European Food Research and Technology

, Volume 231, Issue 1, pp 143–150 | Cite as

Estimation of the homoplasmy degree for transplastomic tobacco using quantitative real-time PCR

  • Huifeng Shen
  • Bingjun Qian
  • Litao Yang
  • Wanqi Liang
  • Weiwei Chen
  • Zhenhua Liu
  • Dabing Zhang
Original Paper

Abstract

Estimation of the homoplasmy of transgene in chloroplast is a necessary step in chloroplast transformation. This task is usually achieved by Southern blot analysis, which is laborious, time-consuming, requires large amounts of plant materials and needs hazardous radioisotopes in some cases. To develop a fast, sensitive, stable and effective technique for determining the homoplasmy of transgene in chloroplast, one real-time PCR system based on TaqMan probe technique was developed for evaluating the homoplasmy degree. In the real-time PCR system, one assay targets the exogenous faeG gene, and another one the tobacco chloroplast reference gene, maturase gene within the trnK intron (matK). The homoplasmy of the transgene was determined by the comparison of copy number of faeG and matK. The analyzed results of 17 transplastomic tobacco lines using the developed real-time PCR system were consistent with those from Southern blot analysis, indicating that the real-time PCR method is suitable for estimating the homoplasmy degree of transplastomic plant with the advantages of high efficiency and throughput, low cost and saving time.

