20.500.12556/RUL-167328 Analiza utišanja tarčnih genov hmelja (Humulus lupulus), posredovanega z glivnimi molekulami mikroRNA Analysis of fungal microRNA-mediated silencing of target gene expression in hops (Humulus lupulus) during RNA interference V sklopu magistrskega dela smo preučevali mehanizem RNA interference med glivo Verticillium nonalfalfae in hmeljem (Humulus lupulus). Želeli smo dokazati utišanje gena gostiteljske rastline hmelja, katero povzroči patogena gliva s svojimi milRNA preko mehanizma imenovanega »cross-kingdom« RNAi. S pomočjo metode RT-qPCR smo želeli določiti relativno raven izražanja tarčnega transkripta tr_001131F v steblih hmelja neokuženih in okuženih rastlin z glivo V. nonalfalfae, ali je ta manj izražen v okuženih rastlinah. Z različnimi pari začetnih oligonukleotidov smo želeli ugotoviti ali pride do utišanja izbranega tarčnega transkripta. Pri odpornem kultivarju Wye Target smo dobili statistično značilne razlike v izražanju tarčnega transkripta tr_001131F med okuženimi in neokuženimi rastlinami pri vseh treh parih začetnih oligonukleotidov, s tem da je bilo utišanje transkripta prisotno v okuženih rastlinah. Naknadno smo z metodo 5' RLM-RACE želeli potrditi mesto razreza tarčnega transkripta po delovanju glivnih milRNA. Razreza tarče z omenjeno metodo nismo potrdili. Rezultati naloge bomo v prihodnje prispevali k boljšemu razumevanje interakcije hmelja ter patogene glive in njune izmenjave malih RNA preko mehanizma »cross-kingdom« RNAi. Prav tako so pridobljeni rezultati odskočna deska za nadaljne študije razvoja odpornih rastlin in postopkov za zaščito pridelka pred različnimi patogeni. As part of our master's thesis, we studied the mechanism of RNA interference between the fungus Verticillium nonalfalfae and hop (Humulus lupulus). Our goal was to demonstrate the silencing of a host plant gene in hop, induced by the pathogenic fungus through its milRNA via a mechanism known as "cross-kingdom" RNAi. Using the RT-qPCR method, we aimed to determine the relative expression level of the target transcript tr_001131F in the stems of non-infected and V. nonalfalfae-infected hop plants, to assess whether its expression was lower in infected plants. By testing different primer pairs, we sought to confirm whether the selected target transcript was silenced. In the resistant cultivar Wye Target, we observed statistically significant differences in the expression of the target transcript tr_001131F between infected and non-infected plants for all three primer pairs, with transcript silencing present in infected plants. Subsequently, we used the 5' RLM-RACE method to verify the cleavage site of the target transcript following the action of fungal milRNA. However, we did not confirm target cleavage using this method. The results of this study will contribute to a better understanding of the interaction between hop and the pathogenic fungus, as well as their exchange of small RNAs through the "cross-kingdom" RNAi mechanism. Additionally, the findings serve as a stepping stone for future studies on developing resistant plants and strategies to protect crops from various pathogens. Verticillium nonalfalfae hmelj milRNA RT-qPCR 5' RLM-RACE utišanje genov Verticillium nonalfalfae hop milRNA RT-qPCR 5' RLM-RACE gene silencing true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-02-15 07:15:45 2025-02-15 07:15:52 2025-02-17 12:15:50 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 256064 226340099 45956.pdf 45956.pdf 1 187C3DD1B140F96289224AB4981ED97C 51065e6978cf19d65ce0cdd2bfdbded00e4b60a53a509c8283fb14aea5ba8e03 6bc8f7a9-eb63-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=199682 Biotehniška fakulteta 0 0 0