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Techniques for Comparing Biological Sequences
Sladký, Roman ; Křivka, Zbyněk (referee) ; Burgetová, Ivana (advisor)
This work presents the building up of basic biological units DNA, RNA and proteins as well as their function. Provided data are kept in biological databases which are connected worldwide to supply preferable communication along with all kinds of available information to be used in the scientific research. The secret of alive is hidden in genes coded in sequences of nucleotides. Genes enable the creation of proteins which are made of sequences of amino-acids. The wide-spread methods of comparing these sequences are FASTA and BLAST algorithms. Their base is used for the PSProt program which is described in this work. PSProt program is the tool for comparing the sequences of proteins. First it is necessary to synthesise the protein from the DNA oligonucleotide because it codes the surveyed protein. The most similar proteins are searched out by heuristic of hitpoints, then their final score that is essential for aligning is modified by semiglobal alignment algorithm.
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Vliv nízké teploty na propustnost buněčných membrán a přežití rostlin
Kubovčíková, Anna
Plants are naturally exposed to low temperatures in nature. During the winter, the temperatures are the lowest and in certain places they even reach very low values. Jasmonic acid plays an important role in the plant's resistance to low temperatures. In the thesis, we have focused on the effect of the PLIP genes that produce precursors of jasmonic acid. The role of the genes in cold resistance in Arabidopsis thaliana was studied after acclimation in different light conditions. The observed parameters in plants acclimated to low temperatures were the parameters of photosynthesis, intactness of cell membranes and survival after exposure to freezing. The results of the experiments proved that the mutation of the PLIP genes cause a decrease in the integrity of the cell membranes when exposed to freezing, and conversely, the ectopically increased expression of the PLIP3 gene results in an increase in plant resistance. Based on ion leakage, we determined LT50 values for each variant, and it was shown that the difference between the most resistant transgenic line plip3 ox and the mutant line in all three PLIP genes is more than 4 °C. Subsequently, a plant survival test after freezing was performed, where it was demonstrated that the LT50 value correlated with plant survival. Chlorophyll fluorescence analysis showed that after acclimation to low temperature, there are no large differences in photosynthetic parameters between individual genotypes with modified PLIP level. The light that was applicated during acclimation had a more pronounced effect on the plants. Plants acclimated at low intensity showed higher values of non-photochemical quenching as well as greater maximum quantum yield of photosystem II. The results prove that the PLIP genes are significantly involved in the resistance of plants to low temperatures.
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Targeted editing of the Arabidopsis CYP98A3 gene using the CRISPR/Cas9 method
Šamaj, Matej
Lignin is an abundant biopolymer, which fulfils several important functions including facilitation of polar water transport and mechanical strengthening of plant body. It is required for proper growth and development of plants but also for plant defence responses. Complex lignin biosynthesis involves the activity of numerous enzymes catalysing diverse chemical reactions that are stepwise generating various precursors for lignin formation. One such enzyme of lignin biosynthetic pathway, P‐COUMAROYL SHIKIMATE 3′‐HYDROXYLASE (C3′H), is encoded by the gene CYP98A3/ C3′H and convert p-coumaroyl shikimate to caffeoyl shikimate. CRISPR/Cas systems adapted from prokaryotes, where they provide adaptive immunity, have very quickly evolved to unique and powerful tool for precise gene editing. The CRISPR/Cas9 technology allows to specifically target desired sites within the genomes to subsequently perform very precise mutagenesis of selected gene(s). The purpose of this thesis was to employ the CRISPR/Cas9 method to perform targeted editing of the CYP98A3/C3′H gene in transformed Arabidopsis thaliana plants. Selected transformed plants were verified to confirm the presence of CRISPR/Cas9-mediated gene editing. The verified T2 generation putative mutant plant shows altered phenotypes of leaves, leaf rosettes, stems and inflorescences accompanied by lower deposition of lignin as revealed by histochemical staining by basic fuchsin and microscopic observations.
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AGO-hook domains in RNA-directed DNA methylation in Arabidopsis thaliana
Teznerová, Kateřina ; Čermák, Vojtěch (advisor) ; Moravec, Tomáš (referee)
RNA-directed DNA methylation (RdDM) is an important pathway that regulates gene expression by inducing DNA methylation and is involved in regulation of gene expression and defence against invading DNA elements (especially transposons). Argonaut (AGO) proteins with small RNAs (sRNAs) that have sequence complementarity to the target DNA play a key role in the RdDM pathway. Domains called AGO-hooks are able to interact with Argonaut proteins. In plants, two proteins with AGO-hook domains are involved in the RdDM pathway: NRPE1 and SPT5L. Recently, a third protein, SPT6L, has been discovered at the investigator's site to be part of the Pol V complex (as well as the two proteins mentioned above). The role of SPT6L has not yet been described, but we hypothesize that it also plays a role in the RdDM pathway. This work focuses on the study of all three AGO-hook domains in Pol V complex and their involve in the RdDM pathway in Arabidopsis thaliana, from the preparation of mutants lacking different combinations of these AGO-hook domains to the study of their role and substitution in DNA methylation at different loci. Key words AGO-hook, Arabidopsis thaliana, NRPE1, SPT5L, SPT6L, siRNA, epigenetic chromatin labelling, Argonaut protein
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Nuclear Transport Signals in the proteins of the ARP2/3 complex
Němcová, Barbora ; Bellinvia, Erica (advisor) ; Cvrčková, Fatima (referee)
The intracellular movement of macromolecules such as proteins and RNA is essential for maintaining cellular homeostasis and coordinating various cellular processes. In eukaryotic cells, the transport of these molecules between the cytoplasm and the nucleus is carefully regulated. Nuclear transport signals (NTS) play a key role in facilitating the import and export of proteins across the nuclear envelope. The ARP2/3 complex, which is an important regulator of activity dynamics, has been studied mainly for its functions in the cytoplasm, such as cell movement and cell division. However, new findings suggest that the ARP2/3 complex might also have nuclear functions in plants. Plants are unique multicellular organisms that rely on precisely coordinated cellular activities for growth, development, and response to stimuli. Understanding the molecular mechanisms behind nuclear processes in plants has recently become a focus of research. The ARP2/3 complex, which consists of seven subunits, is known for its ability to branch actin filaments and thereby control cellular processes requiring actin remodeling. However, recent studies have revealed a potential link between the ARP2/3 complex and nuclear functions in plants. Proteins associated with the ARP2/3 complex have been found to localize within the plant...
