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Potrjevanje in silico napovedane tarče miR319 pri okužbi hmelja (Humulus lupulus) z glivo Verticillium nonalfalfae
ID Žnidaršič, Emanuela (Author), ID Štajner, Nataša (Mentor) More about this mentor... This link opens in a new window, ID Stajič, Ester (Comentor)

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Abstract
V okviru magistrske naloge smo analizirali interakcijo med mikro RNA (miRNA) miR319 ter in-silico napovedano tarčo tcp2 pri odpornem kultivarju hmelja Wye Target ob okužbi z glivo Verticillium nonalfalfae. Da bi optimizirali postopek izolacije kakovostne RNA, smo iz korenin in listov hmelja izolirali celokupno RNA z različnimi metodami. Z metodo RT-qPCR smo poskušali kvantitativno opredeliti količino tarčne mRNA na treh delih sekvence (levo, desno in okoli mesta razreza) z različnimi pari začetnih oligonukleotidov (ZO). Izvedli smo tudi metodo 5' RLM-RACE, s katero smo želeli ugotoviti mesto na tarčni mRNA, kjer pride do razreza ob interakciji z miRNA. Ugotovili smo, da so pri vzorcih okuženih rastlin prisotne statistično značilne razlike v količini RNA med regijami pomnoženimi z različnimi pari ZO. Z metodo 5' RLM-RACE smo potrdili razrez tarčne mRNA pri kontrolnem vzorcu, vendar je do razreza prišlo 50 bp stran od pričakovanega mesta razreza. V okviru naloge smo optimizirali določene procese izolacije RNA in kvantifikacije tarčnih regij. S pridobljenimi rezultati bomo prispevali k boljšemu razumevanju mehanizmov pri imunskem odzivu hmelja ob okužbi z glivo, ki lahko v prihodnosti pripomorejo k razvoju postopkov za obvladovanje verticilijske uvelosti.

Language:Slovenian
Keywords:hmelj, Verticillium nonalfalfae, odpornost rastlin, miRNA, RNAi, 5' RLM-RACE, sekvenciranje
Work type:Master's thesis/paper
Typology:2.11 - Undergraduate Thesis
Organization:BF - Biotechnical Faculty
Year:2024
PID:20.500.12556/RUL-162316 This link opens in a new window
COBISS.SI-ID:208763395 This link opens in a new window
Publication date in RUL:21.09.2024
Views:79
Downloads:22
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Secondary language

Language:English
Title:Confirmation of in-silico predicted miR319 targets in Verticillium nonalfalfae-infected hop plants (Humulus lulupus)
Abstract:
In this master thesis we investigated the interaction between the microRNA (miRNA) miR319 and its in-silico predicted target tcp2 in the resistant hop cultivar Wye Target during infection with the Verticillium nonalfalfae fungus. To optimize the isolation of high-quality RNA, we extracted total RNA from roots and leaves of hop plants using various methods. We used RT-qPCR to quantify target mRNA levels in three different regions of the sequence (left, right and around the cleavage site) with different primer pairs. Additionally, we used the 5' RLM-RACE technique to identify the specific cleavage site on the target mRNA that occurs due to interaction with the miRNA. Our analysis revealed statistically significant differences in RNA levels between primer pairs in the infected plant samples. Using the 5' RLM-RACE method we confirmed cleavage of the target mRNA in the control sample. However, the cleavage occurred 50 bp away from the expected cleavage site. As part of this master's thesis, we optimized certain processes for RNA isolation and the quantification of target regions.These results will contribute to a better understanding of the mechanisms involved in the hop's immune response to fungal infection and may contribute to the development of strategies to control Verticillium wilt in the future.

Keywords:hops, Verticillium nonalfalfae, plant resistance, miRNA, RNAi, 5' RLM-RACE, sequencing

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