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Dissertations / Theses on the topic 'Plant gene silencing'
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McMaster, S. "Studies on Gene Silencing in Plant Parasite Nematodes." Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501370.
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George, Gavin M. (Gavin Mager). "Virus induced gene silencing for the study of starch metabolism." Thesis, Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/4024.
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Thesis (PhD (Plant Biotechnology))--University of Stellenbosch, 2010.ENGLISH ABSTRACT: Virus Induced Gene Silencing (VIGS) was optimized to allow for the study of starch metabolism. The plastidial inorganic pyrophosphatase gene, for which a mutant has never been identified, was studied using VIGS and it was found to have a broad role in this subcellular compartment. The accumulation of inorganic pyrophosphate limited the production of starch, carotenoids, chlorophyll, and increased the plants susceptibility to drought stress. These effects highlight the importance of this enzyme in maintaining a low intraplastidial concentration of PPi providing an environment which facilitates these anabolic processes. Several genes involved in starch synthesis and degradation were also targeted with the aim of establishing a system of multiple gene silencing for the study of metabolic pathways. One, two and three genes were successfully silenced using this system which was validated based on previously published data. Interestingly, simultaneous silencing of the two isoforms of disproportionating enzyme led to a novel phenotype as a large reduction in starch instead of the expected increase was observed.
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Dalzell, J. J. "The development of gene silencing strategies for plant parasitic nematodes." Thesis, Queen's University Belfast, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546037.
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Chau, Ling Bess. "Capacity of plant-derived siRNA for gene silencing in mammalian cells." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B36778874.
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Chau, Ling Bess, and 周玲. "Capacity of plant-derived siRNA for gene silencing in mammalian cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B36778874.
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Davies, Gareth John. "Co-suppression of chalcone synthase genes in Arabidopsis thaliana." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318010.
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Raponi, Mitch Biochemistry & Molecular Genetics UNSW. "Antisense RNA-mediated gene silencing in fission yeast." Awarded by:University of New South Wales. Biochemistry and Molecular Genetics, 2001. http://handle.unsw.edu.au/1959.4/18277.
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The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.
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Starkus, Laura. "Virus-induced gene silencing of putative Diuraphis noxia (Kurdjumov) resistance genes in wheat." Thesis, Manhattan, Kan. : Kansas State University, 2010. http://hdl.handle.net/2097/4193.
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Gammelgård, Elin. "Interactions of potato virus A with host plants : recombination, gene silencing and non-hypersensitive resistance /." Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2007. http://epsilon.slu.se/2007111.pdf.
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Buchmann, Cody. "Reversal of RNA-mediated gene silencing pathways by geminivirus AL2 and L2 proteins." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1221847080.
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Mendu, Venugopal. "ROLES OF MICRORNAS IN PLANT ABIOTIC STRESS, DEVELOPMENT AND VIRAL INFECTION." UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/663.
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Plant microRNAs play important roles in plant growth and development. Here we investigated the roles of miRNAs in the plant abiotic stress, development and viral infection. MicroRNA membrane array analysis using five different abiotic stress treatments resulted in the identification of 8 novel stress inducible miRNA-families. Functional studies on novel stress inducible miR168 revealed its functional relation with abiotic stress. Over expression of miR168 in Arabidopsis showed upregulation of four stress related miRNAs (miR163, miR167, miR398 and miR408). Analysis of 9 independent transgenic lines showed induction of miR398, an oxidative stress responsive miRNA with a corresponding down regulation of its target genes. Heavy metal oxidative stress tolerance bioassays confirmed the susceptibility of transgenics compared to the wild types indicating the fact that the miR168 is indirectly involved in plant abiotic stress by inducing other stress responsive miRNAs.
MicroRNAs are highly conserved across the plant kingdom. A miRNA atlas was drafted for different tomato organs and fruit stages using the known miRNA sequences from different plants species. A large variation in both number and level of miRNA expression pattern was observed among different organs as well as among fruit stages. In the present investigation, we have found a window of expression for different miRNAs during the fruit development. A gradual decrease in the expression levels of miR160h, miR167a and miR399d and a gradual increase in miR164a have been noticed towards the fruit maturation while miR398b showed dual peaks during fruit development indicating a potential role of various miRNAs in fruit development and maturation.
Sonchus yellow net virus (SYNV) infected Nicotinana benthamiana leaves showed severe disease symptoms at two weeks post infection (WPI) and gradually recovered from the SYNV infection after 4-5 WPI correlating with the overall miRNA levels. The miRNA array and northern analysis showed an overall reduction of miRNA biogenesis during 2WPI followed by restoration to normal levels supporting the idea that the SYNV indeed interfered with the host miRNA levels which caused the symptoms and recovery phenotypes. Overall studies on plant abiotic stress, development and viral infection showed important roles of miRNAs in different aspects of plant life.
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Fultz, Dalen R. "The Silencing of Endogenous and Exogenous Transposable Elements in Arabidopsis." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492468003210374.
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Creasey, Kate M. "Investigating the roles of arabidopsis polycomb-group genes in regulating flowering time and during plant development by (I) challenging silencing and (II) developing approaches to dissect Pc-G action." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4025.
