A Guide to Choosing Vectors for Transformation of the Plastid Genome of Higher Plants1[C][W][OA]
Plastid transformation, originally developed in tobacco (Nicotiana tabacum), has recently been extended to a number of crop species enabling in vivo probing of plastid function and biotechnological applications. In this article we report new plastid vectors that enable insertion of transgenes in the...
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| Published in: | Plant physiology (Bethesda) Vol. 145; no. 4; pp. 1201 - 1210 |
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| Main Authors: | , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Rockville
American Society of Plant Biologists
01-12-2007
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| Series: | Focus Issue on Vector Systems for Plant Research and Biotechnology |
| Subjects: | |
| Online Access: | Get full text |
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| Summary: | Plastid transformation, originally developed in tobacco (Nicotiana tabacum), has recently been extended to a number of crop species enabling in vivo probing of plastid function and biotechnological applications. In this article we report new plastid vectors that enable insertion of transgenes in the inverted repeat region of the plastome between the trnV and 3'rps12 or trnI and trnA genes. Efficient recovery of transplastomic clones is ensured by selection for spectinomycin (aadA) or kanamycin (neo) resistance genes. Expression of marker genes can be verified using commercial antibodies that detect the accumulation of neomycin phosphotranseferase II, the neo gene product, or the C-terminal c-myc tag of aminoglycoside-3''-adenylytransferase, encoded by the aadA gene. Aminoglycoside-3''-adenylytransferase, the spectinomycin inactivating enzyme, is translationally fused with green fluorescent protein in two vectors so that transplastomic clones can be selected by spectinomycin resistance and visually identified by fluorescence in ultraviolet light. The marker genes in the new vectors are flanked by target sites for Cre or Int, the P1 and phiC31 phage site-specific recombinases. When uniform transformation of all plastid genomes is obtained, the marker genes can be excised by Cre or Int expressed from a nuclear gene. Choice of expression signals for the gene of interest, complications caused by the presence of plastid DNA sequences recognized by Cre, and loss of transgenes by homologous recombination via duplicated sequences are also discussed to facilitate a rational choice from among the existing vectors and to aid with new target-specific vector designs. |
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| Bibliography: | Some figures in this article are displayed in color online but in black and white in the print edition. www.plantphysiol.org/cgi/doi/10.1104/pp.107.106963 This work was supported by the U.S. Department of Agriculture Biotechnology Risk Assessment Research Grant Program (grant no. 2005–33120–16524) and the National Science Foundation Eukaryotic Genetics Program (grant no. MCB–039958 to P.M.). Kerry Lutz was the recipient of a Busch Predoctoral Fellowship. Present address: Johns Hopkins University, School of Medicine, Department of Neurology, 600 N. Wolfe Street, Pathology 631, Baltimore, MD 21287. The online version of this article contains Web-only data. Corresponding author; e-mail maliga@waksman.rutgers.edu. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Pal Maliga (maliga@waksman.rutgers.edu). Open Access articles can be viewed online without a subscription. |
| ISSN: | 0032-0889 1532-2548 |
| DOI: | 10.1104/pp.107.106963 |