20.500.12556/RUL-151638 Analiza vpliva proteina HcPro virusa Y krompirja na transkripcijski faktor PTI5 v tobaku Nicotiana benthamiana in proizvodnja dvoverižne RNA za uporabo kot pesticid proti koloradskemu hrošču The analysis of the influence of PVY HcPro protein on PTI5 transcription factor in Nicotiana benthamiana and the synthesis of Mesh dsRNA and its use as a pesticide against colorado potato beetle Krompir je glede na svetovno porabo ena izmed pomembnejših poljščin, vendar njegov pridelek desetkata koloradski hrošč, ki postaja zmeraj bolj odporen proti obstoječim pesticidom, in okužba z virusom PVY, ki v naravi zelo hitro mutira. Namen naših poskusov je bil preizkusiti okolju prijaznejšo alternativo obstoječim pesticidom proti koloradskemu hrošču in raziskati mehanizem obrambe rastlin proti PVY. Dvoverižna RNA je zelo obetaven kandidat za pesticid proti koloradskemu hrošču zaradi svoje selektivnosti, hkrati pa se v naravi hitro razgradi na neškodljive produkte. Preizkusili smo pesticidno aktivnost dsRNA, ki cilja gen Mesh. Gen Mesh ima vlogo pri tvorbi septatnih stikov v črevesju koloradskega hrošča. Njegovo utišanje deluje pesticidno tudi proti koruznemu hrošču. dsRNA iz gena Mesh (dsMesh) smo pridobili in vivo, in sicer smo gen Mesh vstavili v plazmid L4440 za izražanje dsRNA, z njim transformirali E. coli z onesposobljeno funkcijo razgradnje dsRNA ter pripravili bakterijski ekstrakt, s katerim smo hranili ličinke koloradskega hrošča. Izkazalo se je, da je dsMesh, pridobljen in vivo, uspešen pri zatiranju ličink koloradskega hrošča in ima primerljivo učinkovitost kot pesticid spinosad ter in vitro pridobljen dsMesh. Za raziskavo mehanizmov rastlinske obrambe rastlin proti virusu PVY smo opazovali vpliv virusnega proteina HcPro, znanega zaviralca utišanja RNA, na rastlinski transkripcijski faktor obrambnih genov PTI5. S PCR smo pripravili konstrukt HcPro z vezanim reporterjem TFP. Z uporabo sistema Gateway smo pripravili ekspresijski vektor, s katerim smo transformirali agrobakterije, s temi pa nato v kombinaciji s PTI5-YFP prehodno transformirali tobak. Iz opazovanj transformiranih listov rastlin pod konfokalno lupo smo prišli do zaključka, da HcPro ne sproži obrambne reakcije rastlin prek PTI5, saj ne sproži njegovega izražanja in ne povzroči njegove stabilizacije ali spremembe njegove lokalizacije. Iz fenotipizacije transformiranih rastlin smo zaključili, da HcPro v krompirju sorte Rywal ne sproži preobčutljivostnega odziva in ne razvije nekrotičnih lezij kot posledica obrambe s posredovanjem obrambnega gena Nytbr. Potato is one of the most consumed crops in the world. However, its world yield is severely affected by the Colorado potato beetle that is becoming resistant to existing pesticides, as well as by the infections caused by the quickly mutating PVY virus. The goal of our experiments was to test more ecological pesticidal alternatives against the colorado potato beetle, as well as to analyze plant defense response against PVY. Double stranded RNA has a great pesticidal potential because of its selectivity and its natural ability to degrade quickly into harmless products. The pesticidal activity of dsRNA targeting Mesh gene has been tested. Mesh gene has a role in formation of septate junctions in the midgut epithelium of Colorado potato beetle and has been proven in the past to be an efective pesticide against Western corn rootworm. Double stranded Mesh RNA (dsMesh) was produced by inserting the Mesh gene into L4440 plasmid enabling the production of dsRNA. The construct was used to transform E. coli with a knockout in the dsRNA degrading enzyme. We prepared an extract of the transformed bacteria which was then used in the feeding trials. Our research shows an effective pesticidal acitivity of in vivo dsMesh against colorado potato beetle larvae, comparable to in vitro produced dsMesh and the most commonly used pesticide against the Colorado potato beetle, spinosad. In the research of plant defense mechanisms against PVY, the influence of PVY´s known silencing supressor HcPro on the plant transcription factor PTI5, that activates the transcription of defense genes, was investigated. By PCR the HcPro-TFP phusion was produced and afterwards inserted into a binary plant expression vector via Gateway cloning approach. This contruct was used to transiently cotransform Nicotiana benthamiana plants in combination with a plant vector containing PTI5 gene fused with the yellow fluorescent protein. From our observation of the transformed leaves using confocal microscopy, we came to the conclusion that HcPro does not trigger plants´ defense response via PTI5, for it does not cause its expression, stabilization or change of localization. Furthermore, from the phenotypization of transiently transformed plants we determined that HcPro does not trigger the formation of necrotic lesions, a common consequence of hypersensitive response, via Nytbr defense gene in potato plants cv. Rywal. krompir utišanje RNA pesticid na osnovi dsRNA obramba rastlin proti PVY potato RNA silencing dsRNA-based pesticide plant defense against PVY true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-10-13 15:20:01 2023-10-13 15:20:12 2023-11-30 11:18:47 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 22617 174326787 Ferenc_Rok_-_Analiza_vpliva_proteina_HcPro_virusa_Y_krompirja_na_transkripcijski_faktor_PTI5_v_t.pdf Ferenc_Rok_-_Analiza_vpliva_proteina_HcPro_virusa_Y_krompirja_na_transkripcijski_faktor_PTI5_v_t.pdf 1 F19279E5624EEA52ECFA40D35C09B1ED 3005feeffad1709faca5c25c3b892499ca718b15e4ac3d8cd088f100b514496d 3e4c3517-69cb-11ee-9f22-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=176658 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0