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Polycomb-group (Pc-G) proteins regulate homeotic gene silencing associated with the repressive covalent histone modification, trimethylation of histone H3 lysine 27 (H3K27me3). Pc-G mediated silencing is believed to remodel chromatin, rendering target genes inaccessible to transcription factors. Pc-G mediated silencing might result in irreversible changes in chromatin structure, however, there has been little analysis addressing whether Pc-G mediated silencing is reversible. In this work we focused on CURLY LEAF (CLF), the first Pc-G homologue discovered in Arabidopsis. CLF mediated repression of the floral homeotic gene AGAMOUS (AG) was challenged during early and late leaf development. AG was activated by the late leaf promoter, revealing that Pc-G mediated silencing can be overcome in old leaves in the presence of CLF. AG was also activated in young leaf primordia, yet did not persist in older leaves, revealing that transient activation of a Pc-G target is not epigenetically stable. To address the mechanism of Pc-G action within an endogenous environment, the histone dynamics at the APETALA1 (AP1) locus were characterized by Chromatin Immunoprecipitation. Unexpectedly, we found that the activation of AP1 in leaves did not require the removal of H3K27me3, questioning whether H3K27me3 is sufficient to silence. The roles of CLF in leaf and flower development are masked due to partial redundancy with SWINGER (SWN). clf- swn- mutants form a callus-like mass on sterile-tissue culture with no distinguishable plant organs. The role of CLF in regulating flowering time in natural populations of A. thaliana was investigated by complementing clf- mutants with CLF alleles from two accessions. We found that natural variation in CLF did not affect flowering time. To dissect the roles of CLF and SWN in late leaf and flower development, two approaches were developed for targeted expression. Firstly, CLF was introduced into the LhG4/ pOp transactivation system to provide CLF during early plant development. For mosaic analysis, CLF was introduced into the CRE lox recombination system in order to create clf- sectors surrounded by CLF+ SWN+ and CLF+ swn- cells.
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Nauth, Brittany J. "Soybean QTL Mapping and Candidate Gene Identification for Pythium irregulare and Phytophthora sojae Partial Resistance; and Root-Knot Nematode Induced Suppression of Gene Silencing." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1406151869.
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He, Jie. "Isolation of An ARGONAUTE Gene in Pelargonium and Identification Of Candidate Genes Regulated Through ARGONAUTE4-Dependent RNA-Dependent DNA Methylation In Arabidopsis." Connect to full text in OhioLINK ETD Center, 2009. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=toledo1260812913.
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Dissertation (Ph.D.)--University of Toledo, 2009.Typescript. "Submitted as partial fulfillment of the requirements for the Doctor of Philosophy degree in Biology." Bibliography: leaves 54-56, 91-95, 118-119, 133-139.
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El-Habbak, Mohamed H. "OVEREXPRESSION/SILENCING OF SELECTED SOYBEAN GENES ALTERS RESISTANCE TO PATHOGENS." UKnowledge, 2013. http://uknowledge.uky.edu/plantpath_etds/8.
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Plant diseases remain a major obstruction to meeting the world’s increased demand for soybean oil and protein. Reducing the losses caused by diseases in order to improve crop production is a high priority for agricultural research. The need for novel strategies for plant disease control cannot be overstated. In the present study, selected defense-related genes were silenced and/or overexpressed in soybean using a virus-based vector and the resultant plants were tested for their responses to pathogens. The first part of the study focused on Rps1k (Resistance to Phytophthora sojae) gene. The two conserved domains encoding ‘P-Loop NTPase’ and ‘PLN03210’ of Rps1k were independently overexpressed. Stem inoculation assays for the overexpressing plants showed significant resistance to virulent races; 90% standing plants compared to 10% in controls. Lesion length was greatly restricted only in case of plants overexpressing ‘PLN03210’. Simultaneous silencing of Rps1k-1 and Rps1k-2 resulted in remarkable susceptibility to avirulent races when tested by a detached-leaf assay. The second part of the study entailed silencing/overexpression of the chlorophyllase genes GmCLH1 and GmCLH2 and testing the responses of the silenced/overexpressing plants to the sudden death pathogen Fusarium virguliforme. Four weeks post root inoculation, GmCLH2-silenced plants showed enhanced resistance while the GmCLH2-overexpressing plants exhibited markedly increased susceptibility when compared to empty vector control. RT-PCR assay of PR genes revealed elevated expression of PR2 and PR4 in GmCLH2-silenced plants. In the third part of the study, soybean plants silenced for a leucine-rich repeat receptor-like kinase (GmRLK3) gene were examined for their responses to different pathogens. Silencing of GmRLK3 enhanced susceptibility to infection with Alternaria tenuissima or Sclerotinia sclerotiorum as revealed by rapid disease progress on treated leaves. Surprisingly, silencing of GmRLK3 in known susceptible soybean cultivars rendered the silenced plants resistant to P. sojae. The ensuing partial resistance to P. sojae was consistent with results of RT-PCR assays that showed a significant increase in the transcript level of the osmotin-encoding gene (PR5a) in the GmRLK3-silenced plants. PR5a is considered a marker for systemic acquired resistance.
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Chakrabarti, Manohar. "EVOLUTIONARY PERSPECTIVE OF NICOTINE TO NORNICOTINE CONVERSION, ITS REGULATION AND CHARACTERIZATION OF EIN2 MEDIATED ETHYLENE SIGNALING IN TOBACCO." UKnowledge, 2010. http://uknowledge.uky.edu/gradschool_diss/88.
