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ISOLATION OF TRANSGENIC PLANTS NICOTIANA TABACUM AND SILENE VULGARIS
Kováčová, Viera ; Doškař, Jiří (referee) ; Vyskot,, Boris (advisor)
This project is focused on transformation of Silene vulgaris mediated by Agrobacterium tumefaciens and A. rhizogenes. S. vulgaris is a good model plant to study gynodioecy, an evolutionary step from bisexuality to dioecy. Gynodioecious plants form in some individuals bisexual flowers, while the others possess only female flowers. The aim of this research is do develop a technique to introduce foreign genes into this plant to study its developmental consequences. Using A. rhizogenes we successfuly prepared hairy root cultures, which unfortunately do not form shoot regenerants. We have prepared a protocol to induce plant regenerants from S. vulgaris leaf fragments. The first results do not confirm that A. tumefaciens infected plant regenerants harbor reporter transgenes. We used Nicotiana tabacum as a positive control.
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Health risk and profit of genetically modified golden rice
KUČEROVÁ, Kateřina
People in developing countries very often suffer from severe vitamin A deficiency due to an insufficient and unbalanced diet. This vitamin is necessary, among other things, for the production of the visual pigment rhodopsin. According to the World Health Organization, up to half a million children go blind each year because of this deficit. Its deficiency also weakens the immune system and thus significantly increases the risk of death from various infectious diseases. The best solution to this deficiency would be if the vitamin was contained directly in the only food that these people get - in husked rice. There is already a special, genetically modified rice, into which has been introduced by a complicated and extensive modification of its DNA the entire metabolic pathway, which ensures the production of betacarotene in this crop. In the theoretical part I deal with the golden rice project and the origin of this genetically modified crop by the method of transgenesis, using bacteria of the genus Agrobacterium tumefaciens. Plasmids of this bacterium are able to incorporate parts of their genetic information into the target organism. Thanks to restriction enzymes, we can insert genes selected by us into plasmids, which we then introduce into a specific plant. But are these products safe? Opinions on these crops are very diverse, but one thing interests everyone: how to safely identify a genetically engineered plant? In the laboratory, these experiments can best be mastered on model material. Therefore, in the experimental part of my bachelor thesis I deal with the transgenesis of the model plant Nicotiana tabacum using a selected strain of Agrobacterium tumefaciens. These bacterial strains were provided to me from the private collections of the Institute of Molecular Plant Biology in České Budějovice. The aim of my work in the laboratory was to master the practical methodology of preparation of genetically modified plant and subsequent verification of the presence of introduced genes in the examined samples of the model organism. Specifically, genes for antibiotic resistance. DNA isolation, PCR amplification and electrophoretic assays were used for this purpose. The signal gene used in agroinfection was verified too. Also, it was monitored how many copies of the transgene were integrated into the research material during agroinfection.
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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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Functional analysis of plant Arp2/3 complex subunits
Kukla, Jakub ; Schwarzerová, Kateřina (advisor) ; Honys, David (referee)
1. Abstract ARP2/3 complex is well studied in case of animals, it plays key roles in motility of cells and intracellular organels. It's malfunctions result in severe growth disorders and even lethality of affected cells. On the contrary, plant cells do not exhibit such dramatic phenotype of ARP2/3 complex mutations like it is by animals. It is possible that just the different life strategies of plants and animals contribute to differences in a way how animal and plant cells use their cytoskeleton, where ARP2/3 complex is it's part as well. It is highly conserved 7 protein complex from yeast to human. His main functions are creation of new "de novo" actin filaments, actin branched filaments network. Some of the parasite organisms are capable of missusing its nucleator activity to actively move inside of host cell. Because of the plant cells are sourounded by the cell wall, which give them support in creating various shapes and also hinders active movement of the whole cell body, it is likely that ARP 2/3 complex could be possibly involved in novel plant specific functions as well. If we think about the different life strategy of plants and animals we can not ignore all the things these two kingdoms have in common regarding to cytoskeleton processes. That is the need both for vesicular transport and...
