6.1 POVZETEK
Škodljivci predstavljajo v kmetijstvu veliko težavo, ki močno zmanjša količino pridelka kulturnih rastlin. Izguba pridelkov zaradi škodljivcev se zmanjša z uporabo pesticidov ali z uporabo odpornih sort kulturnih rastlin, vzgojenih s pomočjo klasičnega križanja ali z uporabo tehnologije genskega inženirstva. Le-ta že omogoča razvoj sort odpornih proti različnim žuželkam. Najuspešnejši primer so GSR, ki vsebujejo insekticidne toksine iz talne bakterije B. thuringiensis. Kljub velikemu naboru pesticidnih sredstev in uspehu GSR odpornih proti škodljivcem je povpraševanje po novih insekticidnih učinkovinah še vedno zelo aktualno. Proti že obstoječim insekticidom se namreč lahko pojavi odpornost, prav tako se pojavlja potreba po iskanju naravnejših insekticidnih sredstev.
Glive predstavljajo bogat, a slabo raziskan vir številnih naravnih substanc, ki jih lahko uporabimo za izboljšavo rastlin. Med drugim so na Odseku za biotehnologijo IJS iz gliv, ki oblikujejo plodišča, izolirali več genov, ki kodirajo proteine z insekticidno aktivnostjo.
Čeprav zelo obetavni, ti in podobni proteini do sedaj še niso bili uporabljeni za izboljšavo rastlin, zato so rezultati objavljeni v okviru te doktorske naloge zelo pomembni tako za temeljno kot aplikativno znanje na tem področju. V doktorski nalogi smo proučili primernost nekaterih nedavno odkritih proteinov iz gliv za izboljšavo odpornosti krompirja proti koloradskemu hrošču, ki predstavlja najhujšega škodljivca na krompirju. Proučili smo, ali so ti proteini primerni za zaščito krompirja pred koloradskim hroščem.
V preliminarnih testih z delno očiščenimi proteinskimi ekstrakti iz prostotrosnice poprhnjena livka (C. nebularis) so na modelnem organizmu koloradskem hrošču pokazali insekticidne učinke ali upočasnjeno rast in razvoj ličink koloradskega hrošča. Iz poprhnjene livke so nato izolirali in okarakterizirali inhibitor cisteinskih proteaz klitocipin (Clt) in lektin CNL. Iz bazidiomicetne glive orjaški dežnik (M. procera) pa je bila izolirana in okarakterizana družina inhibitorjev cisteinskih proteaz, imenovana makrocipini, ki so strukturno podobni klitocipinu iz poprhnjene livke. Vsi trije proteini so bili natančno okarakterizirani na genetskem in biokemijskem nivoju.
Koloradski hrošč, modelni organizem na področju zaščite rastlin proti žuželkam, se prehranjuje s krompirjem, zato smo ga uporabili za testno rastlino. Poleg tega je transformacija krompirja v tkivni kulturi dobro uveljavljena, regeneracija in razmnoževanje transgenih rastlin pa relativno preprosta, saj se krompir razmnožuje vegetativno.
Da bi dosegli čim večjo produkcije glivnih proteinov v testni rastlini, smo zaporedja preoblikovali glede na uporabo kodonov pri krompirju. Naročili smo sintetične gene in jih z rekombinantnim kloniranjem prenesli v Gateway vektorje za ekspresijo v rastlinah.
Uporabili smo konstitutuivni promotor 35S in nos terminator, kot selekcijski marker pa uporabili zapis za odpornost proti antibiotikom.
Tako pripravljene konstrukte smo s stabilno transformacijo z bakterijo A. tumefaciens vnesli v krompir. Izražanje rekombinantnih genov v transgenih rastlinah smo določili z metodo RT-qPCR, nadalje pa smo s prenosom western določili še izražanje na proteinskem nivoju.