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Nicotine, nornicotine, anabasine and anatabine are four major alkaloids in tobacco, of which nicotine is predominant. In many tobacco cultivars and also in other Nicotiana species, nicotine is converted to nornicotine, which in turn gives rise to potent carcinogen NNN. Nicotine to nornicotine conversion via nicotine-N-demethylation is mediated by the CYP82E family of P450 enzymes. Tobacco (Nicotiana tabacum) converts in senescing leaves, while its diploid progenitors N.tomentosiformis and N.sylvestris convert in both green and senescing and only in senescing leaves, respectively. Previously it has been shown that N.tomentosiformis has different active conversion loci in green and senescing leaves. The green leaf conversion enzyme CYP82E3 is inactivated in tobacco by a single amino acid substitution, while the senescing leaf converter enzyme CYP82E4 is active in tobacco, which gave tobacco a ‘senescing leaf converter’ phenotype. In nonconverter tobacco, CYP82E4 shows transcriptional silencing.
The nicotine-N-demethylase gene NsylCYP82E2 involved in nicotine to nornicotine conversion in senesced leaves of N. sylvestris was isolated. NsylCYP82E2 is active in N. sylvestris, but it has become inactivated in tobacco through mutations causing two amino acid substitutions. The conversion factor from N.sylvestris was characterized and a model for the alkaloid profile evolution in the amphidiploid N.tabacum from its diploid progenitors was proposed.
Regulation of conversion phenomenon was tested under different spatio-temporal conditions and various stresses. The promoter region for NtabCYP82E4 was isolated and promoter-reporter construct was used to determine that NtabCYP82E4 is specifically induced only during senescence. This pattern correlates with the nornicotine accumulation as measured by alkaloid profiling. Thus the regulatory regions of NtabCYP82E4 represent a senescence specific promoter.
In another project functional characterization of tobacco EIN2 (NtabEIN2) was undertaken. EIN2 from tobacco and N.sylvestris were cloned, their genomic structure was deduced and NtabEIN2 was silenced using RNAi approach. Silenced plants showed significant delay in petal senescence and abscission; as well as anther dehiscence, pod maturation, pod size, seed yield and defense against tobacco hornworm. Mechanism of delayed petal senescence phenotype, including possible cross-talk with Auxin Response Factor 2 and potential involvement of tasiRNA3 were also investigated.
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Biba, Angeliki. "Allele specific silencing of proteins at the neuromuscular junction." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:92aeab4d-1339-4e69-ad3f-eab2213ed401.
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RNA interference (RNAi) is a post transcriptional gene silencing mechanism that allows potent and specific silencing of cognate mRNA transcripts. Selective silencing can be used to dissect complex polygenic diseases, elucidate the function of known genes and provide a tool for genetic therapy. Its use in the case of dominant inherited disorders including disorders of the central nervous system, depends on its ability to confer single nucleotide discrimination between normal and mutant gene alleles. In this thesis the ability of RNAi effector molecules to provide single nucleotide specificity was examined by targeting two dominant inherited mutations of the acetylcholine receptor that cause slow-channel syndrome. Allele-specific silencing was achieved for one mutation. The other mutation was also silenced but not in an allele specific way despite employing known techniques for increasing single-nucleotide specificity. The model used in this thesis is the congenital myasthenia slow-channel syndrome. This is a dominant inherited disorder of the neuromuscular junction which is both well-characterised and more readily accessible compared to the central nervous system, thus provides a prototype for development of allele-specific RNAi therapeutics. Here we describe a new transgenic animal model of the slow-channel syndrome and show good representation of the human disorder. The need for defining the characteristics that determine the effectiveness and the specificity of RNAi effectors at single-nucleotide level, along with the future uses of the newly described animal model are discussed.
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Rupp, Jessica Lynn Shoup. "RNA interference mediated virus resistance in transgenic wheat." Diss., Kansas State University, 2015. http://hdl.handle.net/2097/20387.
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Doctor of PhilosophyPlant Pathology
John P. Fellers
Harold N. Trick
Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are two viruses affecting wheat in the Great Plains region of the United States. Genetic resistance is severely limited, requiring management methods focusing on the deployment of resistant varieties and various cultural practices. Evaluation of resistance is complicated by the lack of a standard rating scale. The objective of this work was to develop new avenues to mitigate these challenges. A standardized virus symptom rating scale was developed using historical Kansas rating scales, and validated using multiple wheat populations. Two independent RNA interference (RNAi) expression vectors targeting portions of viral coat protein (CP) of WSMV and TriMV were previously transformed into wheat. T₂ plants and beyond were evaluated using PCR, reverse transcription-PCR and bioassays in which plants were challenged with their respective virus. These lines were evaluated for resistance through the T₆ generation. Crosses were made with the susceptible winter wheat cultivars, ‘Overley’ and ‘Karl 92.’ Real-time PCR results show viral titer was up to 20-fold lower in the T₆ transgenic lines, the F₁, and the BC₁F₁ compared to control plants. This provides evidence that this RNAi silencing method is stable in wheat over multiple generations. WSMV and TriMV use host eukaryotic initiation factors (eIF) in order to facilitate replication of their genomes. Previously created RNAi expression vectors were derived from the sequences of the wheat genes eIF(iso)4E-2 and eIF4G. Evaluation of these lines began in the T₁ generation. Resistance has been demonstrated in three lines of eIF(iso)4E-2 and four lines of eIF4G, derived by single seed descent. T₆ progeny co-infected with WSMV and TriMV continue to be resistant. Crosses have been performed with the winter wheat ‘Karl 92’ and three Kansas elite lines, KS030887K-6, KS09H19-2-3, and KS10HW78-1-1. RNAi construct effectiveness was evaluated using real-time PCR. Results show up to 18-fold reduction in viral titer in the transgenic lines, the F₁, and the BC₁F₁ in comparison to control plants. This research provides the first evidence that a single host transgene can provide resistance to multiple viruses and has great potential benefits to both breeders and producers.
