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Identifier 000407882
Title Δημιουργία φυτών Nicotiana benthamiana με πλήρη καταστολή των DCL γονιδίων με την χρήση του συστήματος CRISPR/Cas9
Alternative Title Production of DCL knock-out Nicotiana benthamiana plants using CRISPR/Cas9 system
Author Μήττα, Ελένη Σ.
Thesis advisor Καλαντίδης, Κρίτων
Reviewer Τσαγρή, Ευθυμία
Βόντας, Ιωάννης
Abstract RNA silencing (or RNA interference) is a RNA depended gene regulatory mechanism that inhibits via different pathways the transcription or the translation of a gene and thus reduces protein production. Small RNA molecules (siRNA and miRNA) which are produced by Dicer-like (DCL) enzymes, play a leading role in these processes. In Nicotiana benthamiana (N. benthamiana) four DCL proteins have been detected, each producing small RNAs with specific size and function. The interest in studying these proteins, alongside with the establishment of CRISPPR/Cas9 system as a gene editing tool, prompted us on producing plants with complete loss of function (knock-out) for the DCL proteins. A CRISPR/Cas9 system needs a Cas9 nuclease that is guided to the target sequence by small guide RNA (sgRNA) that provides targeting specificity. Then Cas9 causes a double strand break within the DNA target. We used this system and we have produced plasmids that contain a sgRNA sequence targeting DCL2, DCL3 or DCL4 genes. These plasmids, combined with a Cas9 plasmid were used for transient expression of all the necessary compounds in order to confirm functionality of CRISPR/Cas9 in-vivo. In addition, these plasmids were used for plant transformation producing plants that stably overexpress one of the designed sgRNA or Cas9 and they were crossed to each other. Next step was the detection and identification of induced mutations in F1 and F2 generation. In this work, we provide evidences that the generated plants contain mutations in DCL2 and DCL3 genes. Furthermore, plants which carry mutations for DCL4 genes were also generated, and screened but further analysis is required. All the DCL knock-out plants that will be produced throughout this project should be compared with plants showing partial suppression of such proteins (DCLi plant lines). Therefore, in this study we also show the production of small RNA in the transient expression of a transcript with hairpin structure and the infectivity difference of two viruses (CMVGR21 and TRV-GFP ) on DCLi plant lines (and in plants resulting from their crosses). These experiments will serve as the standard of comparison to plants with complete suppression of DCL proteins being formed. Together, in this work, we describe the CRISPR targeted genome mutagenesis method for obtaining plants with mutations on the desired genes. These plants will be an excellent tool for studying a variety of biological functions like DCL tasks, like their role in viral infections and in silencing of transgenes.
Language Greek
Subject RNA silencing
RNA σίγηση
Issue date 2017-03-17
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Biology--Post-graduate theses
  Type of Work--Post-graduate theses
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