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Characterization of the PTEN domain of selected Arabidopsis class II formins
Přerostová, Sylva ; Cvrčková, Fatima (advisor) ; Havelková, Lenka (referee)
Formins are proteins facilitating formation of actin filaments. They affect structure of cytoskeleton and participate in cytokinesis and tip growth. There are 2 classes of formins in Arabidopsis thaliana, which include FH1 and FH2 (Formin Homology 1 and 2) domain. Formins of the class I have usually a transmembrane domain on N-terminus. Due to this fact they can interact with membranes. Some formins from the class II include PTEN domain (Phosphatase and Tensin Homolog) derived from sequences of PTEN proteins which has lost the function of phosphatase. It is assumed this domain can bind on a membrane via the phosphatase section or C2 domain. This thesis was focused on the formin AtFH13 from the class II in Arabidopsis thaliana and on its PTEN domain. There were analyzed differences between mutants and wild-types in length of roots in seedlings and in size of seeds and seed coats, and observed the effect of dexamethasone on the length of roots on AtFH13. PTEN domain of the formin was isolated from cDNA, cloned to a vector and fused with YFP. The tagged protein was visualized by the method of transient expression in epidermal cells in the leaves of Nicotiana benthamiana. No big differences were observed between plants mutant in the gene AtFH13 and wild-type in choice parameters. Dexamethasone did't influence...
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Impact of elevated carbon dioxide concentration on Rubisco content in Fagus sylvatica and Picea abies
Hamříková, Dominika ; Vítová, Eva (referee) ; Hrstka, Miroslav (advisor)
In this bachelor’s thesis content of Rubisco in beech (Fagus sylvatica) and Norway spruce (Picea abies) was studied. The plants were cultivated in conditions with ambient carbon dioxide concentration (350 mol·mol-1) and elevated carbon dioxide concentration (700 mol·mol-1). The content of Rubisco in exposed and shaded leaves or needles was compared. The content of Rubisco was determined by SDS-PAGE method. A significant decrease in Rubisco content was demonstrated in conditions E versus A in shaded beech leaves. In other cases, the decrease was not so significant. A comparison of exposed and shaded needles also did not show a statistically significant difference, but in shaded beech leaves against exposed leaves Rubisco content was significantly lower, especially in conditions E.
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Elicitins impact on the proteome of tobacco
Dokládal, Ladislav
Title in English: Elicitins impact on the proteome of tobacco Abstract in English: Cryptogein is a proteinaceous elicitor secreted by an oomycete Phytophthora cryptogea that can induce resistance to P. parasitica in tobacco plants. On the basis of previous computer modeling, a series of cryptogein mutants was prepared with altered abilities to bind sterols, phospholipids or both. The sterol binding and phospholipid transfer activities corresponded to expectations based on the structural data reported previously. Induction of synthesis of reactive oxygen species (ROS) in tobacco cells suspension and proteomic analysis of intercellular fluid changes in tobacco leaves triggered by these mutant elicitins were not proportional to their ability to bind or transfer sterols and phospholipids. However, changes in the intercellular proteome corresponded to transcription levels of defense genes and resistance to P. parasitica and structure-prediction of mutants did not reveal any significant changes in protein structure. These results suggest, contrary to previous proposals, that the sterol-binding ability of cryptogein and its mutants, and the associated conformational change in the -loop, might not be principal factors in either ROS production or resistance induction. Nevertheless results support importance of...
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Development and optimalization of methodology for detection of GMO potatoes
ČERMÁKOVÁ, Jitka
Genetically modified (GM) or transgenic crops, now more often called ``Biotech crops{\crqq} they are commercially cultivated since 1996. And also since 1996, the first year of commercialization of biotech crops, GM potatoes were cultivated in USA, Mexico, Canada and later in South Africa, China and India. The global area of approved biotech crops in 2006 was 102 million hectares and 22 countries grew biotech crops, 11 developing countries and 11 industrial countries. The Czech Republic is on of the six EU countries where biotech crops are cultivated at present. The most compelling case for biotechnology, and more specifically biotech crops, is their capability to contribute to: increasing crop productivity and stability of productivity and production; conserving biodiversity, as a land-saving technology; the production of renewable resource based bio-fuels. This diploma paper was focused on developing of fast, precise and cheap method based on PCR to detect the presence of transgenes in potatoes {--} tubers and leaves, allows monitoring the presence of GM potatoes in market, environment, etc. and to quantify ``contamination{\crqq} of ware potatoes (tubers) with GM ones.