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Ho, Thien Xuan. "Antiviral gene silencing in plants." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509958.
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Sternberger, Anne Lauren. "Figuring out Flowers: Insights Into the Mixed Breeding System of Viola pubescens." Ohio University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1584452029880175.
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Unver, Turgay. "Detection And Characterization Of Plant Genes Involved In Various Biotic And Abiotic Stress Conditions Using Ddrt-pcr And Isolation Of Interacting Proteins." Phd thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/12609805/index.pdf.
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The main objective of this thesis dissertation is functionally characterizing the genes involved in biotic and abiotic stresses of plants at molecular level. Previously, upon pathogen attack Rad6 gene expression was found to be changed in wheat and barley plants. To functionally characterize the Rad6 gene, VIGS (Virus induced gene silencing) system was used. HR (Hypersensitive response) like symptoms was detected in every silenced barley and wheat plants. To figure out, transcriptomes and proteomes of Rad6 silenced plants were analyzed. 2-D PAGE analysis was also performed on silenced and control wheat plants. No pathogen growth was observed in Rad6 silenced barley lines. Additionally, the susceptible wild type Arabidopsis plants showed resistant phenotype when any of the Rad6 gene copies is mutated. This suggests that Rad6 gene has a negative regulatory role in plant disease resistance which was proved for the first time. Yeast two hybrid protein interaction study suggests that RAD6 carrying out its function by interacting with SGT1 protein and regulating resistance related genes. It has been first time reported in this thesis that E2 (Ubiquitin conjugating enzyme) takes role in plant disease resistance.
Boron which is the other consideration in the scope of thesis as an abiotic stress factor at a very limited amount is necessary for the normal development of plants. This study is conducted on highly boron tolerant Gypsophila perfoliata L. collected from a location in the boron mining area. The plant samples were tested in the presence of high boron (35 mg/kg) concentrations. The transcriptomes of the plant samples treated with the excess levels of boron to that of the samples grown under normal concentration were compared using differential display PCR method. Thirty bands showing differential expression levels at varying time points were analyzed. 18 of them were confirmed via qRT-PCR.
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Cruickshanks, Hazel Anne. "Conserved chromatin-mediated gene silencing in yeast and plants." Thesis, University of Edinburgh, 2003. http://hdl.handle.net/1842/26421.
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Cells must regulate gene expression to control development, differentiation, and respond to changes in the environment. The simplistic view of gene regulation states that an activator or repressor molecule binds to a specific sequence in DNA and exerts its effects upon transcription. However, the situation becomes more complicated when we consider that eukaryotic DNA is packaged into chromatin. Chromatin must be modified in order to control gene expression. Cells have adopted many ways of achieving this including: chromatin remodelling, DNA methylation and histone modification. The exact contribution of each of these need to be elucidated in order to fully understand gene regulation. Many common themes run through gene regulation between species, suggesting there are conserved mechanisms of gene control. Using the simple model organism, Saccharomyces cerevisiae, I have studied two types of gene repression found in plant species to compare and further determine their molecular bases. A repetitive DNA fragment previously found to induce de novo methylation and expression variegation in Petunia hybrida, was found to cause gene silencing in S. cerevisiae in a methylation independent manner. The possible mechanisms of this were dissected using gene replacement and protein expression studies. In a separate series of experiments, putative homologues of the S. cerevisiae transcriptional co-repressor, TUP1, were tested for chromatin remodelling ability in yeast. A TUP1 homologue from Arabidopsis thaliana was shown to repress transcription in S. cerevisiae but in a different manner from TUP1 indicating mechanistic similarities and differences between their functions. By using yeast as a tool to study gene regulation in higher eukaryotes, the principles of gene repression can be explored and we can speculate the roles of the individual features such as chromatin remodelling and DNA methylation.
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Bovi, Aurelie. "Investigation of novel genes, short RNAs and suppressor proteins involved in post-transcriptional gene silencing in plants." Thesis, University of East Anglia, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522400.
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Alphuche-Solis, Angel Gabriel. "Analysis of transgenic tomato plants with acc oxidase suppressed by sense constructs." Thesis, University of Nottingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297991.
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Lu, Rui. "High throughput virus induced gene silencing for the analysis of disease resistance in plants." Thesis, University of East Anglia, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398556.
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Buglass, Surahanil Katrin. "Regulating stem cell fate within microenvironmental niches." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:75f9498c-30f0-4983-84b2-dd58f2ccf52b.