Pri tako izbranih transgenih rastlinah smo v prehranskih poskusih testirali njihovo učinkovitost pri zaščiti proti koloradskemu hrošču. V prehranjevalnih testih smo ugotovili, da sta oba inhibitorja cisteinskih proteaz iz družine mikocipinov, torej makrocipin iz orjaškega dežnika in klitocipin iz poprhnjene livke, primerna za zaščito rastlin pred koloradskim hroščem. Učinkovitosti lektina CNL za zaščito proti koloradskemu hrošču zaradi težav pri postopku transformacije in zaradi posttranslacijskih modifikacij v krompirju nismo mogli dodobra okarakterizirati. Uporabo makrocipinov, kot zaščitne snovi proti herbivorim žuželkam, smo zaščitili tudi s patentno prijavo.
6.2 SUMMARY
Insect pests pose one of the major threats in agriculture. Crop losses due to pest infestations are reduced by use of resistant varieties and pesticides. Resistant varieties can be obtained using classical breeding techniques or technology of genetic engineering.
Insect resistant genetically modified plants have been among the first commercialized genetically modified plants. Insect resistant genetically modified plants that produce insecticide proteins which originate from bacterium Bacillus thuringiensis have been on the market since 1995 and are among the most successful genetically modified plants.
Demand for new insecticidal agents is still highly topical although many synthetic pesticides have already been produced and although Bt toxin producing transgenic plants are insect resistant. New insecticidal agents are needed because insect resistance against existing insecticide may develop and also new natural insecticides with fewer side effects are preferred.
Fungi represent a rich, but poorly explored source of many natural substances, which could be used for crop improvement. At the Department of Biotechnology at Institute “Jožef Stefan” many mushroom genes with insecticide activity have been isolated and biochemically characterized. Although very promising, those proteins have hitherto not been used for crop improvement. That is why the results published in this doctoral dissertation are important for basic and applicative knowledge on this field. In this dissertation the suitability of some recently discovered protein for protection of potato against Colorado potato beetle was examined.
In preliminary studies it was shown that crude protein extracts from clouded agaric (Clitocybe nebularis) showed insecticide activity towards model organism Colorado potato beetle and retardation in growth and development of its larvae. Further on lectin CNL and cysteine protease inhibitor clitocypin have been isolated from clouded agaric. From parasol mushroom (Macrolepiota procera) family of cysteine protease inhibitors structurally similar to clitocypin was isolated. It was termed macrocypins. All three mentioned fungal proteins (CNL, clitocypin and macrocypin) have been precisely investigated on the genetic and biochemical level.
Colorado potato beetle is one of the model organisms in the area of plant protection against insect herbivores. It primary feeds on potato, that is why potato was chosen for testing of insecticide activity of selected fungal genes. Another argument for selection of potato as testing plant is that plant transformation of potato in tissue culture is well established and plant regeneration and vegetative reproduction is relatively simple.
To achieve high expression of fungal genes in transgenic potato plants, sequences of selected genes were adjusted regarding to potato codon usage. Synthetic genes were ordered and cloned into Gateway compatible vectors appropriate for plant expression.
Genes were expressed under the constitutive 35S promoter, nos terminator was used and antibiotic resistance gene was used as a selection marker. Constructs were introduced into potato explants with Agrobacterium tumefaciens mediated transformation. Expression of inserted genes in transgenic plants was determined using RT-qPCR. Among transgenic lines with highest RNA expression recombinant protein levels were determined with western blot.
Transgenic plants with highest expression of recombinant proteins were used in feeding trials of Colorado potato beetle larvae. In bioassays it was shown that both cysteine protease inhibitors macrocypin and clitocypin are suitable for plant protection against Colorado potato beetle, as they both cause growth and development retardation of Colorado potato beetle larvae. Use of macrocypins as a protection agent against insect herbivores was protected by a patent application. The efficiency of dietary lectin CNL in a form of transgenic plants against Colorado potato larvae was not fully characterized due to problems in the transformation procedure.