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Transcriptional response of plants to genotoxic stress and water deficit
Libus, Jiří ; Štorchová, Helena (advisor) ; Cvrčková, Fatima (referee) ; Kovařík, Aleš (referee)
7 Conclusions 1) cDNA array was prepared and used to assay transcript abundances of 376 selected Ara- bidopsis transcripts following various treatments with MMS. a) LoTrEC, clustering algorithm based on local trends of expression profiles, was de- signed and applied to the data. It succeeded to discover functionally rellevant clusters of expression profiles. b) Transcriptional responses to various MMS treatment regimes were investigated. While high MMS concentration seemed only to induce nonspecific stress reaction, the low and combined MMS resulted in a set of more specific expression changes. c) Expression levels of five transcripts were estimated by qRT-PCR. Trends of most of the profiles were confirmed. 2) Expression of 3 genes related to drought stress and/or response to cytokinin were mea- sured by qRT-PCR in wild type and ZOG1 transgenic plants. Transcript levels of all the genes were altered by water deficit. a) Although there are no significant macroscopic differences between wild type and ZOG1 transgenic plants, the mRNA abundances appeared to be influenced by the genotype. b) Leaf position (age) significantly influenced the expression of cig1 and ZOG1 driven by SAG12 promoter. c) RT primed with oligo-dT appeared more eficient than random hexanucleotide-primed reaction for 3 out of 5 mRNAs...
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Identification and expression of genes engaged in flowering of a model plant Chenopodium rubrum.
Cháb, David ; Štorchová, Helena (advisor) ; Kovařík, Aleš (referee) ; Smýkal, Petr (referee)
Summary: Study presentedin this PhD thesis focusedon the molecularbasis of flowering inductionin a short-dayplantChenopodiumrubrum.We lookedfor respectivehomologsof CONSTANS (CO), FLOWERING LOCUS T (FZ) and LEAFT (LF"I) genes,which act as crucialregulatorsin thephotoperiod-dependentsignalpathvtayinArabidopsisthaliana. We identifiedtwoFT-like (FTL) genesCTFTL| aCTFTL2 ditreringin theirexpression pattemsin tefraploidC. rubrun. CTFTLI showedrhythmicexpressionpeakingin midday. ElevatedexpressionoÍCrFTL| wascorrelatedwithfloweringunderpermissivephotoperiodic treatnents,whereasit was not expressedat all undernon inductivephotoperiďic regimes' CrFTL2 showedconstitutiveexpression.CrFTL| verylikely playsa Íoleas a floral inducer, butthefunctionof CTFTL2remarnsunknown. Two CO-lil&e (COZ) genes CTCOLI a CTCOL2 identified inC. rubrum are altemativelysplicedandproducetwovariantsoftranscripts.Oneofthem wasstandardwith oneintronlocatedin conservativesite,theotheronehadanadditionalintronconespondingto the90bp regionofthe first exon.This typeofaltemativesplicinghasnotbeendescribedin other lnown CoL gercs. All forms of fanscripts show allmost identicď rhythmic Eanscriptionalpattsmspeakingat the end of the night and differ only in the level of individualmRNA. Light stronglyinhibitednanscriptionofboth CrCOtr genes. We have ďso...
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The influence of RDR6 activity and mode of RNAi induction on dynamics and mechanism of silencing of the reporter GFP gene in tobacco cell line BY-2
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee)
RNA interference (RNAi) is a process mediated by small RNAs (sRNA), which is significantly involved in the regulation of gene expression in plants. Diverse RNAi pathways can be divided into two basic mechanisms, which are post-transcriptional and transcriptional gene silencing (PTGS and TGS). Production of sRNAs is dependent on the presence of a double-stranded RNA molecule (dsRNA), which is cleaved by one of DCL proteins to produce sRNAs usually of 21-24 nt in length. One strand of the sRNA is subsequently loaded onto AGO protein. During PTGS, the AGO-sRNA complex interacts with the target RNA based on its sequence complementarity to the sRNA and cleaves it or blocks its translation. In the case of TGS, AGO interacts with plant-specific RNA Pol V and its transcripts, which are again complementary to the sRNA. This interaction allows assembling of a protein complex facilitating DNA and histone methylation inhibiting RNA Pol II transcription. There are numerous ways the dsRNA can arise. A significant part of dsRNA cell production is dependent on synthesising the complementary strand of the dsRNA by RDR6 (RNA-dependent RNA polymerase 6). RDR6 is also involved in the process of the secondary sRNA formation. The significance of RDR6 during PTGS was examined using a GFP reporter gene either during...
