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The role of alternative splicing in plants
Földi, Marek ; Klodová, Božena (advisor) ; Fischer, Lukáš (referee)
Alternative splicing is a mechanism of gene expression regulation that maintains, regulates, and creates genomic diversity and tissue specificity in plants. It involves the differential joining of exons in precursor mRNAs, leading to multiple mRNA isoforms from a single gene. The formation of these isoform variants and their subsequent translation leads to subfunctionalization of proteins, generating diversity in structure and function. Therefore, alternative splicing is often important in various biological processes in plants, such as development, stress response, immunity, and reproduction. Key types of alternative splicing events include intron retention, exon skipping, alternative 5'/3' splice sites, and mutually exclusive exons. Regulation of alternative splicing involves cis-regulatory elements and trans- acting protein factors such as serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). This thesis aims to summarise the mechanisms and consequences of alternative splicing in plant development, including maturation of male and female gametophytes, meiosis, stress, and cell differentiation. It also describes methodological approaches that allow for a genome-wide study of alternative splicing, including microarrays, RNA-seq, and PCR. A better understanding of...
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Photosynthetic characteristics of potato plants with decreased expression of BEL11 transcription factor
Dybová, Magdaléna ; Mašková, Petra (advisor) ; Fischer, Lukáš (referee)
The potato (Solanum tuberosum) is one of the most important crops in the world. Tubers form during morphogenic process called tuberization, which timing is controlled by a complex regu- latory network involving both environmental conditions and internal factors. For the study of this process, model genotype of subspecies S. t. andigena is mostly used. It requires a short photoperiod. Vast majority of cultivated potatos are of spieces S. t. tuberosum that tuberize even under long-day conditions. Regulation of tuber initiation involves the action of endogenous factors, mainly phytohormones, carbohydrates and specific mobile signals, including BEL transcriptional factors - StBEL5, StBEL11 and StBEL29 - which transcripts are produced in leaves and transported into stolons. StBEL5 stimulates tuberization, whereas StBEL11 and -29 suppress it. The aim of this thesis was to contribute to understanding the role of StBEL11 in the regulation of tuberization by characterization of mutants with decreased StBEL11 levels. A sub- objective was to derive of transgenic lines S. t. andigena with decreased StBEL11 levels using a construct with part of StBEL11 coding region in antisense orientation under 35S CaMV pro- motor. I was able to derive forty two stable transgenic lines, which are prepared for StBEL11...
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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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RNA interference in plants
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Kulich, Ivan (referee)
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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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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Transformation of potato with protease inhibitor gene SPI-2
Říhová, Barbora ; Fischer, Lukáš (advisor) ; Vítámvás, Pavel (referee)
The subject of my thesis was to genetically modify a potato for increased resistance against its pathogens and pests. In developing a resistant plant, it is quite common to use the same type of molecules that plants use themselves in their defense reactions. In this work I used the gene SPI-2 originating from a honeycomb moth (Galleria mellonella). The protein SPI-2 is a member of serine protease inhibitors. Since the previous attempts of the team to detect the protein in transformed plants haven't been successful, the basic form of the gene was modified by adding a Kozak sequence near the start codon, which should have increased the translation initiation and hence increase the level of the protein. Two constructs were prepared for the transformation: SPI-2-T a SPI-2-Y. They differ by one amino acid, which slightly changes their inhibitory activity. First, the construct SPI-2-T was used for a transient transformation of tobacco Nicotiana benthamiana by agroinfiltration of its leaves. Then both constructs were used for a stable transformation of Solanum tuberosum cv. Desireé. The detection of the protein has not been successful, although the inserted gene was transcribed and his sequence was verified by sequencing. It is therefore most likely that the protein has a low stability in the cytoplasm....
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Modulation of BELL transcription factors balance as a tool for tuberisation strengthening in selected potato cultivars
Zounková, Andrea ; Mašková, Petra (advisor) ; Fischer, Lukáš (referee)
Potato (Solanum tuberosum) is the third most important crop in terms of human consumption. Hence understanding regulatory mechanisms controlling tuber initiation is significant not only from a theoretical but also practical point of view. Tuberization of S. tuberosum is a complex morphogenic process regulated by both, environmental conditions and inner plant status. Environmental conditions regulating the onset of tuberization have been known for a long time, but the molecular basis of tuber initiation has only recently been discovered, in particular using wild subspecies S. tuberosum subsp. andigena. BELL transcription factors are among the important components of the regulatory signalling network. The aim of this study was to modulate balance between the BELL transcription factors that induce and repress tuberization towards strengthening of tuber induction using two cultivars of Solanum tuberosum subsp. tuberosum, cv. Kamýk, resp. cv. Korela. To achieve this, transgenic lines carrying construct containing a part of coding sequence StBEL11, resp. StBEL29 in antisense orientation (AS) under control of constitutive promoters and lines carrying complete sequences of StBEL5 and StPTB6 genes under control of their native promoters were derived. We used the StBEL11 and StBEL29 AS constructs with the aim to...
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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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Size matters - siRNAs biogenesis and function in Arabidopsis
Přibylová, Adéla ; Fischer, Lukáš (advisor) ; Honys, David (referee)
RNA interference (RNAi) play a key role in various biological processes including regulation of gens and transposons, phylogenetic of part plant body, stress response, chromatin remodeling and antiviral mechanism. The ground of RNAi is short RNA molecules (small RNA, sRNA). In plants they are produced in range from 21 to 24 nucleotides (nt) and on the basis of being complementary they recognize target molecule of RNAi. It is possible to divide small RNA in two basic classes: microRNAs (miRNA) and small interfering RNAs (siRNA). To product and put small RNA into activate needs proteins from several gene family. DICER-LIKE (DCL) proteins create small RNAs from double-strand RNA precursors, which are often created by RNA dependent RNA polymerase (RDR) activity. With these small RNAs interact ARGONAUTE (AGO) proteins and together create RNA-Induced Silencing Complex (RISC). Those complexes play a key role in recognizing target molecule in active phase of RNAi. Structure and biogenesis of sRNAs has decisive influence on RISC complex and its next way in biogenesis. RNAi cause effect on post-transcriptional level (PTGS), as degradation of target molecule or repression of translation. And on transcriptional level (TGS) as sRNA intermediate histone and DNA methylation.
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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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