Keywords

Transplastomic tobacco Real-time quantitative PCR Homoplasmy degree 

References

  1. 1.
    Maliga P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol 21:20–28CrossRefGoogle Scholar
  2. 2.
    Verma D, Daniell H (2007) Chloroplast vector systems for biotechnology applications. Plant Physiol 145:1129–1143CrossRefGoogle Scholar
  3. 3.
    Scott SE, Wilkinson MJ (1999) Low probability of chloroplast movement from oilseed rape (Brassica napus) into wild Brassica rapa. Nat Biotechnol 17:390–392CrossRefGoogle Scholar
  4. 4.
    Nawrath C, Poirier Y, Somerville C (1994) Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation. Proc Natl Acad Sci USA 91:12760–12764CrossRefGoogle Scholar
  5. 5.
    Zhang ZL, Ren YG, Shen YX, Shan S, Fan GC, Wu XF, Qian KX, Shen GF (2000) Expression of Bacillus thuringiensis (Bt) crystal toxin gene in the chloroplast of tobacco. Yi Chuan Xue Bao 27:270–277Google Scholar
  6. 6.
    Zhang ZL, Chen X, Qian KX, Shen GF (1999) Studies on inset resistance of Bt transplastomic plants and the phenotype of their progenies. Acta Bot Sin 41:947–951Google Scholar
  7. 7.
    McBride KE, Svab Z, Schaaf DJ, Hogan PS, Stalker DM, Maliga P (1995) Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Biotechnology (N Y) 13:362–365CrossRefGoogle Scholar
  8. 8.
    Kota M, Daniell H, Varma S, Garczynski SF, Gould F, Moar WJ (1999) Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc Natl Acad Sci USA 96:1840–1845CrossRefGoogle Scholar
  9. 9.
    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 19:71–74CrossRefGoogle Scholar
  10. 10.
    Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N, Paradkar V, Schlittler M, Carroll JA, Spatola L, Ward D, Ye G, Russell DA (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 18:333–338CrossRefGoogle Scholar
  11. 11.
    Daniell H, Lee SB, Panchal T, Wiebe PO (2001) Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol 311:1001–1009CrossRefGoogle Scholar
  12. 12.
    Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, Fairweather N, Ytterberg J, van Wijk KJ, Dougan G, Maliga P (2003) Expression of tetanus toxin Fragment C in tobacco chloroplasts. Nucleic Acids Res 31:1174–1179CrossRefGoogle Scholar
  13. 13.
    Li Y, Sun M, Liu J, Yang Z, Zhang Z, Shen G (2006) High expression of foot-and-mouth disease virus structural protein VP1 in tobacco chloroplasts. Plant Cell Rep 25:329–333CrossRefGoogle Scholar
  14. 14.
    Hasunuma T, Miyazawa S, Yoshimura S, Shinzaki Y, Tomizawa K, Shindo K, Choi SK, Misawa N, Miyake C (2008) Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering. Plant J 55:857–868CrossRefGoogle Scholar
  15. 15.
    Scotti N, Alagna F, Ferraiolo E, Formisano G, Sannino L, Buonaguro L, De Stradis A, Vitale A, Monti L, Grillo S, Buonaguro FM, Cardi T (2009) High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts. Planta 229:1109–1122CrossRefGoogle Scholar
  16. 16.
    Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917CrossRefGoogle Scholar
  17. 17.
    Guda C, Lee SB, Daniell H (2000) Stable expression of a biodegradable protein-based polymer in tobacco chloroplasts. Plant Cell Rep 19:257–262CrossRefGoogle Scholar
  18. 18.
    Soria-Guerra RE, Alpuche-Solis AG, Rosales-Mendoza S, Moreno-Fierros L, Bendik EM, Martinez-Gonzalez L, Korban SS (2009) Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer. Planta 229:1293–1302CrossRefGoogle Scholar
  19. 19.
    Kanamoto H, Yamashita A, Asao H, Okumura S, Takase H, Hattori M, Yokota A, Tomizawa K (2006) Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res 15:205–217CrossRefGoogle Scholar
  20. 20.
    Kumar S, Dhingra A, Daniell H (2004) Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance. Plant Physiol 136:2843–2854CrossRefGoogle Scholar
  21. 21.
    Mason G, Provero P, Vaira AM, Accotto GP (2002) Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnol 2:20CrossRefGoogle Scholar
  22. 22.
    Yang L, Pan A, Jia J, Ding J, Chen J, Cheng H, Zhang C, Zhang D (2005) Validation of a tomato-specific gene, LAT52, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic tomatoes. J Agric Food Chem 53:183–190CrossRefGoogle Scholar
  23. 23.
    Yang L, Ding J, Zhang C, Jia J, Weng H, Liu W, Zhang D (2005) Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep 23:759–763CrossRefGoogle Scholar
  24. 24.
    Ding J, Jia J, Yang L, Wen H, Zhang C, Liu W, Zhang D (2004) Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes. J Agric Food Chem 52:3372–3377CrossRefGoogle Scholar
  25. 25.
    Huang Y, Liang W, Pan A, Zhou Z, Huang C, Chen J, Zhang D (2003) Production of FaeG, the major subunit of K88 fimbriae, in transgenic tobacco plants and its immunogenicity in mice. Infect Immun 71:5436–5439CrossRefGoogle Scholar
  26. 26.
    Verma D, Samson NP, Koya V, Daniell H (2008) A protocol for expression of foreign genes in chloroplasts. Nat Protoc 3:739–758CrossRefGoogle Scholar
  27. 27.
    Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325CrossRefGoogle Scholar
  28. 28.
    Wakasugi T, Sugita M, Tsudzuki T, Sugiura M (1998) Updated gene map of tobacco chloroplast DNA. Plant Mol Biol Rep 16:231–241CrossRefGoogle Scholar
  29. 29.
    Selvaraj D, Sarma RK, Sathishkumar R (2008) Phylogenetic analysis of chloroplast matK gene from Zingiberaceae for plant DNA barcoding. Bioinformation 3:24–27Google Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Huifeng Shen
    • 1
  • Bingjun Qian
    • 2
  • Litao Yang
    • 1
  • Wanqi Liang
    • 1
  • Weiwei Chen
    • 1
  • Zhenhua Liu
    • 1
  • Dabing Zhang
    • 1
  1. 1.GMO Detection Laboratory, SJTU-Bor Luh Food Safety Center, School of Life Science and TechnologyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China
  2. 2.Department of Food Science and Engineering, School of Agriculture and BiologyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China

Personalised recommendations