Plant Molecular Biology

, Volume 24, Issue 2, pp 341–351 | Cite as

Expression of mouse metallothionein-I gene confers cadmium resistance in transgenic tobacco plants

  • Aihua Pan
  • Meizhu Yang
  • Feng Tie
  • Lingyua Li
  • Zhangliang Chen
  • Biggen Ru
Research Article


Transgenic tobacco plants containing a mouse metallothionein-I (MT-I) gene fused to the cauliflower mosaic virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming tobacco leaf discs with an Agrobacterium tumefaciens strain carrying the chimaeric gene. Transformants were directly selected and rooted on medium containing cadmium and kanamycin. A total of 49 individual transgenic tobacco plants were regenerated. Among them 20% showed a very high expression level and their growth was unaffected by up to 200 μM cadmium, whereas the growth of control plants was severely affected leaf chlorosis occurred on medium containing only 10 μM cadmium. The concentration of MT-I in leaves of control and transgenic tobacco was determined with Cd/haemoglobin saturation assay, a polarographic method and western blotting. In addition, seeds from self-fertilized transgenic plants were germinated on medium containing toxic levels of cadmium and scored for tolerance/susceptibility to this heavy metal. The ratio of tolerant to susceptible plants was 3:1 indicating that the metallothionein gene is inherited as a single locus.

Key words

tobacco transgenic mouse metallothionein gene cadmium resistance 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Beavington F: Heavy metal contamination of vegetables and soil in domestic gardens around a complex. Envir Pollut 9: 211–217 (1975).Google Scholar
  2. 2.
    Bevan MW: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).Google Scholar
  3. 3.
    Briggs RW, Armitage IM: Evidence for site-selective metal binding in calf liver metallothionein. Biol Chem 257: 1259–1262 (1982).Google Scholar
  4. 4.
    Burnette WN: Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to antibody and radioiodinated protein A. Anal Biochem 112: 195–203 (1981).Google Scholar
  5. 5.
    Doyle JJ, Doyle JL: Isolation of plant DNA from fresh tissue. Focus 12: 13–15 (1988).Google Scholar
  6. 6.
    Durmam DM, Perrin F, Gannon F, Palmiter RD: Isolation and characterization of the mouse metallothionein-I gene. Proc Natl Acad Sci USA 77: 6511–6515 (1980).Google Scholar
  7. 7.
    Fillati JJ, Kiser J, Rose R, Comai L: Efficient transfer of a glyphosate tolerance gene in tomato using a binary Agrobacterium tumefaciens vector. Bio/technology 5: 726–730 (1987).Google Scholar
  8. 8.
    Hofgen R, Willmitzer L: Storage of competent cells for Agrobacterium transformation. Nucl Acids Res 16: 9877 (1988).Google Scholar
  9. 9.
    Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).Google Scholar
  10. 10.
    Kagi JHR, Kojima Y: Metallothionein II. Experirientia Suppl. 52: 26–61 (1987).Google Scholar
  11. 11.
    Kagi JHR, Nordberg M (eds) Metallothionein. Birkhauser Verlag, Basel (1979).Google Scholar
  12. 12.
    Kagi JHR, Schaffer A: Biochemistry of metallothionein. Biochemistry 27: 8509–8515 (1988).Google Scholar
  13. 13.
    Little S: Plasmid DNA separations in high performance vertical tube rotors. Effect of speed on run times. Applications Data DS-726. Beckman Instruments, Spinco Division, Palo Alto, CA (1988).Google Scholar
  14. 14.
    Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).Google Scholar
  15. 15.
    Matzke AJM, Matzke MA: A set of novel Ti plasmid-derived vectors for the production of transgenic plants. Plant Mol Biol 7: 357–365 (1986).Google Scholar
  16. 16.
    Onosaka S, Cherian MG: Comparison of metallothionein determination by polarographic and cadmium-saturation methods. Toxic Appl Pharm 63: 270–274 (1982).Google Scholar
  17. 17.
    Rauser WE: The amount of cadmium associated with Cd binding protein in roots of Agrostis gigantea, maize and tomato. Plant Sci 43: 85–91 (1986).Google Scholar
  18. 18.
    Robinson NJ, Barton K, Naranjo CM, Sillerud LO: Characterization of metal binding peptides from cadmium resistant plant cells. Experientia Suppl 52: 323–327 (1987).Google Scholar
  19. 19.
    Robinson NJ, Jackson PJ: ‘Metallothionein-like’ metal complexes in angiosperms: their structure and function. Physiol Plant 67: 499–506 (1986).Google Scholar
  20. 20.
    Ru BG, Pan AH, Wang ZX, Zhang LX: Isolation and characterization of mouse liver metallothionein. Chin J Biochem 7: 284–289 (1991).Google Scholar
  21. 21.
    Sanders PR, Winter JA, Barnason AR, Rogers SG, Fraley RT: Comparison of cauliflower mosaic virus 35S and nopaline synthase promoters in transgenic plants. Nucl Acids Res 15: 1543–1558 (1987).Google Scholar
  22. 22.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).Google Scholar
  23. 23.
    Sherlock JC: Cadmium in foods and the diet. Experientia 40: 152–156 (1984).Google Scholar
  24. 24.
    Spitz M, Spitz L, Thorpe R, Eugui E: Intraplenic primary immunization for the production of monoclonal antibidies. J Immunol Meth 70: 39–43 (1984).Google Scholar
  25. 25.
    Vaeck M, Reynaerts A, Hofte H, Jansens S, DeBeuckeleer M, Dean C, Zabeau M, VanMontagu M, Leemans J: Transgenic plants protected from insect attack. Nature 328: 33–37 (1987).Google Scholar
  26. 26.
    Winge DR, Nielson KB, Gray WR, Hamer DH: Yeast metallothionein sequence and metal-binding properties. J Biol Chem 260: 14464–14470 (1985).Google Scholar
  27. 27.
    Zhang LX, Zhang TF, Li LY: Methods of biochemical experiments. People's Education Press, Beijing (1982).Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Aihua Pan
    • 1
  • Meizhu Yang
    • 1
  • Feng Tie
    • 1
  • Lingyua Li
    • 1
  • Zhangliang Chen
    • 1
  • Biggen Ru
    • 1
  1. 1.National Laboratory of Protein Engineering and Plant Genetic Engineering, Department of BiologyPeking UniversityBeijingChina

Personalised recommendations