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Stable transformation of phaC2 gene in tobacco chloroplast genome

Published online by Cambridge University Press:  20 March 2007

Wang Yu-Hua ,
Wu Zhong-Yi ,
Zhang Xiu-Hai ,
Wang Yong-Qin ,
Huang Cong-Lin  and
Yang Qing
Wang Yu-Hua
Affiliation:
College of Life Science, Northwest University, Xian 710069, China Beijing Agro-biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China
Wu Zhong-Yi
Affiliation:
Beijing Agro-biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China
Zhang Xiu-Hai
Affiliation:
Beijing Agro-biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China
Wang Yong-Qin
Affiliation:
Beijing Agro-biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China
Huang Cong-Lin*
Affiliation:
Beijing Agro-biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100089, China
Yang Qing*
Affiliation:
College of Life Science, Nanjing Agricultural University, Nanjing 210095, China
*
*Corresponding authors. E-mail: [email protected]; [email protected]
*Corresponding authors. E-mail: [email protected]; [email protected]
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Abstract

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The key enzyme for mcl-PHA biosynthesis is type II PHA synthase. The gene phaC2 encoding type II PHA synthase was placed under the control of psbA-pro and psbA-ter of rice (Oryza sativa) to construct a phaC2 cassette, which was ligated with the screening marker gene aadA cassette (prrn–aadA–TpsbA-ter). These recombined fragments were cloned between the plastid rbcL and accD genes for targeting to the large single copy region of the chloroplast genome. A chloroplast transformation vector, pTC2, was constructed and introduced into the tobacco (Nicotiana tobacum) chloroplast genome by particle bombardment. PCR and Southern blot analysis confirmed stable integration of phaC2 into the chloroplast genomes of T0 and T1 transgenic plants, and T1 transgenic plants exhibited homoplasmy. The expression of phaC2 at transcription level was detected by reverse transcriptase–polymerase chain reaction (RT-PCR). Recombinant transgenes in the tobacco chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. To our knowledge, this is the first report on the stable transformation of phaC2 encoding type II PHA synthase in tobacco via chloroplast genetic engineering.

Keywords

chloroplast genetic transformationmaternal inheritancemedium-chain-length polyhydroxyalkanoatestobaccotransgenic safety
Type
Research Article
Information
Chinese Journal of Agricultural Biotechnology , Volume 3 , Issue 3 , December 2006 , pp. 201 - 207
Copyright
China Agricultural University and Cambridge University Press 2006

