Ekspresija gena za inhibitor serinskih proteinaza (BvSTI) šećerne repe (Beta vulgaris L.) i uloga u otpornosti na insekte

Abstract

Biljni inhibitori proteinaza aktivno učestvuju u odbrani biljaka od insekata štetočina inhibirajući insekatske digestivne proteinaze. In planta analiza ekspresije BvSTI gena koji kodira inhibitora serinskih proteinaza urađena je sa ciljem otkrivanja uloge ovog inhibitora u otpornosti biljaka šećerne repe na insekte, kao i radi utvrđivanja potencijala ovog gena kao pogodnog kandidat-gena koji bi se biotehnološkim metodama mogao uvesti u osetljive biljne genome, čime bi se povećala njihova otpornost prema insektima štetočinama. Ekspresija BvSTI gena praćena je kod tri genotipa šećerne repe koji se odlikuju umerenom otpornošću prema larvama štetočine korena Tetanops myopaeformis Roder, F1016, F1015 i UT-8, kao i kod jednog osetljivog genotipa, F1010. Kod svih otpornih genotipova mehaničko povređivanje indukovalo je ekspresiju ovog gena, a nivo transkripcije bio je povišen u poređenju sa nivoom kod osetljivog genotipa. Najintenzivniji odgovor na povređivanje zabeležen je kod otpornih genotipova F1016 i UT-8. U listovima osetljivog F1010, ali i trećeg otpornog genotipa F1015, registrovan je samo neznatni porast u intenzitetu BvSTI transkripcije, dok je u korenovima ova dva genotipa mehaničko povređivanje dovelo do blage početne supresije u aktivnosti BvSTI gena. Akumulacija BvSTI transkripata u listovima i korenovima otpornog F1016 i osetljivog F1010 kojima su se hranile larve Spodoptera frugiperda J.E. Smith pokazala je sličan obrazac ekspresije kod oba genotipa. U poređenju sa transkripcionim obrascima dobijenim nakon mehanilčkog povređivanja, ishrana insekata dovela je do znatno sporije indukcije i slabijeg intenziteta transkripcije. Analize na proteinskom nivou pokazale su da nakon povređivanja listova dolazi do akumulacije proteina veličine 30 kDa za koji su se vezala poliklonalna BvSTI specifična antitela kako kod otpornog F1016, tako i kod osetljivog F1010 genotipa. Aktivnost BvSTI inhibitora protiv tripsina pokazana je kod F1016 korenova i listova, kao i kod F1010 listova. U F1010 korenovima aktivnost BvSTI inhibitora nije detektovana. U biotestu u kome je ispitivana otpornost pojedinačnih genotipova na larve S. frugiperda korišćena su sva četiri genotipa šećerne repe kod kojih je pokazano da povređivanje utiče na ekspresiju BvSTI gena. Larve koje su se hranile listovima sva tri otporna genotipa bile su statistički značajno lakše od larvi koje su hranile osetljivim F1010 listovima...

Plant proteinase inhibitor genes are among the prime candidates suitable for insect resistance improvement in plants. Expression pattern of a sugar beet serine proteinase inhibitor gene, BvSTI, was characterized in response to mechanical and fall armyworm (Spodoptera frugiperda J.E. Smith) induced wounding. BvSTI expression was analyzed in three breeding lines moderately resistant to sugar beet root maggot (Tetanops myopaeformis Roder.) and in a susceptible line, F1010. Increased mechanical wounding induced levels of BvSTI expression were observed in all resistant lines as compared to F1010. The most intensive response to wounding was observed in resistant lines, F1016 and UT-8, with a maximum up to 4- and 2,5-fold increase of BvSTI transcript levels over non-wounded roots and leaves, respectively. In contrast, slight increase of BvSTI transcript levels in leaves and even an initial decrease in roots were observed in sensitive F1010, but also in the third resistant line, F1015. BvSTI transcript accumulation in F1016 and F1010 tissues wounded by FAW showed a similar gene expression pattern, but it was delayed and less intense than the response incited by abiotic wounding. On the protein level, BvSTI-specific polyclonal antibodies confirmed increased accumulation of the 30 kDa BvSTI protein in wounded leaves but not in roots of F1016 and F1010. Using trypsin inhibition assays, the activity of BvSTI was confirmed in F1016 roots and leaves and F1010 leaves. In F1010 roots BvSTI activity was completely lacking. To confirm the potential role of the BvSTI gene in defending mechanisms to insect pests in sugar beet the same analyzed germplasm were bioassayed for resistance to fall armyworm insects. Larvae fed sugar beet leaves from all three resistant germplasms (F1016, F1015 and UT-8) had significant reductions in larval weights as compared to larvae fed on sensitive F1010 leaves. The observed daily weight increase was also the highest in larvae from sensitive vs. resistant leaves. As the larvae entered the pupal stage, pupal sizes did not reflect the overall larval weights and all developed pupae were similar. Larvae fed on roots were almost double lighter than larvae from leaves for all analyzed gemplasms. Some developmental abnormalities of the pupae fed on F1016 and F1015 leaves were noted...