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Identifier 000417594
Title Χρήση του συστήματος CRISPR/Cas9 για τη δημιουργία φυτών Nicotiana benthamiana με πλήρη καταστολή των DCL γονιδίων
Alternative Title Using CRISPR/Cas9 technology to create DCL knock-out Nicotiana benthamiana plants
Author Μπαρδάνη, Ειρήνη Ε.
Thesis advisor Καλαντίδης, Κρίτων
Reviewer Τσαγρή, Ευθυμία
Κοτζαμπάσης, Κυριάκος
Abstract RNA silencing refers to the involvement of RNA molecules in mechanisms of gene regulation, leading either to the cleavage of mRNA-targets, or to inhibition of translation. RNA silencing includes different pathways, with different functions and proteins involved. However, they all share the production of 20-24nt long small RNAs (sRNAs) by Dicer-like (DCL) proteins. In the model plant Nicotiana benthamiana (N. benthamiana) four DCLs have been identified, each of which produced sRNAs of specific length, involved in specific pathways. Studying the role of these proteins is of great interest. The recently evolved CRISPR/Cas9 system was chosen, in order to knock-out the DCL genes. As a gene editing tool, CRISPR/Cas9 includes the Cas9 endonuclease and a single-guide RNA (sgRNA), which shows homology to the target sequence and guides the Cas9 to produce double-strand breaks in the DNA. For the purpose of this project, in the past there have been designed plasmids which express guide RNAs targeting the DCL genes (DCL2, DCL3, DCL4) at specific sites, whereas at the same time a plasmid expressing Cas9 was obtained. The plasmids were used for the creation of transgenic plants and then through crosses there have been obtained plants expressing simultaneously the Cas9 endonuclease and one or two sgRNAs targeting a specific DCL. After the creation of these plants follows a prosess of screening for mutant plants and analyzing the mutations in the next generation. Through already completed analysis from previous work in the lab, combined with the results of this work, plants with genetic modifications in the genes of DCL2, DCL3 and DCL4 have been created. For DCL3, the functional test based on the production of 24nt long small RNAs has been completed, whereas homozygous knock-out mutants have been obtained. For DCL2, the analysis has been completed until the functional test and putative knock-out plants with no 22nt sRNA production have been selected for further screening. Finally, for DCL4 the functional test is ongoing, in order to find plants that do not produce 21nt long small RNAs. The knock-out DCL plants have to be compared in the future with the respective DCLi plants. For that purpose, DCLi lines as well as DCLi crossed lines were used for the study of CMV infectivity. Studies like this will allow the comparison between DCLi and DCL knock-out plants. In the context of the above comparison, DCL3 knock-out plants were selected for experiments of viral/viroidal infections. In this work there has been an attempt to compare PSTVd (Potato Spindle Tuber Viroid) infectivity between DCL3i and DCL3 knock-out plants. Summing up, this work is part of the process that will lead to the creation of DCL knockout plants. These plants will be useful tools in studying the role of DCL proteins, especially during viral infections.
Language Greek
Subject RNA silencing
RNA σίγηση
Issue date 2018-07-20
Collection   School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
  Type of Work--Post-graduate theses
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