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Phototropic memory in early plant development
Šišková, Michaela ; Konrádová, Hana (advisor) ; Krtková, Jana (referee)
This thesis was inspired by controversial published results on the existence and reliability of a memory trace of the direction of the last illumination in young dicots. The theoretical part first covers phototropism, it briefly summarizes current knowledge about this growth movement, phototropins, and the mechanism of phototropic bending, including the role of auxin and its transporters. It also discusses the effect of previous light exposure on hypocotyl bending. Another section of the theoretical part is focused on the question of the existence of memory in plants. The experiments performed in the practical part are based on the assumption that plants are able to store a memory trace of previous light exposure. After the establishment of a system that made it possible to obtain a robust phototropic response to blue light, seedlings of the model plant Lepidium sativum were tested under different conditions to see if and how the direction of light affected the plants' response to subsequent phototropic stimulation. The results suggest that in at least one case, the final phototropic response could be interpreted as a formation of a specific memory trace in the tested plants. However, based on the results of this work, the existence of a memory trace of previous light exposure cannot be...
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Diversity of Polycomb complexes and their function
ŘÍHA, Luboš
The aim of this bachelor thesis is testing and further development of a vector system that should help clarify the alleged functional redundancy of Polycomb repressive complex 2 (PRC2) subunits. The theoretical part introduces the field of epigenetics and the role of Polycomb group complexes (PcGs) in Arabidopsis thaliana (mouse-ear cress) is explained. The issue of PRC2 subunits SWN and CLF redundancy is set in context and the tested hypothesis is explained. Genetic engineering tools relevant to this study are presented. Finally, the background of the promoter and marker vectors developed in the practical part is explained. In the practical part vectors with markers and promoters are developed and transgenic plants were grown on selection and genotyped. Results are presented and discussed.
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Rozhovory přes kutikulu
KOUTKOVÁ, Simona
Leaves of the plants communicate with their environment on the interface formed by epidermis with stomata and cuticle. Cuticle layer, composed of cutin and waxes, represents a barrier, which protects a plant from excessive water loss, but not only this. The changes of these boundaries are dependent on the environmental conditions, especially light (PPFD) and ambient CO2 concentration (Ca). This work tries to answer these questions: How is the availability of CO2 inside the leaf (Ci) altered? How is the development of epidermis related to cuticle formation?
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Characterisation of Arabidopsis thaliana mutants psbo1 and psbo2
Nykles, Ondřej ; Duchoslav, Miloš (advisor) ; Hála, Michal (referee)
The PsbO protein is necessary for the function of the electron-transport chain of the thylakoid membrane in higher plants. In most of the angiosperms, including Arabidopsis thaliana, this protein has two isoforms termed as PsbO1 and PsbO2. Many authors tried to reveal the fundamental difference between the PsbO1 and PsbO2 with the help of the mutant lines which lack one of the isoforms. The problem is that the mutants in psbO isoforms do not possess the same level of PsbO as WT does. So we made psbo1isoL mutants. These lines contain only one isoform but their level of the whole PsbO is comparable to the level of the whole PsbO of WT. Results from these experiments suggest that if a psbo1isoL plant has the same amount of PsbO as WT does, there is no observable phenotype difference. Thus we were not able to identify, in the usual cultivation conditions, if there are any functional differences between PsbO1 and PsbO2 Following the above mentioned results, we would like to know conditions (if there are any) in which T-DNA insertion mutants psbo2 (respectively psbo2cr which are made with the use of the CRISPR/Cas9), which have only PsbO1 isoform, could be phenotypically distinguished from WT. With the use of usual cultivation conditions, we are unable to tell apart the psbo2 and psbo2cr from WT by the...
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Role of TCTP1 in plant reproduction
Pitoňak, Oliver ; Hafidh, Said (advisor) ; Lafon Placette, Clément (referee)
TCTP is a conserved eukaryotic protein involved in regulation of multiple cellular processes, such as translation, cell cycle and cell death. Apart from its intracellular functions, TCTP is secreted and participates in human immune response. Dimerization via a terminal cysteine residue is critical for its extracellular function. Multiple sequence alignment of eukaryotic TCTPs revealed that terminal cysteine residue is conserved not only in animals but also in green plant lineage. In contrast to animal model organisms, knowledge about TCTP in plants is limited. Arabidopsis thaliana genome harbors two TCTP paralogs, TCTP1 and TCTP2. TCTP1 expression is highly upregulated in pollen. Previously published studies pointed to its role in embryo development, pollen tube growth and targeting. Using mutant characterization and analysis of subcellular localization, the role of TCTP1 in plant reproduction was investigated in this thesis. Recombinant Arabidopsis thaliana TCTP1 protein was produced along with its potential interacting partners BRL2 and TTL3 in heterologous Escherichia coli system. tctp-1 T- DNA line characterization pointed to TCTP1 role in pollen tube growth. It was expressed in mature pollen and pollen tube under native promoter and localized to the cytoplasm. Recombinant AtTCTP1 formed dimers...
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