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Improving the repopulation potential of human umbilical cord blood (UCB) haemopoietic stem cells (HSCs) remains a paramount goal in HSC transplantation (HSCT) therapy. This implies enhancing the homing and engraftment potential of UCB-CD34+CD133+ cells to the bone marrow (BM). Although an array of molecules continues to be identified as ‘key’ homing molecules, the molecular mechanisms controlling HSC homing are still not fully understood. The regulatory implications of hypoxia in the BM, with the concomitant stabilisation of hypoxia inducible transcription factor-1α (HIF-1α), are becoming more apparent, yet at the commencement of this thesis no study had explored whether hypoxia induced signalling can be adopted to regulate the homing and engraftment of transplanted HSCs. The aim of this DPhil project was thus to investigate whether hypoxic conditions as detected in the BM influence the adhesion of UBC-CD133+ cells to osteoblasts, BM stromal cells and BM endothelial cells-60 (BMEC-60), as well as their transmigration towards chemokine SDF-1α across BMEC-60. Increasing the exposure of UCB-CD133+ cells to 1.5% O2 doubled the percentage of transmigrating cells (p<0.05), and while hypoxia stimulated UCB-CD133+ cells preferentially adhered to IL-1β stimulated BMEC-60, their adhesion to non-stimulated (BMEC-60) was significantly improved (p<0.001). To help unravel the underlying molecular mechanisms, we attempted to examine the potential involvement of hypoxia regulated scaffolding protein HEF-1/NEDD9/Cas-L (HEF-1) in the increased percentage of migrating UCB-CD133+ cells after hypoxia pre-conditioning. The role of HEF-1 in HSCs is unexplored, and its multifunctional contribution in a variety of processes including cell migration, attachment and invasion make HEF-1 a prime candidate as a contributing homing molecule. After identifying a suitable short-hairpin RNA (shRNA) sequence to knockdown HEF-1, generating lentiviral (LV)-particles in house and optimising transduction protocols, HEF-1 knockdown was achieved in haemopoietic model cell lines KG-1 and KG-1A (KG-1/KG-1A–HEF1). Significantly decreased KG-1A–HEF1 cell adhesion to non-stimulated BMEC-60 was detected. Together, these studies provide a promising platform to further explore the role of HEF-1 in hypoxia induced UCB-CD133+ cell transmigration towards the key homing molecule SDF-1α.
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Wang, Nai, and 王鼐. "Construction of a high-throughput vector for inducible gene suppression in plants and its application in control of floweringtime." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30064958.
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Van, Eeden C. (Christiaan). "The construction of gene silencing transformation vectors for the introduction of multiple-virus resistance in grapevines." Thesis, Stellenbosch : Stellenbosch University, 2004. http://hdl.handle.net/10019.1/53764.
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Thesis (MSc)--University of Stellenbosch, 2004.ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.
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Raja, Priya. "Methylation of Geminivirus Genomes: Investigating its role as a host defense and evaluating its efficacy as a model to study chromatin methylation in plants." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1274728228.
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Camargo, Roberto de Almeida. "RNAi para o controle de Tuta absoluta em tomateiro." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/11/11151/tde-08042014-095355/.
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Desde seu descobrimento, o fenômeno de silenciamento gênico por RNA (RNAi) rapidamente se tornou uma técnica amplamente estudada e utilizada nos mais diversos aspectos da biologia molecular. Uma destas possibilidades é sua aplicação no campo da entomologia agrícola, mais especificamente para o controle de insetos-praga como uma alternativa de alta eficiência, especificidade e com impacto ambiental reduzido. Por meio da geração de plantas transgênicas expressando RNAi para genes essenciais de insetos-praga específicos, a ingestão destas moléculas de RNAi pelo inseto mediante herbivoria pode resultar no silenciamento do respectivo gene, resultando em fenótipos que podem variar entre perda de apetite, infertilidade ou até a morte. Neste contexto, o presente trabalho teve como objetivo provar a viabilidade de aplicação desta técnica para a interação Tomateiro x Tuta absoluta, cultura de grande expressão econômica e social no mercado nacional e internacional e que é amplamente atacada por esta praga, com prejuizos que podem alcançar a ordem dos 100% da produção. Por meio da clonagem de genes ortólogos essenciais descritos na literatura e de genes altamente expressos nos primeiros estádios larvais, após a caracterização transcriptômica em escala do inseto, foram realizados ensaios de alimentação contendo moléculas de dsRNAs que possuíam estes genes como alvo. Também, foi realizado a transformação genética de tomateiro cultivar \"Micro-Tom\" com dois destes genes (V-ATPase A e Arginina kinase) para a realização de ensaios de herbivoria. Com os resultados obtidos nestes experimentos, foram mostradas sólidas evidências da viabilidade da técnica de RNAi para o controle de Tuta absoluta, evidenciado pelo silenciamento gênico específico observado no inseto e consequentemente os efeitos nocivos deste silenciamento.Since their discovery, the phenomenon of gene silencing by RNA ( RNAi ) has rapidly become a widely studied and used technique in the molecular biological field. One of these possible applications is in the entomology field, more specifically for the control of insect pests, as a high efficiency, specificity and with reduced environmental impact alternative. Through the generation of transgenic plants expressing dsRNA targeting essential insect genes, their ingestion by the insect and consequently the uptake of the silencing RNA, may result in specific gene silencing, resulting in a variety of phenotypes that can range from loss of appetite, infertility to death. In this context, this study aimed to prove the feasibility of this technique to control tomato leaf miner (Tuta absoluta) in tomatoes plant, a major crop worldwide and seriously attacked by this pest, with losses that can reach 100%. For the present work, orthologous genes from successfully cases of insect gene silence described in the literature, was selected together with highly expressed genes in the early larval stages of T. absoluta, chosen after the insect molecular characterization and used in feeding assays with dsRNAs molecules to targeted these genes. Also, genetic transformation of the \"Micro-Tom\" tomato cultivar with two of these genes (V-ATPase and Arginine kinase) was conducted for testing in an herbivore assay. With these two approaches was possible to get solid evidences of the feasibility of the RNAi technique to control this insect, evidenced by specific gene silencing observed and its consequent effect on pest phenotype.
