd, Anil Day

    Research output: Contribution to journalArticlepeer-review

    111 Downloads (Pure)

    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. Highyield 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. © 2012 American Society of Plant Biologists. All Rights Reserved.
    Original languageEnglish
    Pages (from-to)2219-2226
    Number of pages7
    JournalPlant Physiology
    Volume160
    Issue number4
    DOIs
    Publication statusPublished - Dec 2012

    Keywords

    • chloroplast transformation, D-amino acid oxidase, genetic containment, herbicide tolerance, negative selection, tobacco

    Fingerprint

    Dive into the research topics of 'Growth of transplastomic cells expressing D-amino acid oxidase in chloroplasts is tolerant to D-alanine and inhibited by D-valine'. Together they form a unique fingerprint.

    Cite this