Applied Biochemistry and Biotechnology

, Volume 171, Issue 1, pp 209–224 | Cite as

Protease Inhibitor from Insect Silk-Activities of Derivatives Expressed In Vitro and in Transgenic Potato

  • Dalibor Kodrík
  • Barbara Kludkiewicz
  • Oldřich Navrátil
  • Oxana Skoková Habuštová
  • Vendulka Horáčková
  • Zdeňka Svobodová
  • Konstantin S. Vinokurov
  • František Sehnal
Article
  • 253 Downloads

Abstract

Several recombinant derivatives of serine protease inhibitor called silk protease inhibitor 2 (SPI2), which is a silk component in Galleria mellonella (Lepidoptera, Insecta), were prepared in the expression vector Pichia pastoris. Both the native and the recombinant protease inhibitors were highly active against subtilisin and proteinase K. The synthetic SPI2 gene with Ala codon in the P1 position was fused with mGFP-5 to facilitate detection of the transgene and its protein product. A construct of the fusion gene with plant regulatory elements (promoter 35S and terminator OCS) was inserted into the binary vector pRD400. The final construct was introduced into Agrobacterium tumefaciens that was then used for genetic transformation of the potato variety Velox. The transgene expression was monitored with the aid of ELISA employing polyclonal antibody against natural SPI2. In vitro tests showed increased resistance to the late blight Phytophthora infestans in several transformed lines. No effect was seen on the growth, mortality, life span or reproduction of Spodoptera littoralis (Lepidoptera, Insecta) caterpillars, while feeding on transformed potato plants expressing the fusion protein, indicating that the transformed potatoes may be harmless to non-target organisms.

Keywords

Genetically modified organism Silk protease inhibitor Protease Transgene Insect Resistance Potato 

Notes

Acknowledgments

This study was supported by project no. QI91A229 from the Ministry of Agriculture of the Czech Republic. The authors thank Misses J. Zralá and R. Tanzer Fabiánová for their technical assistance, and Mr. Richard Klee (University of South Bohemia) for the English corrections.

References

  1. 1.
    Barrett, A. J., & Salvesen, G. (1986). Proteinase inhibitors. Amsterdam: Elsevier.Google Scholar
  2. 2.
    Kurioka, A., Yamazaki, M., & Hirano, H. (1999). European Journal of Biochemistry, 259, 120–126.CrossRefGoogle Scholar
  3. 3.
    Sehnal, F., & Akai, H. (1998). International Journal of Insect Morphology and Embryology, 19, 79–132.CrossRefGoogle Scholar
  4. 4.
    Žurovec, M., Yang, C., Kodrík, D., & Sehnal, F. (1998). Journal of Biological Chemistry, 273, 15423–15428.CrossRefGoogle Scholar
  5. 5.
    Nirmala, X., Kodrík, D., Žurovec, M., & Sehnal, F. (2001). European Journal of Biochemistry, 268, 2064–2073.CrossRefGoogle Scholar
  6. 6.
    Nirmala, X., Mita, K., Vanisree, V., Žurovec, M., & Sehnal, F. (2001). Insect Molecular Biology, 10, 437–445.CrossRefGoogle Scholar
  7. 7.
    Hněvsová, V., Kodrík, D., & Weyda, F. (2011). European Journal of Entomology, 108, 711–715.Google Scholar
  8. 8.
    Kludkiewicz, B., Kodrík, D., Grzelak, K., Xavier, N., & Sehnal, F. (2005). Protein Expression and Purification, 43, 94–102.CrossRefGoogle Scholar
  9. 9.
    Gvozdeva, E. L., Ievleva, E. V., Gerasimova, N. G., Ozeretskovskaya, O. L., & Valueva, T. A. (2004). Applied Biochemistry and Microbiology, 40, 165–169.CrossRefGoogle Scholar
  10. 10.
    Bradford, M. M. (1976). Analytical Biochemistry, 72, 248–254.CrossRefGoogle Scholar
  11. 11.
    Laemmli, U. K. (1970). Nature, 227, 680–685.CrossRefGoogle Scholar
  12. 12.
    Wedde, M., Weise, C. H., Kopáček, P., Franke, P., & Vilcinskas, A. (1998). European Journal of Biochemistry, 255, 535–543.CrossRefGoogle Scholar
  13. 13.
    Chou, J., & Chou, T. C. (1987). Dose-effect analysis with microcomputers. Cambridge: Biosoft.Google Scholar
  14. 14.
    Ortego, F., Novillo, C., & Castanera, P. (1996). Archives of Insect Biochemistry and Physiology, 32, 163–180.CrossRefGoogle Scholar
  15. 15.
    Houseman, J. G., & Downe, E. R. (1982). Insect Biochemistry, 12, 651–655.CrossRefGoogle Scholar
  16. 16.
    Novillo, C., Castanera, P., & Ortego, F. (1997). Archives of Insect Biochemistry and Physiology, 36, 181–201.CrossRefGoogle Scholar
  17. 17.
    Christeller, J. T., Laing, W. A., Shaw, B. D., & Burgess, E. P. J. (1990). Insect Biochemistry, 20, 157–164.CrossRefGoogle Scholar
  18. 18.
    Lee, K. Y., Valaitis, A. P., & Denlinger, D. L. (1998). Archives of Insect Biochemistry and Physiology, 37, 97–205.Google Scholar
  19. 19.
    Haseloff, J., Siemering, K. R., Prasher, D. C., & Hodge, S. (1997). Proceedings of National Academy of Sciences of the USA, 94, 2122–2127.CrossRefGoogle Scholar
  20. 20.
    Datla, R. S. S., Hammerlindl, J. K., Panchuk, B., Pelcher, L. E., & Keller, W. (1992). Gene, 122, 383–384.CrossRefGoogle Scholar
  21. 21.
    Dietze, J., Blau, A., & Willmitzer, L. (1995). In I. Potrykus & G. Spangenber (Eds.), Gene transfer to plants (pp. 24–29). Berlin: Springer.CrossRefGoogle Scholar
  22. 22.
    Stoscheck, C. M. (1990). In M. P. Deutscher (Ed.), Methods in enzymology (Vol. 182, pp. 50–68). London: Academic Press.Google Scholar
  23. 23.
    Horáčková, V., Domkářová, J., & Kreuz, L. (2007). Scientific works. Potato Research Institute Havlíčkův Brod, 15, 101–109.Google Scholar
  24. 24.
    Vrbová, M., Horáček, J., Seidenglanz, M., Smýkalová, I., Sehnal, F., Navrátil, O., & Griga, M. (2010). In Vitro Cellular & Development Biology–Animal, 46, 118.Google Scholar
  25. 25.
    Hartl, M., Giri, A. P., Kaur, H., & Baldwin, I. T. (2010). The Plant Cell, 22, 4158–4175.CrossRefGoogle Scholar
  26. 26.
    Brown, E. S., & Dewhurst, C. F. (1975). Bulletin of Entomological Research, 65, 221–226.CrossRefGoogle Scholar
  27. 27.
    Jongsma, M. A., Bakker, P. L., Peters, J., Bosch, D., & Stiekema, W. J. (1995). Proceedings of National Academy of Sciences of the USA, 92, 8041–8045.CrossRefGoogle Scholar
  28. 28.
    Brioschi, D., Nadalini, L. D., Bengtson, M. H., Sogayar, M. C., Moura, D. S., & Silva-Filho, M. C. (2007). Insect Biochemistry and Molecular Biology, 37, 1283–1290.CrossRefGoogle Scholar
  29. 29.
    Martins, T., Oliveira, L., & Garcia, P. (2005). Biocontrol, 50, 761–770.CrossRefGoogle Scholar
  30. 30.
    Broadway, R. M. (1997). Journal of Insect Physiology, 43, 855–874.CrossRefGoogle Scholar
  31. 31.
    Hilder, V. A., & Boulter, D. (1999). Crop Protection, 18, 177–191.CrossRefGoogle Scholar
  32. 32.
    Gatehouse, J. A., Gatehouse, A. M. R., & Bown, D. P. (2000). In D. Michaud (Ed.), Recombinant protease inhibitors in plants (pp. 9–26). Georgetown: Landes Bioscience Eurekan Com.Google Scholar
  33. 33.
    Dunse, K. M., Stevens, J. A., Lay, F. T., Gaspar, Y. M., Heath, R. L., & Anderson, M. A. (2010). Proceedings of National Academy of Sciences of the USA, 107, 15011–15015.CrossRefGoogle Scholar
  34. 34.
    Navrátil, O., Kodrík, D., Kludkiewicz, B., Vinokurov, K. S., Sehnal, F., & Horáčková, V. (2012). In J. Romeis, M. Meissle, & F. Álvarez-Alfageme (Eds.), GMOs in Integrated Plant Production, IOBC wprs Bulletin, 73 (pp. 61–67). Darmstadt, Germany.Google Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Dalibor Kodrík
    • 1
  • Barbara Kludkiewicz
    • 1
  • Oldřich Navrátil
    • 3
  • Oxana Skoková Habuštová
    • 1
  • Vendulka Horáčková
    • 4
  • Zdeňka Svobodová
    • 2
  • Konstantin S. Vinokurov
    • 1
  • František Sehnal
    • 1
  1. 1.Institute of EntomologyBiology Centre ASCRČeské BudějoviceCzech Republic
  2. 2.Faculty of ScienceUniversity of South BohemiaČeské BudějoviceCzech Republic
  3. 3.Institute of Experimental Botany, ASCRPraha 6Czech Republic
  4. 4.Potato Research Institute Ltd.Havlíčkův BrodCzech Republic

Personalised recommendations