Plastid marker gene excision by the phiC31 phage site-specific recombinase

Chokchai Kittiwongwattana, Kerry Lutz, Mark Clark, Pal Maliga

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

Abstract

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.

Original languageEnglish (US)
Pages (from-to)137-143
Number of pages7
JournalPlant Molecular Biology
Volume64
Issue number1-2
DOIs
StatePublished - May 2007

All Science Journal Classification (ASJC) codes

  • Agronomy and Crop Science
  • Genetics
  • Plant Science

Keywords

  • Nicotiana tabacum
  • Plastid marker excision
  • Plastid transformation
  • phiC31 phage recombinase

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