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References

Anderson, AJ and Dawes, EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiological Reviews 54: 450–472.CrossRefGoogle ScholarPubMed
Arai, Y, Shikanai, T, Doi, Y, Yoshida, S, Yamaguchi, I and Nakashita, H (2004) Production of polyhydroxybutyrate by polycistronic expression of bacterial genes in tobacco plastid. Plant and Cell Physiology 45(9): 1176–1184.CrossRefGoogle ScholarPubMed
Boynton, JE, Gillham, NW, Harris, EH, et al. (1988) Chloroplast transformation in chlamydomonas with high velocity microprojectiles. Science 240: 1534–1538.CrossRefGoogle ScholarPubMed
Daniell, H (2002) Molecular strategies for gene containment in transgenic crops. Nature Biotechnology 20: 581–586.CrossRefGoogle ScholarPubMed
Daniell, H, Khan, MS and Alison, L (2002) Milestones in chloroplast genetic engineering: An environmentally friendly era in biotechnology. Trends in Plant Science 7: 84–91.CrossRefGoogle ScholarPubMed
Daniell, H, Chebolu, S, Kumar, S, Singleton, M and Falconer, R (2005) Chloroplast–derived vaccine antigens and other therapeutic proteins. Vaccine 23: 1779–1783.CrossRefGoogle ScholarPubMed
DeGray, G, Rajasekaran, K, Smith, F, Sanford, J and Daniell, H (2001) Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiology 127: 852–862.CrossRefGoogle ScholarPubMed
Gong, XS and Yan, LF (1991) Improvement of chloroplast DNA isolation from higher plant. Chinese Science Bulletin 36(6): 467–469 (in Chinese).Google Scholar
Gray, MW (1993) Origin and evolution of organelle genomes. Current Opinion in Genetics and Development 3: 884–890.CrossRefGoogle ScholarPubMed
Hahn, JJ, Eschenlauer, AC, Sleytr, UB, Somers, DA and Srienc, F (1999) Peroxisomes as sites for synthesis of polyhydroxyalkanoates in transgenic plants. Biotechnology Progress 15(6): 1053–1057.CrossRefGoogle ScholarPubMed
Houmiel, KL, Slater, S, Broyles, D, et al. (1999) Poly (β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus. Planta 209: 547–550.CrossRefGoogle ScholarPubMed
Kang, TJ, Loc, NH, Jang, MO, et al. (2003) Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization. Transgenic Research 12: 683–691.CrossRefGoogle Scholar
Lee, SB, Kwon, HB, Kown, SJ, et al. (2003) Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Molecular Breeding 11: 1–13.CrossRefGoogle Scholar
Lee, SY (1996) Bacterial polyhydroxyalkanoates. Biotechnology and Bioengineering 49: 1–14.3.0.CO;2-P>CrossRefGoogle ScholarPubMed
Lutz, KA, Knapp, JE and Maliga, P (2001) Expression of bar in the plastid genome confers herbicide resistance. Plant Physiology 125: 1585–1590.CrossRefGoogle ScholarPubMed
McBride, KE, Svab, Z, Schaaf, DJ, et al. (1995) Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Biotechnology 13(4): 362–365.Google Scholar
Nawrath, C, Poirier, Y and Somerville, C (1994) Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation. Proceedings of the National Academy of Sciences, USA 91: 12760–12764.CrossRefGoogle Scholar
Palmer, JD (1990) Contrasting modes and tempos of genome evolution in land plant organelles. Trends in Genetics 6: 115–120.CrossRefGoogle ScholarPubMed
Poirier, Y, Dennis, DE, Klomparens, K and Somerville, C (1992) Polyhydroxybutyrate, a biodegradable thermoplastic, produced in transgenic plants. Science 256: 520–523.CrossRefGoogle ScholarPubMed
Romano, A, van der Plas, LHW, Witholt, B, et al. (2005) Expression of poly-3-(R)-hydrozyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs. Planta 220(3): 455–464.CrossRefGoogle Scholar
Shinozaki, K, Ohme, M, Tanaka, M, et al. (1986) The complete nucleotide sequence of the tobacco chloroplast genome: Its gene organization and expression. EMBO Journal 5: 2043–2049.CrossRefGoogle ScholarPubMed
Su, N, Yang, B, Meng, K, et al. (2002) The expression of Bt and OC gene cotransformation in tobacco chloroplast. Scientia Agricultura Sinica 35(4): 394–398 (in Chinese).Google Scholar
Svab, Z and Maliga, P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proceedings of the National Academy of Sciences, USA 90: 913–917.CrossRefGoogle ScholarPubMed
Wakasugi, T, Sugita, M, Tsudzuki, T and Sugiura, M (1998) Updated gene map of tobacco chloroplast DNA. Plant Molecular Biology Reporter 16: 231–241.CrossRefGoogle Scholar
Watson, J, Koya, V, Leppla, SH and Daniell, H (2004) Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop. Vaccine 22: 4374–4384.CrossRefGoogle ScholarPubMed
Zhang, JY, Su, N, Zhang, ZL, Zhao, HY, Zhu, SW and Song, YR (2002) Expressing of poly-3-hydroxybutyrate synthetic genes through chloroplast genetic engineering. Chinese Science Bulletin 47(11): 845–849.Google Scholar