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Influence of genotype on rutin content in plants from genus \kur{(Fagopyrum)}
KRÁLOVÁ, Martina
The bachelor work is focused on evaluation of the influence of buckwheat species and variety on rutin containt in particular plant parts, on the total rutin yield per hectare production and on comparison varietal differences between both species, common buckwheat \kur{(Fagopyrum)}esculentum)} Moench) and tartary buckwheat \kur{(Fagopyrum tataricum)}. Rutin is a flavonol (quercetin-3-O- rutinosid) which is also called P vitamin. As well as it is known as the permeability factor or factor allowing permeability of the blood vessel. Rutin stimulates the C vitamin effect. Rutin has as well a high influence on hypertension, hypercholesterolemia and other cardiovascular diseases due to antioxidant properties. Buckwheat is the most important source of rutin. It is annual plant which belongs to Polygonaceae family and has a beneficial effect on human body. The common grown type is common buckwheat \kur{(Fagopyrum)}esculentum)} Moench) and less used type is buckwheat tartary \kur{(Fagopyrum tataricum)}. Tartary buckwheat \kur{(Fagopyrum tataricum)} contains several times higher amount of rutin than common buckwheat \kur{(Fagopyrum)}esculentum)} Moench). The highest amount of rutin is presented in the leaves and blossoms and the lowest one in seeds. Tartary buckwheat \kur{(Fagopyrum tataricum)} has much higher amount of rutin in seeds than common buckwheat \kur{(Fagopyrum)}esculentum)} Moench). The varietal differences in rutin content are considerable.
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Study of acid phosphatase in pea and tobacco
Šandová, Sandra ; Ryšlavá, Helena (advisor) ; Tichá, Marie (referee)
Phosphatases catalyze the hydrolytic fission of orthophosphoric acid ester. They are generally divided into acid and alkaline phosphatases according to their pH optimum. There was preparated the extract of leaves Nicotiana tabacum L. and Pissum sativum. Furthermore, there were determinated pea and tobacco phosphatases's rate constants. Reaction speed katalyzed by tobacco phosphatase is 10,0 mol/min.ml and by pea phosphatase 38,0 mol/min.ml using paraNP - phosphate as a substrate. Using a substrate phospho - L serine the reaction speed katalyzed by phosphatase of tobacco is 1,8 mol/min.ml and by phosphatase of pea is 0,4 mol/min.ml. Michaelis constant Km is 1,8 mM for tobacco phosphatase and 8,5 mM for pea phosphatase using paraNP - phosphate. Using a substrate phospho - L serine Michaelis constant Km of phosphatase of tobacco and pea is the same - 4,0 mM. The pH optimum of pea phosphatase is 5,0 using paraNP - phosphate as a substrate and 6,0 using phopho - L serine. pH optimum of tobacco phosphatase is 5,4 using paraNP - phosphate and 7,0 using phospho - L serine. Temperature optimum for pea phosphatase is 60 C and for tobacco phosphatase 55C.
