Abstract
To analyze the suitability of Gateway® vectors for transformation of chloroplasts, we converted a standard plastid transformation vector into a Gateway® destination vector containing the necessary recombination sites attR1 and attR2. Insertion of the green fluorescent protein (GFP) coding sequence with associated T7g10 ribosome binding site into this destination vector created the expression vector for transformation of tobacco chloroplasts with the biolistic method. Correct integration of the transgene into the plastid genome was verified by PCR and the homoplasmic nature of the transformed plants was confirmed by Southern Blot analysis. Expression of the GFP reporter protein was monitored by confocal laser scanning microscopy (CLSM) and quantification by western blot analysis showed a GFP accumulation level of 3 % total soluble protein (TSP). The presented results clearly demonstrate that the Gateway® recombination sites are compatible with all steps of plastid transformation, from generation of transplastomic plants to expression of GFP. This is the first report of a plastid transformation vector made by the Gateway® recombinant cloning technology, which proves the suitability of this system for use in chloroplasts.
References
Bernard P (1995) New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers. Gene 162:159–160
Bock R (2007) Plastid biotechnology: prospects for herbicide and insect resistance, metabolic engineering and molecular farming. Curr Opin Biotechnol 18:100–106
Daniell H (1997) Transformation and foreign gene expression in plants mediated by microprojectile bombardment. Recomb Gene Expr Protoc 62:463–489
Dubin M, Bowler C, Benvenuto G (2008) A modified gateway cloning strategy for overexpressing tagged proteins in plants. Plant Methods 4:3
Eibl C, Zou Z, Beck A, Kim M, Mullet J, Koop H-U (1999) In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency. Plant J 19:333–345
Golds T, Maliga P, Koop H-U (1993) Stable plastid transformation in PEG-treated protoplasts of Nicotiana tabacum. Nat Biotech 11:95–97
Hajdukiewicz PT, Allison LA, Maliga P (1997) The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids. EMBO J 16:4041–4048
Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795
Karimi M, Inzé D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated planttransformation. Trends Plant Sci 7:193–195
Karimi M, Depicker A, Hilson P (2007) Recombinational cloning with plant gateway vectors. Plant Physiol 145:1144–1154
Kuroda H, Maliga P (2001) Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol 125:430–436
Landy A (1989) Dynamic, structural, and regulatory aspects of lambda sitespecific recombination. Ann Rev Biochem 58:913–949
Maliga P (2004) Plastid transformation in higher plants. Annu Rev Plant Biol 55:289–313
Murray M, Thompson W (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4326
Studier WF, Rosenberg AH, Dunn JJ, Dubendorff JW, David VG (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917
Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci 87:8526–8530
Verma D, Samson NP, Koya V, Daniell H (2008) A protocol for expression of foreign genes in chloroplasts. Nat Protocols 3:739–758
Waheed MT, Thönes N, Müller M, Hassan S, Razavi N, Lössl E, Kaul H-P, Lössl A (2011a) Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effective second-generation vaccines. Transgenic Res 20:271–282
Waheed MT, Thönes N, Müller M, Hassan SW, Gottschamel J, Lössl E, Kaul H-P, Lössl AG (2011b) Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes. Plant Biotechnol J 9:651–660
Ye GN, Hajdukiewicz PTJ, Broyles D, Rodriguez D, Xu CW, Nehra N, Staub JM (2001) Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 25:261–270
Zou Z, Eibl C, Koop HU (2003) The stem-loop region of the tobacco psbA 5′UTR is an important determinant of mRNA stability and translation efficiency. Mol Genet Genomics 269:340–349
Acknowledgments
This work was supported by the GLOBVAC program, subprogram Vaccination Research, part II (Project 192510) and funded by the Research Council of Norway (RCN). The authors also acknowledge the collaboration with the EU COST action FA0804.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Gottschamel, J., Waheed, M.T., Clarke, J.L. et al. A novel chloroplast transformation vector compatible with the Gateway® recombination cloning technology. Transgenic Res 22, 1273–1278 (2013). https://doi.org/10.1007/s11248-013-9726-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-013-9726-3