Kodrík D., Kludkiewicz B., Navrátil O., Skoková Habuštová O., Horáčková V., Svobodová Z., Vinokurov K.S., Sehnal F.
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
171:
209-224,
2013
Klíčová slova:
Genetically modified organism, Silk protease inhibitor, Protease, Transgene, Insect, Resistance, Potato
Abstrakt:
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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