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Gene activation in plastids by the CRE site-specific recombinase

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Abstract

We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.

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Abbreviations

aadA :

gene encoding aminoglycoside 3′′-adenylytransferase

CRE:

cyclization recombination, the P1 phage site-specific recombinase

DTT:

dithiothreitol

gfp :

gene encoding green fluorescent protein

GFP:

green fluorescent protein

IPTG:

Isopropyl-1-thio-β-d-galactopyranoside

loxP :

locus of X-over of P1 sites for CRE

MW:

Molecular Weight

PMSF:

Phenylmethanesulfonyl fluoride

Prrn:

rrn operon promoter, rrn, plastid rRNA operon

T7g10:

T7 phage gene 10

TpsbA:

psbA gene 3′-UTR

trnI :

trnV, rrn16 and 3′-rps12/7, plastid genes

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Authors and Affiliations

  1. Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ, 08854-8020, USA

    Tarinee Tungsuchat, Hiroshi Kuroda & Pal Maliga

  2. Department of Biotechnology, Faculty of Science, Mahidol University, Prayathai, Bangkok, 10400, Thailand

    Tarinee Tungsuchat & Jarunya Narangajavana

  3. Department of Plant Biology, Rutgers, the State University of New Jersey, 59 Dudley Road, New Brunswick, NJ, 08901, USA

    Pal Maliga

  4. Graduate School of Natural Sciences, Nagoya City University, Yamanohata, Mizuho-ku, Nagoya, 467-8020, Japan

    Hiroshi Kuroda

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  1. Tarinee Tungsuchat
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  2. Hiroshi Kuroda
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  3. Jarunya Narangajavana
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  4. Pal Maliga
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Correspondence to Pal Maliga.

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Tungsuchat, T., Kuroda, H., Narangajavana, J. et al. Gene activation in plastids by the CRE site-specific recombinase. Plant Mol Biol 61, 711–718 (2006). https://doi.org/10.1007/s11103-006-0044-5

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