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Comparison of methods of isolating double-stranded RNA from plant tissue with a focus on viral fraction yield.
MATYÁŠOVÁ, Alena
High-throughput sequencing is one of methods used for diagnostics of plant pathogens and has advantage of unspecific unbiased detection of all nucleic acids present in a sample. Input material for HTS can be prepared using by different approaches that reflect purpose of the planned task. During viral infections of plants, viral double stranded RNAs are generated as replication intermediates or transcription products. Thus, they are often used for HTS. Nevertheless, such preparations contain large amount of plant RNAs. The work aimed for comparison of two methods of double stranded RNA enrichment - widely used cellulose chromatography and differential centrifugation with lithium chloride. Both qualitative and quantitative profiles of viral nucleic acids were estimated. An isolate HZ2 of red clover (Trifolium pratense L.) was used for the study. Previously, there were detected eight different RNA viruses with HTS-aimed analyses in the plant. To compare qualitative profile, RNA was extracted by each method, transcribed into cDNA, and specific viral fragments were amplified using PCR, followed by agarose gel electrophoresis. Quantitative profiles were analyzed using three selected viruses with single- and double-stranded RNA genomes. Their relative quantification was estimated using RT-qPCR approach. Further, normalized (to 26S rRNA as a reference) expressions were calculated using Bio-rad CFX Manager 3.1 software. All eight viruses were successfully detected using RNA material obtained by each method. After evaluating the quantification data and performing statistical tests (F-test, T-test; with a significance level of = 0.05), no significant difference was found between the compared methods.
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Detekce fytopatogenů jahodníku
MICHÁLKOVÁ, Barbora
Strawberries were tested for presence of selected phytopathogens (Agrobacterium, Rhodococcus, phytoplasmas, SMYEV, SMoV, SPV1, SCV and SCRh1) by the PCR. At the same time was researched the transmission of mentioned phytopathogens by grafting. The DNA of the strawberries with positive results was sequenced.
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Coat protein-RNA interaction in vivo and the biotechnological use of VLPs
Kratochvílová, Kateřina ; Moravec, Tomáš (advisor) ; Hála, Michal (referee)
The Tobacco mosaic virus (TMV) is a simple and frequently used model virus which has been studied already more over than 130 years. Due to the intensive study of this virus the details of its infectious cycle, genomic information and also the structure of the created viral particle as well as the mechanism of its creation are known today. The process of encapsidation (viral particle formation) is sufficiently described in the in vitro conditions. In the in vitro conditions the origin of assembly (OAS) was also described. The OAS was identified in the coding sequence of the gene for the movement protein (MP). The importance of replication centers (replication factories) has also been supposed. The aim of the diploma thesis was to study the specificity of the interaction of RNA and coat protein in the process of the particle assembly taking place directly inside the plants. The experiments were performed to verify the necessity of presence of OAS sequence in process of initiation of viral encapsidation. The effect of the cell compartmentation on this process has also been studied. Based on several viral systems (the Tobacco mosaic virus, the Potato virus X, the Bean yellow dwarf virus and Cowpea mosaic virus) gene constructs were created. These constructs enables to study this idea at the molecular...
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The use of plants for the expression of Human papillomavirus vaccine
Dlabalová, Lucie ; Moravec, Tomáš (advisor) ; Fischer, Lukáš (referee)
Papillomaviruses are causing various diseases from skin warts to the lesions leading to malignant tumours and are widespread among people. For this reason, the current research is trying to develop methods for the production of inexpensive and effective vaccines against both Papillomaviruses and against all other infectious diseases. Currently animal and microbial expression systems are most frequently used for the production of biopharmaceuticals which have several drawbacks and their capacity is limited. This opens up the doors for plants - potentially very efficient producers of biopharmaceuticals. Currently there is rapid development towards the optimization and improvement of the results of plant expression systems and establishing the best and safest methods of their use. This paper summarizes and compares the advantages and disadvantages of different methods of plant transformation, leading either to stable production of the protein of interest in transgenic plants or to transient expression of recombinant virus infecting non-transgenic plants. Furthermore it analyzes the most appropriate plant species, which provide high yields combined with a transformation method and ease of cultivation, describes few basic ways of optimizing expression levels and outlines the future of plant expression systems.
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