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Tsao, Theresa Tsun-Hui. "Towards the development of transgenic banana bunchy top virus (BBTV)-resistant banana plants : interference with replication." Queensland University of Technology, 2008. http://eprints.qut.edu.au/17031/.
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Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions.
A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated.
The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos.
The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV.
In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.
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Boehm, Christian Reiner. "Gene expression control for synthetic patterning of bacterial populations and plants." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267842.
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The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.
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Raponi, Mitch. "Antisense RNA-mediated gene silencing in fission yeast /." 2000. http://www.library.unsw.edu.au/~thesis/adt-NUN/public/adt-NUN20020131.041016/index.html.
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Jordan, Chad Victor. "Geminivirus-induced gene silencing as a method to determine the role of essential cell cycle genes in plant development." 2005. http://www.lib.ncsu.edu/theses/available/etd-05182005-011034/unrestricted/etd.pdf.
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Shittu, Hakeem Olalekan. "PLANT-ENDOPHYTE INTERPLAY PROTECTS TOMATO AGAINST A VIRULENT VERTICILLIUM DAHLIAE." Thesis, 2010. http://hdl.handle.net/10214/2271.
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When tomato Craigella is infected with Verticillium dahliae Dvd-E6 (Dvd-E6), a tolerant state is induced with substantial pathogen load, but few symptoms. Unexpectedly, these plants are more robust and taller with Dvd-E6 behaving as an endophyte. Some endophytes can protect plants from virulent pathogens. This research was undertaken to improve understanding of the cellular and molecular nature of Verticillium tolerance in tomato, especially whether infection by Dvd-E6 can protect Craigella from virulent V. dahliae, race 1 (Vd1). To permit mixed infection experiments a restriction fragment length polymorphism (RFLP)-based assay was developed and used for differentiating Dvd-E6 from Vd1, when present in mixed infections. The results suggested that protection involves molecular interplay between Dvd-E6 and Vd1 in susceptible Craigella (CS) tomatoes, resulting in restricted Vd1 colonization. Further studies showed a dramatic reduction of Vd1 spores and mycelia. To examine genetic changes that account for these biological changes, a customized DNA chip (TVR) was used to analyze defense gene mRNA levels. The defense gene response was categorized into four groups. Group 1 was characterized by strong induction of defense genes followed by suppression. However, Vd1-induced gene suppression was blocked by Dvd-E6 in mixed infections.
These genes included some transcription factors and PR proteins such as class IV chitinases and beta glucanases which are known to target fungal spores and mycelia. Experiments also were repeated with a Craigella resistant (CR) isoline containing a fully active Ve locus (Ve1+ and Ve2+). The biological results showed that the presence of the Ve1+ allele resulted in restricted Vd1 colonization and, in a mixed infection with Dvd-E6, Vd1 was completely eliminated from the plant stem. Surprisingly, there was no significant increase in defense gene mRNAs. Rather, elevated basal levels of defense gene products appeared sufficient to combat pathogen attack. To investigate functional effects of the genetic changes observed, an inducible RNAi knockdown vector for a defense gene (TUS15G8) with unknown function (pMW4-TUS15G8) as well as the Ve2 resistance gene (pMW-Ve2) was prepared as a initial step for future transformation analyses. Taken together the results reveal intriguing but complex biological and molecular changes in mixed infections, which remain a basis for future experiments and potential agricultural benefits.Canadian Commonwealth Scholarship and Fellowship Plan
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Shih, Jie-Ren, and 施傑仁. "Development of transgenic solanaceous plants resistant to multiple viruses via gene silencing." Thesis, 2002. http://ndltd.ncl.edu.tw/handle/90081090820004973680.
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碩士國立中興大學
植物病理學系
90
Abstract The concept of pathogen-derived resistance (PDR) postulated by Sandford and Johnston states that plants transformed with genes from pathogen may confer resistance to homologous or related pathogens. Previous studies of Jan et al. had shown that N gene segments (400 bp) of Tomato spotted wilt virus (TSWV) fused with three shorter N gene segments (200 bp) of TSWV, Groundnut ringspot virus (GRSV), and Impatiens necrotic spot virus (INSV) can induce PTGS by developing resistance against TSWV, GRSV, and INSV in Nicotiana benthamiana plants. Based on these results, Jan et al. formulated a hypothesis stating that transforming plants with transgene constructs having gene segments of different viruses linked to a universal silencer DNA can produce multiple virus resistance. In this study, we transformed N gene segment of Watermelon silver mottle virus (WSMoV) fused with three shorter N gene segments of TSWV, GRSV, and INSV to N. benthamiana plants. The results showed that transgenic plants with this construction can confer resistance to these three tospoviruses. To generate multiple resistance among viruses belonging to different genus and to confirm the use of the genes other then CP gene, nearly 250 bp CP gene segments of Potato virus Y (PVY), Tomato mosaic virus (ToMV), Cucumber mosaic virus (CMV), and segment of ToMV replicase gene were fused with WSMoV N gene segment and transformed to N. benthamiana plants. The R0 resistant lines obtained were challenged with PVY and three lines have shown high level of resistance. The seedlings collected from these will be subjected to select multiple viral resistance progenies.