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Determination of Rubisco enzyme content in leaves
Hlaváček, Viliam ; Vránová, Dana (referee) ; Hrstka, Miroslav (advisor)
The aim of the bachelor thesis was to optimize the conditions of the Rubisco enzyme quantity determination by the SDS-PAGE method and the determination of appropriate protein extraction method from plant tissue. Also the concentration range for which the method is linear was determined. Gels with different density ran under different voltages during the electrophoresis were tested for the aims of optimization. Also two different protein extraction methods were used. These were direct extraction to TRIS buffer and extraction with TCA/acetone method. The best results were obtained with protein separation on 8% gel and voltage of 90 V, while the volume of loaded sample was 5 µl. Under these conditions was the Rubisco great subunit band enough separated from the other protein bands, so that the densitometric evaluation provided better quality peaks with good baseline. The linearity of method under these conditions was in concentration range 0,125–1,0 mg of enzyme in 1 ml of sample. Direct extraction of the enzyme with TRIS buffer was less effective than the precipitation of proteins with trichloracetic acid in acetone followed with dissolution of the precipitate. The best buffer for the protein pellet dissolution was the thiourea buffer prepared according to Amalraj et al. [17]..
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The rutin content in amaranth (Amaranthus sp.)
BERANOVÁ, Kristina
This work was aimed to the determination of the flavonoids of rutin in the leaves, stems and seeds of different kinds and species of amarant. Rutin is a phenolic substance, which belongs to big group of secondary plant metabolites. Most recently lot of attention is paid to quercetin and its glycoside {--} rutin because of its demonstrable positive effect on the human organism, mainly the antioxidant effects which prevents the atherosclerotic plaque and preroxidation of lipids and antiviral activities. The research orients to its potential anti-carcinogen activities. Thanks to these properties it became a common part of the human daily diet. Amarant could be a beneficial source of these substances. Content of rutin was determined by the micelar electrokinetic capillary chromatography (MECC) in the group of seven genotypes of amaranth. For the analysis a samples of leaves, stems and seeds were used, because of its high contents of flavonoids. The highest content of rutin was found in the leaves of the species A. cruentus {--} genotype Olpir (27 400 mg/kg of dry matter) and the lowest in the seeds of A. cruentus {--} genotype Monatana (25,3 mg/kg of dry matter). Results of this research are unique as they were never published before.
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Diurnal changes of Rubisco activity under ambient and elevated concentrations of carbon dioxide
Uhrová, Lucie ; Vítová, Eva (referee) ; Hrstka, Miroslav (advisor)
Diurnal changes of Rubisco activity under the impact of ambient (380 µmol mol-1) and elevated (700 µmol mol-1) concentrations of carbon dioxide were studied in beech (Fagus sylvatica). Samples from leaves were taken in the two-hour intervals from 4:00 to 21:30 on the 22. 7. 2009 and activity of Rubisco was determined spectrophotometrically. No statistically significant differences were found between Rubisco activity of leaves growing in ambient and elevated CO2 concentrations. No increase of Rubisco total activity after daylight shows that nocturnal inhibitor CA1P is not present or plays only negligible role in beech. During the day Rubisco activity showed only statistically insignificant deviations. A slight decrease of Rubisco activation state in the afternoon indicates the afternoon depression of Rubisco initial activity.
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Determination of mercury content in materials in various stages of wine production, geochemistry of mercury in the vineyard
Buchtová, Jana ; Mihaljevič, Martin (advisor) ; Száková, Jiřina (referee)
This work was focused on determination of mobility of mercury and its content in materials from each part of the wine production process. The aim of this work was to establish the most suitable analytical method for determination and evaluation of changes in content of mercury during the production process. Samples of soil, biomass, vine wood, vine leaves, berries, grapes, rough wine and cider sediment of red wine and also four liquid samples of white wine were analyzed. All the samples originated from the area of Malé Žernoseky and Rudoltice nad Bílinou, white wine samples were collected in Chrámce u Mostu in the northern part of the Czech Republic. Analyzer AMA 254 was used for determination of mercury in solid samples and liquid samples had to be prearranged by PSA analyzer. This analytical approach was not so sufficient and mercury from liquid samples had to be captured by gold-coated amalgamator in first step and then analyzed by AMA 254. Obtained results confirmed interception of mercury mostly in leaves and in the grapes peelings. The grapes from Rudoltice contained 0,0022 mg/kg in dry mass and further processed wine grapes contained 0,0068 mg/kg in dry mass. The lowest content of mercury was in cider and completed wine contained less mercury then the rough wine. Unfiltrated cider from Chrámce has...
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