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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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Influence of cultivation medium composition on metabolism of tobacco plants
Minářů, Kateřina ; Ryšlavá, Helena (advisor) ; Müller, Karel (referee)
Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) plays many roles in plants; it connects the metabolism of saccharides and amino acids. PEPC is regulated at many levels including phosphorylation of serine residue near N-terminus. The aim was to found out, if the composition of cultivation medium affects the activity of PEPC and metabolically related enzymes such as NADP-malic enzyme (EC 1.1.1.40), pyruvate, phosphate dikinase (EC 2.7.9.1; PPDK) and enzymes participating in nitrogen assimilation, thus nitrate reductase (EC 1.7.1.1; NR), glutamine synthetase (EC 6.3.1.2; GS), glutamate synthase (EC 1.4.1.14; GOGAT), NAD-glutamate dehydrogenase (1.4.1.2; NAD-GDH) and NADP-glutamate dehydrogenase (1.4.1.4; NADP-GDH). Tobacco plants (Nicotiana tabacum L., cv. Petit Havana SR1) were grown in vitro in Murashige-Skoog agar. The effect of the presence of 2% succrose, reduced content of nitrogen compounds or phosphate and limited supply of CO2 was followed. Sucrose added to Murashige-Skoog medium caused the increase of enzyme activity of PEPC, NADP-ME and most of enzymes related to nitrogen uptake and metabolism - NR, GS, GOGAT, NAD-GDH a NADP-GDH. Reduced content of nitrogen compounds and phosphate in cultivation medium decreased activity of all monitored enzymes. Enzyme activities were also affected by the...
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Characterization of membrane protein DREPP
Vosolsobě, Stanislav ; Schwarzerová, Kateřina (advisor) ; Cvrčková, Fatima (referee)
Proteins of DREPP family (20-25 kDa, syn. PCaP1 in Arabidopsis thaliana) first appeared in ferns and we have shown that several independent duplications of DREPP protein occurred during evolution of large families (Poaceae, Brassicaceae, Solanaceae and Asteraceae) and in group Coniferophyta. Secondary losses of one paralogue occurred in subfamilies Pooideae and Solanoideae.We have also detected two large-scale modification of DREEP protein in Asparagales and Brassicaceae (this divergent paralogue was previously described as MAP18 protein). We have examined colinearity of chromosome fragments in vicinity both PCaP1 and MAP18 paralogues in Arabidopsis thaliana and we hypothesize that MAP18 gene arose during genome duplication on the origin of Brassicaceae family. DREPP protein was previously identified in detergent-resistant membrane microdomain fraction and a myristyl anchor was shown to be necessary for their membrane localization. Membrane association was shown to be modified by the interaction of unique N-terminal domain with PtdInsPs, which was inhibited by binding of Ca-calmodulin (Nagasaki et al., 2008). The mutation of Gly2 by Ala in the myristilation site, or C-terminal GFP-fusion (GFP-DREPP), affect membrane association in Arabidopsis thaliana (Nagasaki et al., 2008). Several DREPP paralogues in...
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ISOLATION OF TRANSGENIC PLANTS NICOTIANA TABACUM AND SILENE VULGARIS
Kováčová, Viera ; Doškař, Jiří (referee) ; Vyskot,, Boris (advisor)
This project is focused on transformation of Silene vulgaris mediated by Agrobacterium tumefaciens and A. rhizogenes. S. vulgaris is a good model plant to study gynodioecy, an evolutionary step from bisexuality to dioecy. Gynodioecious plants form in some individuals bisexual flowers, while the others possess only female flowers. The aim of this research is do develop a technique to introduce foreign genes into this plant to study its developmental consequences. Using A. rhizogenes we successfuly prepared hairy root cultures, which unfortunately do not form shoot regenerants. We have prepared a protocol to induce plant regenerants from S. vulgaris leaf fragments. The first results do not confirm that A. tumefaciens infected plant regenerants harbor reporter transgenes. We used Nicotiana tabacum as a positive control.
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The archeobotanical research of the Early Modern waterlogged cesspit in Prague-Celetná street and possibilities of photography documentation
IRMIŠOVÁ, Jitka
Based on the macroremains analysis from Early Modern waste filling of the well in Prague Celetná street, dietary habits of Prague dwellers and the character of the city environment from the second quarter of the 18th century to the early 20th century were constructed. In total 44 015 macroremains were found, of which only 2 707 diasporas were wild-growing species. Strong predominance of crop confirmed hypothesis of well-into-cesspit transformation at the end of the 18th century. The species such as Nicotiana rustica, Piper nigrum and Ocimum basilicum, not commonly occurring in the archeological objects were recorded. The changes of the diaspora external structure influenced by their long-term deposition under anaerobic conditions were examined by electron microscopy method. A difference in subtle damage to the distinct diaspora structure along with the rate of diaspora organic pollution was noted.
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