Abstract
Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes—aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)—in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.
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Abbreviations
- aadA :
-
aminoglycoside 3'-adenyltransferase
- BA:
-
6-benzyladenine
- gfp :
-
green fluorescent protein
- MS:
-
Murashige and Skoog
- NAA:
-
α-naphthaleneacetic acid
References
Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing. Curr Opin Plant Biol 2:109–113
Daniell H (2002) Molecular strategies for gene containment in transgenic crops. Nat Biotechnol 20:581–586. doi:10.1038/nbt0602-581
Daniell H, Khan MS, Allison L (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci 7:84–91. doi:10.1016/S1360-1385(01) 02193-8
de Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74. doi:10.1038/83559
DeGray G, Rajasekaran K, Smith F, Sanford J, Daniell H (2001) Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol 127:852–862. doi:10.1104/pp.010233
Dufourmantel N, Pelissier B, Garcon F, Peltier G, Ferullo JM, Tissot G (2004) Generation of fertile transplastomic soybean. Plant Mol Biol 55:479–489. doi:10.1007/s11103-004-0192-4
Jeong SW, Jeong WJ, Woo JW, Choi DW, Liu JR (2004) Dicistronic expression of the green fluorescent protein and antibiotic resistance genes in the plastid for tracking and selecting plastid-transformed cells in tobacco. Plant Cell Rep 22:747–751
Khan MS, Maliga P (1999) Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nat Biotechnol 17:910–915
Kumar S, Dhingra A, Daniell H (2004) Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Mol Biol 56:203–216. doi:10.1007/s11103-004-2907-y
Lee SB, Kwon HB, Kwon SJ, Park SC, Jeong MJ, Han SE, Byun MO, Daniell H (2003) Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol Breed 11:1–13. doi:10.1023/A:1022100404542
Lin SS, Henriques S, Wu HW, Niu QW, Yeh SD, Chua NH (2007) Strategies and mechanisms of plant virus resistance. Plant Biotechnol Rep 1:125–134
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x
Raquel H, Lourenco T, Moita C, Oliveira MM (2008) Expression of prune dwarf Ilarvirus coat protein sequences in Nicotiana benthamiana plants interferes with PDV systemic proliferation. Plant Biotechnol Rep 2:75–85. doi:10.1007/s11816-008-0050-y
Ruf S, Hermann M, Berger I, Carrer H, Bock R (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 19:870–875
Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PTJ, Staub JM, Nehra NS (1999) Stable chloroplast transformation in potato: Use of green fluorescent protein as a plastid marker. Plant J 19:209–216. doi:10.1046/j.1365-313X.1999.00508.x
Sugita M, Sugiura M (1996) Regulation of gene expression in chloroplasts of higher plants. Plant Mol Biol 32:315–326. doi:10.1007/BF00039388
Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci 87:8526–8530
Acknowledgments
This work was supported by a grant (Code 20070301034020) to WJJ from the BioGreen 21 Program funded by the Rural Development Administration and by grants to JRL from the Crop Functional Genomics Center of the 21st Century Frontier Research Program; from the Korea Science and Engineering Foundation through the Plant Metabolism Research Center of the Kyung Hee University funded by the Korea Ministry of Education, Science, and Technology; and from the Marine Extreme Genome Research Center funded by the Korean Ministry of Marine Affairs and Fisheries.
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Davarpanah, S.J., Jung, S.H., Kim, Y.J. et al. Stable Plastid Transformation in Nicotiana benthamiana . J. Plant Biol. 52, 244–250 (2009). https://doi.org/10.1007/s12374-009-9023-0
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DOI: https://doi.org/10.1007/s12374-009-9023-0