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Transgene silencing in potato plants and tobacco cell line BY-2
Tyč, Dimitrij ; Fischer, Lukáš (advisor) ; Honys, David (referee)
Accidental discovery of RNA interference in plants a few decades ago and its description by Fire et al. (1998) brought a real revolution in molecular biology and in many other branches of biology. Researchers have uncovered a large and unexpected mechanism of regulation of gene expression at many levels in almost all studied eukaryotes. The first studies have revealed silencing of both exogenous and endogenous genes in plants. So, it soon became clear that this process serves to organisms not only in the defense against viruses, but also against mobile genetic elements as well as in modulation of expression own genes. The key role in all RNAi mechanisms is played by dsRNA that is processed into small RNAs. In association with a number of other proteins, small RNAs can direct either cleavage of complementary or nearly complementary mRNAs or block their translation in the process of posttranscriptional gene silencing (PTGS). Additionaly, sRNAs can affect gene expression at transcriptional level via modifying DNA in a process called RNA dependent DNA methylation (RdDM). In plant transformation, RNAi cause variability amongst transgene lines and instability in the expression of introduced genes, which complicates the use of transgenic lines, because many biotechnological approaches require a defined...
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Proteomic and functional characterization of PsbO isoforms
Duchoslav, Miloš ; Fischer, Lukáš (advisor) ; Hála, Michal (referee)
PsbO (manganese-stabilizing protein) is the largest extrinsic protein of photosystem II, located on the lumen side of photosystem. It is present in all known oxyphototrophic organisms. PsbO facilitates photosynthetic water splitting, which takes place in an oxygen evolving center (Mn4CaO5 cluster) of photosystem II. This work is focused on PsbO of higher plants and its isoforms, particularly their evolution and functions. Bioinformatic analyses revealed that majority of higher plants express exactly two psbO isoforms. A phylogenetic tree of PsbO sequences has an unusual topology. The two paralogous isoforms do not diverge at the base of the phylogenetic tree, as anticipated, but rather at the end of particular branches, at the level of family or lower taxonomic unit. In this work we propose and discuss several hypotheses concerning evolution of PsbO isoforms. The work further includes detailed analysis and identification of protein spots assigned to PsbO on 2D IEF-SDS PAGE gels of potato thylakoid proteins. We identified predominant version of PsbO isoform in most of the spots. We did not succeed to find any posttranslational modification. We optimized a method of psbO expression in E. coli and subsequent purification, which yielded relatively big amount of properly folded recombinant protein. Analysis of...
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Study of the mechanism of posttranscriptional and transcriptional transgene silencing in tobacco BY-2 cell line
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The RNA interference is a mechanism, which allows cells to regulate their genes functions, to establish and maintain heterochromatin and to defend them against invasive nucleic acids. In plants, RNA interference is initiated by double-stranded RNA, which is processed by Dicer into small RNAs, usually 20-24nt long. These small RNAs form a complex with Argonaut protein that participates in different processes based on sequence complementarity. This complex can guide mRNA cleavage, translation blocking and chromatin modifications, resulting either into posttranscriptional silencing (by preventing translation of already existing mRNA, PTGS) or transcriptional silencing (by preventing transcription of mRNA, TGS). The first step of this thesis was to establish different ways of triggering PTGS and to evaluate their functionality and efficiency. The next step was a preparation of a system which would allow to study the transition from posttrancriptional to transcriptional silencing. These so called "indicator lines" should allow to observe the timing and dynamics of this process by utilizing fluorescent proteins. This system is also going to enable to evaluate, how different factors are involved in this process - one of the factors is RNA-dependent RNA polymerase 6 (RDR6) which plays an essential role in...
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Revealing phosphoproteins playing role in tobacco pollen activated in vitro
Fíla, Jan ; Honys, David (advisor) ; Fischer, Lukáš (referee)
5 Abstract Tobacco mature pollen rehydrates in vivo on a stigma tissue, and develops into the rapidly-growing pollen tube. This rehydration process is accompanied by the de-repression of stored mRNA transcripts, resulting in the synthesis of novel proteins. Furthermore, such metabolic switch is also likely to be regulated on the level of post-translational modifications of the already-present proteins, namely via phosphorylation, since it was shown to play a significant regulatory role in numerous cellular processes. Since only a minor part of proteins is phosphorylated in a cell at a time, the employment of various enrichment techniques is usually of key importance. In this diploma project, metal oxide/hydroxide affinity chromatography (MOAC) with aluminium hydroxide matrix was applied in order to enrich phosphoproteins from the mature pollen and the 30-minute in vitro activated pollen crude protein extracts. The enriched fraction was separated by both 2D-GE and gel-free liquid chromatography (LC) approaches with subsequent mass spectrometric analyses. Collectively, 139 phosphoprotein candidates were identified. Additionally, to broaden the number of phosphorylation sites identified, titanium dioxide phosphopeptide enrichment of trypsin-digested mature pollen crude extract was performed. Thanks to the...
