Ekspresija gena za inhibitore cisteinskih proteinaza (OCI i OCII) u transformisanim biljkama krompira (Solanum tuberosum L.)
Expression of cysteine proteinase inhibitor genes (OCI and OCII) in transformed potato (Solanum tuberosum L.) plants
Doctoral thesis (Published version)
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The combination or stacking different genes in transgenic plants to achieve disease and pest control and/or higher crop yield is one of a major method of contemporary biotechnology. Oryzacystatins I and II (OCI and OCII), inhibitors with different specificity, show potential in controlling pests that utilize cysteine proteinases for protein digestion. To strengthen this inhibitory range and, possibly, achieve an additive effect in the overall efficiency of these proteins against pests, both cystatins were co-expressed in three potato cultivars. Oryzacystatin genes pyramiding in Dragačevka and Desiree cultivars were achieved by co-transformation with OCI and OCII genes co-integration frequency of 20-22%. For Jelica cultivar sequential re-transformation was more efficient approach: OCII gene integration frequency following re-transformation of an OCI-expressing line was 91%. Additionally, pyramiding of different oryzacystatin genes, by co- or re-transformation approach, were achieved using the nptII gene as the only selection marker. Wounding induction of OCI and OCII gene transcripts and accumulation of biologically active OCI and OCII recombinant proteins was confirmed in all analyzed OCI/OCII transformed lines. OCI/OCII potato lines did not exhibit morphological abnormalities, indicating low level of somaclonal variation or interference of the recombinant OCI or OCII with host plant metabolism. In the absence of significant mortality, feeding Colorado potato beetle larvae (Leptinotarsa decemlineata Say) on OCI/OCII-expressing foliage had an impact on various aspects of the growth and developmental performances of larvae. Larvae feeding on transformed potato leaves tended to molt earlier and, especially during L2-L3 stages, gain weight up to 29.7% faster and consume leaf material up to 29.1% faster, compared to those on untransformed foliage. Larvae on OCI/OCII foliage were also reach maximum weight gained three days earlier and slow down earlier in preparation for pupation. Despite their faster growth and feeding, with similar efficiencies of conversion of ingested food, L4 larvae reared on transformed foliage were not compensating presence of the recombinant oryzacystatins in the diet. Compared to those on untransformed foliage, maximum weight gained and amount of foliage consumed were up to 19.4% and 18.5%, respectively, lower for the larvae fed on OCI/OCII potato foliage. Larval weight reduction on OCI/OCII foliage resulted in adult emergence with up to 26.3% reduced body mass. Analysis of total digestive proteinases activity showed initially, up to 56%, reduction in digestive capacity of L3 potato beetle larvae, accompanied with inhibition of cysteine proteinase specific activity up to 62% (acute effect). However, by continual ingestion of OCI/OCII potato foliage total and cysteine proteinases specific activities were at control level, suggesting compensatory responses of larvae protease system to the presence of recombinant oryzacystatins in the diet. The observed alterations in larval feeding and growth performance can be interpreted as regulatory responses aimed at stabilizing the final body weight despite presence of the recombinant inhibitors. These changes can trigger complex interactions between feeding, food processing and growth regulatory mechanisms, which tend to compensate for the potential fitness loss caused by feeding on transformed foliage.
Keywords:Gene stacking; Co-transformation; Re-transformation; Potato; Cysteine proteinase inhibitors; Oryzacystatin I; Oryzacystatin II; Insect resistance; Colorado potato beetle; Insect compensatory response
Source:University of Belgrade, Faculty of Biology, 2012, 1-233