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Coleman, Rory Tristan. "Making Memories: Modes and Mechanisms of Gene Silencing by the Polycomb Repressive Complexes in Drosophila." Thesis, 2017. https://doi.org/10.7916/D8CC159Q.
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Fundamental to the development of metazoa and plants is the capacity of cells to respond to transient intrinsic and extrinsic signals with permanent changes in gene expression that control cellular fates. A paradigmatic example of this process is observed in the case of the conserved, master regulatory HOX genes. The HOX genes are activated early in embryogenesis in combinatorial ON/OFF codes of expression, which act to specify and maintain segment identity in animals. In Drosophila, the “choice” of HOX code is controlled by transiently expressed transcription factors, while the “memory” of that choice is maintained in all future descendant cells through the action of the evolutionarily ancient Polycomb Group (PcG) gene family. The products of the PcG genes function in large, multimeric enzyme complexes known as Polycomb Repressive Complexes (PRCs) that are targeted to the HOX loci by cis-acting Polycomb Response Elements (PREs). Anchored at PREs, the PRCs catalyze a variety of chromatin modifications, most notably the trimethylation of histone H3 at lysine 27 (H3K27me3) by PRC2. These chromatin modifications are thought to both carry the memory of the HOX OFF code through DNA replication and to maintain transcriptional repression.
In addition to the HOX genes, the PcG regulates hundreds of other important developmental control genes, the majority of which do not adopt heritable patterns of ON/OFF expression but instead are more dynamically expressed. This poses the question of how PcG activities control such diverse modes of gene expression.
To investigate how PRE-anchored PRCs maintain heritable patterns of HOX gene expression, we have generated a transgenic lacZ reporter of the classical HOX gene Ultrabithorax (Ubx). Composed of minimal Ubx enhancer and promoter elements required to recapitulate the regulation of the native Ubx gene, the transgene contains the Ubx PRE embedded within a genetically labeled Flp-out cassette. H3K27me3 is deposited throughout the transgene in a manner that depends on the presence of the PRE. By excising the PRE in the cells of the wing imaginal disc where the transgene, like native Ubx, is heritably repressed, we are able to monitor the consequences of the loss of PRE-anchored PRC2 on both the inheritance of H3K27me3 and OFF state. We demonstrate that loss of the OFF state following PRE excision is correlated with the cell division-coupled dilution of H3K27me3. Further, by directly manipulating PRC2 activity of the H3K27 substrate, we demonstrate a causal relationship between the dilution of H3K27me3 nucleosomes and the number of times a cell can divide while maintaining the OFF state following PRE excision.
In addition, we identified Ubx-lacZ transgene insertions that deviate from the classical patterns of heritable expression characteristic of the HOX genes in novel and informative ways. Our analysis of these insertions supports the view that PcG dependent chromatin modifications impose a quantitative rather than qualitative repressive influence on a gene’s promoter, with the promoter’s activity being determined within the context of other regulatory inputs. Similarly, contrary to classical view, we demonstrate that transcription too plays a quantitative role in determining whether or not a HOX locus adopts the heritable ON state.
Together, our work suggests that the activities of the PcG confer a generic repressive influence on target loci. We posit that this influence is capable of maintaining heritable patterns of repression, as in the case of the HOX genes, because these loci have undergone stringent selection against enhancers capable of overcoming the repression mediated by the PcG. The absence of such strong activating inputs, together with the capacity of H3K27me3 to confer locus specific memory of the OFF state, allows for heritable patterns of repression. We propose that this is a special, albeit essential, attribute of the HOX genes. In contrast, most target genes have evolved to integrate repressive PcG chromatin modifications within the context of activating inputs that can override them. In these contexts, we propose that the PcG performs the role of a more general repressor, ensuring that repression is only overridden in those cells receiving peak activating cues. In this way the system may perform an essential role in conferring spatial and temporal robustness to gene expression programs.
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Cho, Yu-Hsiu, and 卓育秀. "Generation of transgenic plants expressing gene silencing suppressor HC-Pro and analyses of the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/30132412519406268384.
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碩士國立中興大學
生物科技學研究所
96
One of the strategies to increase the expression of foreign gene in plants is to generate transgenic lines that express a replicating virus vector carrying a gene of interest. However, productivity in combination of transgenic and viral vector expression system could be greatly reduced by post-transcriptional gene silencing (PTGS). This limitation could be effectively overcome by crossing stably-transformed plants expressing the suppressor of PTGS with a stably-transformed line carrying a replicating virus vector. In this study, we have generated transgenic N. benthamiana lines expressing HC-Pro gene by Agrobacterium-mediated transformation. At meantime, we also construct P1/HC-Pro mutant by inserting three additional amino acid into P1 region. To investigate the effects of HC-Pro on gene expression using Bamboo mosaic virus-based vector (BaMV vector), N. benthamiana plants were co-infiltrated with Agrobacteria harboring the plasmid clones of wild-type or mutant P1/HC-Pro and BaMV (pKB), or BaMV vector carrying GFP (pKBG) or VP1 epitopes of Foot-and-mouth disease virus (pKBVP1). At 7 and 14 days post infiltration (d.p.i.), plants co-infiltrated with pBIN-P1/HC and pKB or pKBG or pKBVP1 showed higher accumulation of viral coat protein or foreign proteins in both infiltrated and systemic leaves than those without pBIN-P1/HC; but there were no significant difference between the infiltration with and without mutant P1/HC-Pro. P1/HC-Pro transgenic lines 37-5-9 and 37-5-11 were agroinfiltrated with pKB、pKBG or pKBVP1, the virus and foreign gene accumulation of infiltrated and systemic leaves were higher than non-transgenic line. Furthermore, symptom-less transgenic N. benthamiana line B2B12-1 expressing full length BaMV-S genome was agroinfiltrated with wild-type or mutant P1/HC-Pro, the virus accumulation of infiltrated leaves was higher than non-infiltrated one; but there was no significant difference between systemic leaves of the plants infiltrated either with wild-type or mutant P1/HC-Pro or without. We hypothesize that HC-Pro could suppress local and systemic gene silencing in plants and improved the replication of virus and the expression of foreign proteins, but in B2B12-1 line, P1/HC-Pro could just improve the virus accumulation in infiltrated leaves by suppressing local gene silencing.
