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

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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.

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Abbreviations

aadA :

Gene encoding aminoglycoside 3″-adenylytransferase

attB :

Bacterial attachment site

attP :

Phage attachment site

bar :

Gene conferring resistance to the herbicide bialaphos

Cre:

P1 phage site-specific recombinases

Int:

Integrase or phiC31 phage site-specific recombinase

int :

Gene encoding Int

ptDNA:

Plastid genome

PP–BamHI:

Fusion promoter containing the promoter of the plastid rrn operon and the plastid clpP gene

Prrn:

Promoter of the plastid rrn operon

rrn :

Plastid rRNA operon

TpsbA:

psbA gene 3′-UTR

clpP, trnI, trnV, rrn16 and 3′-rps12/7 :

Plastid genes

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Acknowledgements

This research was supported by the USDA Biotechnology Risk Assessment Research Grant Program Award No. 2005-33120-16524 to PM. Chokchai Kittiwongwattana is the recipient of a Ph.D. scholarship of the Thailand Commission on Higher Education. Mark Clark contributed to this project while supported by a Summer Undergraduate Research Fellowship.

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Authors and Affiliations

  1. Waksman Institute, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ, 08854-8020, USA

    Chokchai Kittiwongwattana, Kerry Lutz, Mark Clark & Pal Maliga

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  1. Chokchai Kittiwongwattana
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  2. Kerry Lutz
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  3. Mark Clark
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  4. Pal Maliga
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Correspondence to Pal Maliga.

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Kittiwongwattana, C., Lutz, K., Clark, M. et al. Plastid marker gene excision by the phiC31 phage site-specific recombinase. Plant Mol Biol 64, 137–143 (2007). https://doi.org/10.1007/s11103-007-9140-4

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  • DOI: https://doi.org/10.1007/s11103-007-9140-4

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