caa a22 4500 467894191 CHVBK 20180406152758.0 cr unu---uuuuu 170328e20060701xx s 000 0 eng 10.1007/s11103-006-0044-5 doi (NATIONALLICENCE)springer-10.1007/s11103-006-0044-5 Gene activation in plastids by the CRE site-specific recombinase [Elektronische Daten] [Tarinee Tungsuchat, Hiroshi Kuroda, Jarunya Narangajavana, Pal Maliga] 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. Springer Science+Business Media B.V., 2006 CRE/ loxP site-specific recombination nationallicence Green fluorescent protein (GFP) nationallicence Nicotiana tabacum nationallicence Plastid gene activation nationallicence Plastid transformation nationallicence aadA : gene encoding aminoglycoside 3′′-adenylytransferase nationallicence CRE : cyclization recombination, the P1 phage site-specific recombinase nationallicence DTT : dithiothreitol nationallicence gfp : gene encoding green fluorescent protein nationallicence GFP : green fluorescent protein nationallicence IPTG : Isopropyl-1-thio-β-d-galactopyranoside nationallicence loxP : locus of X-over of P1 sites for CRE nationallicence MW : Molecular Weight nationallicence PMSF : Phenylmethanesulfonyl fluoride nationallicence Prrn : rrn operon promoter, rrn, plastid rRNA operon nationallicence T7g10 : T7 phage gene 10 nationallicence TpsbA : psbA gene 3′-UTR nationallicence trnI : trnV, rrn16 and 3′-rps12/7, plastid genes nationallicence Tungsuchat Tarinee Waksman Institute, Rutgers, the State University of New Jersey, 08854-8020, Piscataway, NJ, USA aut Kuroda Hiroshi Waksman Institute, Rutgers, the State University of New Jersey, 08854-8020, Piscataway, NJ, USA aut Narangajavana Jarunya Department of Biotechnology, Faculty of Science, Mahidol University, 10400, Prayathai, Bangkok, Thailand aut Maliga Pal Waksman Institute, Rutgers, the State University of New Jersey, 08854-8020, Piscataway, NJ, USA aut Plant Molecular Biology Kluwer Academic Publishers 61/4-5(2006-07-01), 711-718 0167-4412 61:4-5<711 2006 61 11103 https://doi.org/10.1007/s11103-006-0044-5 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11103-006-0044-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Tungsuchat Tarinee Waksman Institute, Rutgers, the State University of New Jersey, 08854-8020, Piscataway, NJ, USA aut NATIONALLICENCE 700 1- Kuroda Hiroshi Waksman Institute, Rutgers, the State University of New Jersey, 08854-8020, Piscataway, NJ, USA aut NATIONALLICENCE 700 1- Narangajavana Jarunya Department of Biotechnology, Faculty of Science, Mahidol University, 10400, Prayathai, Bangkok, Thailand aut NATIONALLICENCE 700 1- Maliga Pal Waksman Institute, Rutgers, the State University of New Jersey, 08854-8020, Piscataway, NJ, USA aut NATIONALLICENCE 773 0- Plant Molecular Biology Kluwer Academic Publishers 61/4-5(2006-07-01), 711-718 0167-4412 61:4-5<711 2006 61 11103 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer