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

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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.

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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.

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Authors and Affiliations

  1. Institute of Entomology, Biology Centre ASCR, Branišovská 31, 370 05, České Budějovice, Czech Republic

    Dalibor Kodrík, Barbara Kludkiewicz, Oxana Skoková Habuštová, Konstantin S. Vinokurov & František Sehnal

  2. Faculty of Science, University of South Bohemia, Branišovská 31, 370 05, České Budějovice, Czech Republic

    Zdeňka Svobodová

  3. Institute of Experimental Botany, ASCR, Rozvojová 263, 165 02, Praha 6, Czech Republic

    Oldřich Navrátil

  4. Potato Research Institute Ltd., Dobrovského 2366, 580 01, Havlíčkův Brod, Czech Republic

    Vendulka Horáčková

Authors
  1. Dalibor Kodrík
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  2. Barbara Kludkiewicz
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  3. Oldřich Navrátil
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  4. Oxana Skoková Habuštová
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  5. Vendulka Horáčková
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  6. Zdeňka Svobodová
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  7. Konstantin S. Vinokurov
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  8. František Sehnal
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Correspondence to Dalibor Kodrík.

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Kodrík, D., Kludkiewicz, B., Navrátil, O. et al. Protease Inhibitor from Insect Silk-Activities of Derivatives Expressed In Vitro and in Transgenic Potato. Appl Biochem Biotechnol 171, 209–224 (2013). https://doi.org/10.1007/s12010-013-0325-9

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  • DOI: https://doi.org/10.1007/s12010-013-0325-9

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