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The application of magnetic particles for DNA isolation from selected vegetable products
Akwari, Michala ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
Micromethod of DNA isolation using magnetic particles is one of the modern technological methods used in DNA isolation, and makes the process simpler, more effective and faster. The main aim of this study was to isolate the DNA from various plant (tomato) food products, using different types of magnetic particles. The results were compared and the quantity, purity and the possibility of amplication of the isolated DNA among samples were found to be different. The DNA isolation method using magnetic particles P(HEMA-co-GMA) or HPS B-M-NH2 was shown to be the most effective in achieving the above mentiond parametres. DNAs from the analysed samples of plant food products were isolated in sufficient quantity and quality to be used in the conventional PCR. Differences in the possibility of the amplification of the isolated DNA stored at -20 °C during more than a half year were not found.
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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 application of magnetic nano- and microparticles for the isolation of DNA from selected foods
Ráčková, Lucie ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
In thesis was verified micromethod for isolation of plant DNA from different vegetable (onion and broccoli) and plant food products in quality for application in polymerase chain reaction (PCR). The micromethod allows isolation DNA using magnetic particles from crude lysates of cells obtained by direct homogenization of plant tissues. Various methods of processing homogenates were compared. Homogenization was performed by lysis buffer containing cetyltrimethylammonium bromide (CTAB). The effect of the organic extraction agents was tested (chloroform-octanol and isopropanol). DNA was purified from homogenates by reversible adsorption on magnetic particles (four different types of magnetic particles were tested). The quality of isolated DNA was verified by UV spectrophotometry. The amplificabilty of DNA was tested by polymerase chain reaction (PCR). Specific primers for plant ribosomal DNA (rDNA) were used. PCR products of lenght 700 and 220 bp were detected by agarose gel electrophoresis. Differences in yield and quality of DNA were depended on the homogenate processing and magnetic particles used. The proposed procedure with two magnetic particles was tested for the isolation DNA from plan food products (spreads). DNA was amplified in PCR. Micromethod is suitable for DNA analysis of foods.
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The application of magnetic particles for DNA isolation from thermally processed food products
Hronová, Aneta ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The thesis has been focused on testing of micromethod of DNA isolation using magnetic particles from thermic-managed food products in a quality suitable for polymerase chain reaction (PCR). Currant jams were selected for the analysis. These were homogenized using plastic copist and stomacher in lysis buffer with cetyltrimethylammonium bromide (CTAB). The effect of chloroform-octanol and isopropanol in the preparation of homogenates was tested. Homogenates were used for DNA isolation by magnetic particles. Rough fraction of DNA was purified by binding on the magnetic particles after centrifugation of the CTAB complexes with proteins, polyphenols and polysaccharides. Two types of magnetic particles were tested: microparticles of poly(hydroxyethylmethacrylate-co-glycidylmethacrylate) - P(HEMA-co-GMA) and nanoparticles of iron oxides covered by poly(L-lysine) - PLL. Isolated DNA was analyzed spectrophotometrically - it was assessed its concentration and contamination by polyphenols and proteins. After that, amplification of the DNA was tested in PCR. Primers specific for plant ribosomal DNA were used. PCR products of expected length 700 bp were detected by agarose gel electrophoresis. It was shown that DNA isolated from currant jams using magnetic particles was in PCR-ready quality.
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The use of magnetic particles for DNA isolation from selected spices
Gaňová, Martina ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The isolation of DNA from plant tissue of the required quality is very complicated, especially because of the presence of substances that can interfere during amplification of DNA. These substances are mainly polyphenols, polysaccharides, proteins and various dyes. The chemical diversity of such materials can have a significant effect on the yield and quality of DNA using one isolation procedure. The main aim of the work was to evaluate the use of microisolation protocol for related matrices to the quality of the isolated DNA as well as the evaluation of the effect of inhibitors isolated with the nucleic acid to the amplification in the PCR. DNA was isolated from dried paprika (Capsicum annuum). In the first step, the samples were homogenized using a lysis reagent with cetyltrimethylammonium bromide. Subsequently, the DNA was purified by reversible adsorption on magnetic particles. It was tested six different modified particles. The concentration and purity of the obtained DNA was determined by spectrophotometry measuring the absorbance of the DNA solution in TE buffer. The quality of the DNA was confirmed by amplification in PCR. For the PCR were used primers specific for plant ribosomal DNA (rDNA). The presence of PCR products was detected by agarose gel electrophoresis. It was found out that used microassay is suitable for isolating of the DNA of the corresponding purity that is suitable for the genetic analysis by PCR. The differences were found between the magnetic particles that were tested for DNA isolation.
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Modification of Speech Rate
Kovářík, Aleš ; Schwarz, Petr (referee) ; Szőke, Igor (advisor)
This diploma thesis discusses modification of a speech rate. The PSOLA (Pitch Synchronous OverLap Add) method was used for the rate modification. This algorithm works in time domain. Another method -- phase vocoder, which works in frequency domain is also presented in an overview. This thesis extends the PSOLA method with a phoneme recognition, which allows for better understandability of the speech output by considering characteristics of the phonemes beeing pronounced. To examine this proposed method, an application connecting PSOLA and a phoneme recognizer was developed.
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