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Utilization of biotechnological method in growing of technical hemp
Širl, Marek ; Zelenková, Sylva (advisor) ; Fischer, Lukáš (referee)
Hemp (Cannabis sativa L.) is a multi-use crop, able to provide fibre celulose and hurds for industrial treatment biomass for energy conversion and produces secondary metabolites useful for pharmaceutical application. For its resistence to stress and ability to accumulate high concentration of heavy metals it can be potentialy used for phytoremediation. The aim of current research is the development of new strains, with specialy improved qualities and the enhancement of its applicability. Besides traditional breeding methods, genetic manipulation might be the possible tool. What is currently discussed is finding optimal conditions allowing efective in vitro cultivation as a basic assumption of genetic manipulation. Hemp transformation via Agrobacterium tumefaciens co-cultivation might be the appropriate technic.
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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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Genetically modified plants in practical applications
Říhová, Barbora ; Fischer, Lukáš (advisor) ; Cvrčková, Fatima (referee)
Genetic engineering (GI) of plants is a very current topic, and more and more controversial, since it is becoming an inseparable part of our lives. GI has, among other things, a great potential to help solve the current problem of hunger and malnutrition in certain parts of the world. The goal of this project is to clarify what genetically modified (GM) plants are, to present the possibilities of their practical use, to explain methods of preparation and to consider their advantages and eventual risks. By GM plant we understand a plant whose genetic information has been changed by introducing or removing part of the genetic information (a section of DNA, generally a gene). The most frequently used methods of transformation are the transformation by Agrobacterium tumefaciens and the balistic method. GM plants can be used for production of food or feed in better quality and greater quantity, thanks to an introduction of higher resistance, whether it is to biotic stress (pest, virus or bacterial resistance) or to abiotic stress (drought, salinity, toxic substance in the ground..). In particular, the resistance to abiotic stress is an important issue these days, since through the climate changes some regions suffer more and more from insufficient precipitation and consequent drought. Some of the GM plants grown...
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Variability and silencing of transgene expression in potato plants and in tobacco cell line BY-2
Nocarová, Eva ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee) ; Kovařík, Aleš (referee)
Conclusions In suspension cultures of tobacco BY-2 cell line derived from calli after transformation about 90 % of lines contained cells with various GFP fluorescence level after transformation. Newly introduced cloning method allowed obtaining nearly 50 % of clones with homogeneous GFP expression from primarily heterogeneous BY-2 lines. Heterogeneity of GFP expression in transgenic BY-2 lines had two causes - genetic (primary lines contained cells with different T-DNA insertions) and epigenetic one. Epigenetic heterogeneity of BY-2 lines was connected with transgene silencing, formation of stable epigenetic states early after transformation, and "permanent heterogeneity" with fluctuating levels in GFP expression. Reduction or silencing of transgene expression in potato was predominantly observed in lines with higher number of T-DNA insertions and with higher initial GFP expression. Silencing in potato always gradually affected both introduced genes. Silencing of GFP expression preceded (in months) loss of resistance to kanamycin (silencing of NPTII gene) in all monitored cases that indicates interconnections between silencing of both transgenes. The same sequence of silencing of both transgenes in potato was also observed in silenced lines after reactivation of transgene expression by 5-azacytidine, which...
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The role of manganese-stabilizing protein of photosystem II
Duchoslav, Miloš ; Rothová, Olga (referee) ; Fischer, Lukáš (advisor)
Miloš Duchoslav The role of manganese-stabilizing protein of photosystem II Abstract The appearance of oxygenic photosynthesis was a key event in the evolution of life on the Earth. All molecular oxygen in the atmosphere likely comes from a water-splitting reaction catalysed by the oxygen-evolving center of photosystem II. Photosystem II - a multisubunit protein-cofactor complex with a phylogeneticaly highly conserved structure - is embedded in the thylakoid membrane of chloroplasts and cyanobacteria. The mechanism of the photosynthetic water-splitting reaction, which occurs on the manganese cluster, has been widely investigated; however, it has not yet been fully understood. An essential role in the stabilization of the manganese cluster and in the facilitation of oxygen evolution is played by photosystem II extrinsic proteins that occur in thylakoid lumen. The most important among them is a manganese-stabilizing protein (MSP) that is present in all known oxyphototrophs. This protein is believed to have many functions: besides stabilizing the manganese cluster, it is also carbonic anhydrase activity, GTPase activity and regulation of the turnover of the D1 protein. The functions of the MSP are probably regulated through changes in its dynamic structure. The MSP is likely to take part in the regulation of...
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RNA interference in plants
Čermák, Vojtěch ; Kulich, Ivan (referee) ; Fischer, Lukáš (advisor)
The process of RNA interference allows cells to regulate functions of their genes. This process is usually initiated by the presence of double-stranded RNA within a cell. Such double-stranded RNA is diced by a specific protein called Dicer into duplexes of small RNAs, usually 20-25 nucleotides long. Single-stranded small RNAs, released from the duplexes, are the heart of RNA interference and they can be categorize into several groups according to their biogenesis. There are two groups of small RNAs in plants: miRNA and siRNA. Small RNAs can associate with a protein called Argonaut and guide it to the target molecule on the bases of sequence complementarity. The Argonaut-small RNA complex can act on itself or it can interact with other proteins in a wide spectrum of processes. The complex can slice the target mRNA (which can be handled by the sole Argonaut and small RNA), it can suppress translation or it can direct chromatin modifications. The phenomena of RNA interference can be found in almost all Eukaryotes where it can serve many functions, for example it can control cell differentiation, participate in stress responses, direct changes in chromatin and defend the organism against viruses. A diverse set of operating modes of RNA interference can be found in plants, which we are only at the...
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