Ekspresija gena za inhibitore cisteinskih proteinaza (OCI i OCII) u transformisanim biljkama krompira (Solanum tuberosum L.)

Abstract

Kombinovanje ili “slaganje” različitih gena u transgenim biljkama radi postizanja uspešnije kontrole patogena i štetočina i/ili većeg prinosa predstavlja jednu od glavnih oblasti istraživanja savremene biotehnologije. Orizacistatini I i II (OCI i OCII), proteinazni inhibitori različitih specifičnosti, pokazali su potencijal u kontroli štetočina koje koriste cisteinske proteinaze za digestiju proteina. Da bi se pojačao njihov inhibitorni potencijal i, eventualno, povećala efikasnost ovih inhibitora u kontroli štetočina, oba cistatina su koeksprimirana u transformisanim biljkama tri sorte krompira. “Slaganje” orizacistatinskih gena kod sorti Dragačevka i Dezire ostvareno je postupkom ko-transformacije i zabeležena je frekvenca kointegracije OCI i OCII gena od 20-22%. Kod sorte Jelica sekvencijalna re-transformacija se pokazala kao efikasniji pristup: frekvenca integracije OCII gena nakon re-transformacije OCI-transformisane linije iznosila je 91%. Istovremeno, “slaganje” dva orizacistatnska gena, bilo postupkom ko- ili re-transformacije, postignuto je upotrebom nptII gena kao jedinog selekcionog markera. Ekspresija OCI i OCII gena indukovana povređivanjem i akumulacija biološki aktivnih rekombinantnih OCI i OCII proteina potvrđena je kod svih analiziranih OCI/OCII transformisanih linija krompira. OCI/OCII linije krompira nisu ispoljavale značajna odstupanja od normalnog fenotipa, što ukazuje na nizak nivo somaklonalnih varijacija i odsustvo uticaja rekombinantnih OCI i OCII na metabolizam biljke domaćina. Iako nije uticala na preživljavanje, ishrana larvi krompirove zlatice (Leptinotarsa decemlineata Say) listovima krompira koji eksprimiraju oba orizacistatina imala je značajan uticaj na različite osobine performanse rasta i razvića larvi. Larve hranjene transformisanim listovima su se presvlačile ranije, i tokom L2 i L3 stupnja uvećavale masu do 29,7% brže i konzumirale listove do 29,1% brže u odnosu na one hranjene netransformisanim listovima. Istovremeno, larve na OCI/OCII listovima su do tri dana ranije dostizale maksimum mase i ranije “usporavale” sa ishranom ulazeći u prepupalnu fazu razvića. Uprkos povećanju performansi rasta i ishrane, pri istoj efikasnosti ishrane, L4 larve na transformisanim listovima nisu u potpunosti uspele da kompenzuju negativne efekte prisustva orizacistatina u hrani. U odnosu na larve hranjene netransformisanim listovima, maksimalna masa na kraju larvenog razvića i ukupan stepen oštećenja listova bili su do 19,4% i do 18,5% manji kod larvi krompirove zlatice hranjenih OCI/OCII transformisanim listovima krompira. Smanjenje mase larvi na OCI/OCII listovima dovelo je i do pojave adulta krompirove zlatice sa do 26,3% redukovanom telesnom masom. Analiza ukupne proteinazne aktivnosti kod larvi krompirove zlatice pokazala je inicijalno smanjenje digestivnog kapaciteta L3 larvi do 56%, koje je praćeno inhibicijom specifične aktivnosti cisteinskih proteinaza do 62% (akutni efekat). Sa druge strane, pri hroničnoj ingestiji OCI/OCII listova krompira, ukupna i aktivnost cisteinskih proteinaza kod L3 larvi ne pokazuje značajna odstupanja od kontrolnog nivoa, ukazujući na kompenzatorne odgovore proteaza larvi na prisustvo rekombinantnih orizacistatina u ishrani. Uočene promene u ishrani, rastu i razviću larvi krompirove zlatice mogu biti tumačene kao regulatorni odgovor kojim se postiže maksimum telesne mase uprkos prisustvu rekombinantnih inhibitora. Time se pokreću složene interakcije između ishrane, digestivnih procesa i regulatornih mehanizama rasta i razvića koje mogu da kompenzuju potencijalno smanjenje adaptivne vrednosti usled ishrane transformisanim listovima.

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.