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Wu, Hui-Wen, and 吳惠雯. "Generation of transgenic plants conferring siRNA-mediated resistance against two potyviruses, and analysis of discriminating mutations of potyviral gene-silencing suppressor on pathogenicity and symptom development." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/40229438427144610046.
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博士國立中興大學
植物病理學系所
97
Production of oriental melon (Cucumis melo L.) worldwide is often limited by infection by two potyviruses, the watermelon infecting Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus W type (PRSV W) and. In order to engineer melon lines resistant to these potyviruses, a construct containing the coat protein (CP) sequences of these viruses was generated and used to transform an elite cultivar of oriental melon (Silver light) mediated by Agrobacterium using an improved cotyledon-cutting method. Altogether, our results indicated that RNA-mediated post-transcriptional gene silencing (PTGS) was the underlying mechanism of virus resistance of the transgenic melon lines. In order to further understand the relationship between plant defense and virus counteraction, we investigated the role of gene silencing suppressor, potyviral HC-Pro, which affects small RNA pathways involving in pathogenicity and symptom development. This dissertation is divided into five (including appendix) chapters as described below. Chapter 1, “Literature review” describes references relevant to this study. Chapter 2, 3 and 4 comprise the main text of this thesis, describing the engineering of transgenic melon lines resistant to ZYMV and PRSV W (Chapter 2 and 3) to solve the potyvirus problem in the field. We used binary vector harboring single (ZYMV) or double (ZYMV and PRSV W) CP constructs to transform an elite cultivar of oriental melon (Silver light) mediated by Agrobacterium using an improved cotyledon-cutting method. Removal of 1 mm portion from the proximal end of cotyledon greatly increased the frequency of transgenic regenerants by significantly decreasing the incidence of false positive and aberrant transformants. Southern hybridization analysis of transgenic lines revealed random insertion of the transgene in host genome, with insert numbers differing among transformants. Northern hybridization analysis evidenced an inverse correlation of the levels of accumulation of transgene transcript to the degrees of virus resistance, indicating post-transcriptional gene silencing (PTGS)-mediated transgenic resistance. Chapter 4 describes “Discriminating mutations of HC-Pro of Zucchini yellow mosaic virus with differential effects on small RNA pathways involving viral pathogenicity and symptom development”. It is well known that the potyviral HC-Pro is a gene silencing suppressor. We sought to obtain molecular evidence on the roles of the three highly conserved amino acids, R180I (mutation A), F205L (B), and E396N (C) of HC-Pro in microRNA (miRNA) and small interfering RNA (siRNA) pathways related to viral pathogenicity and symptom development using transgenic Arabidopsis plants system. We demonstrated that amino acid residues 180, 205 and 396 of HC-Pro are critical in suppression of miRNA, trans-acting siRNA (ta-siRNA) and virus induced gene silencing (VIGS) pathways, but not sense-post transcriptional gene silencing (s-PTGS). Because the R180I/ E396N HC-Pro mutant does not interfere with miRNA and tasiRNA pathways the ZGAC mutant elicits only attenuated symptoms. Furthermore, the recovery seen on ZGAC-infected plants likely results from the weak VIGS suppression by the double AC mutations of HC-Pro. The findings of this study are useful to protect high levels of transgene expression and to genereate an attenuated potyvirus for control of virus by cross protection.. Chapter 5 (Appendix) entitled “Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-mediated silencing” was carried out at Prof. Nam-Hai Chua’s laboratory, Rockefeller University, during my Ph.D program (2006-2008). The published paper is included as an appendix, because all the experiments of this project were carried out at Prof. Chua’s laboratory and this study was not directly connected to thesis. The main objective of this chaper was directed to determine, through artificial mutagnesis, the criticality of single nucleotide positions in the amiRNA-targeted sequence of 21 nucleotides, which were constructed in a potyvirual vector. Furthermore, the evolution of the virus through deletion and substitution in amiRNA-targrted sequence to escape amiRNA recognition was also investigated.
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Tran, Van Tuan. "Adhesion of the rapeseed pathogen Verticillium longisporum to its host Brassica napus: Uncovering adhesion genes and the evolutionary origin of the fungus." Doctoral thesis, 2011. http://hdl.handle.net/11858/00-1735-0000-000D-F1D2-9.
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