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.
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