Дисертації з теми "Nicotiana benthamiana Viruses"
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Chewachong, Godwill Mih. "Engineering Plant Virus " Vaccines" Using Pepino mosaic virus as a Model." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1384203201.
Повний текст джерелаValenzuela, Aguila Sofia. "Transformation of Nicotiana benthamiana with different BWYV (Beet western yellows virus) sequences to test for virus resistance Transformation von Nicotiana benthamiana mit verschiedenen Sequenzen des BWYV (Beet western yellows virus) zur Virus-Resistenztestung /." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=959528695.
Повний текст джерелаVarrelmann, Mark. "Begrenzung von heterologer Enkapsidierung und Rekombination bei pathogen-vermittelter Resistenz gegen das Plum pox virus der Pflaume (PPV)." [S.l. : s.n.], 1999. http://deposit.ddb.de/cgi-bin/dokserv?idn=958530033.
Повний текст джерелаLin, Junyan. "NONHOST RESISTANCE TO BEAN POD MOTTLE VIRUS IN NICOTIANA BENTHAMIANA." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1372723537.
Повний текст джерелаFulton, Andrew Dale. "Monoclonal Antibody Expression and Novel Purification in Nicotiana benthamiana." Thesis, Virginia Tech, 2011. http://hdl.handle.net/10919/43361.
Повний текст джерелаMaster of Science
Dieterich, Guido. "Molekularbiologische Untersuchungen zur subzellulären Lokalisierung des putativen Transportproteins - P19,5k - des beet western yellows virus (BWYV) und Erarbeitung der Grundlagen für eine gentechnisch zu erzeugende Resistenz gegen das BWYV." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=960233989.
Повний текст джерелаTorres, Arzayus Maria Isabel. "Engineering yam mosaic virus resistance in Nicotiana benthamiana using genetic transformation techniques." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264199.
Повний текст джерелаMbewana, Sandiswa. "Development of Rift Valley fever virus candidate vaccines and reagents produced in Nicotiana benthamiana." Doctoral thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/25446.
Повний текст джерелаWu, Cheng Ying. "Characterization of innate immune response to «Nicotiana benthamiana»-derived Influenza H5 virus-like particles." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119400.
Повний текст джерелаA l'heure actuelle, la plupart des vaccins contre les infections par le virus influenza sont produits à partir d'œufs de poule fécondés. Ce procédé long et fastidieux constitue l'un des principaux obstacles à la production rapide d'un vaccin lors d'une pandémie. Une solution à ce problème consiste en l'utilisation de plantes afin de générer les antigènes nécessaires à l'élaboration du vaccin. Les pseudovirus ou Virus-like particles (VLP) produites à partir de la plante de tabac Nicotiana benthamiana représentent une alternative moins couteuse et plus rapide pour la production de vaccins antigrippaux. Des études préalables ont démontré qu'une immunisation avec les VLP exprimant l'hémagglutinine (HA) du virus influenza H5N1 (H5-VLP) induisaient une immunité protective lors d'une infection par ce virus chez la souris et le furet. Dans notre étude, nous avons utilisé les cellules mononuclées du sang périphérique humain (PBMC) afin de préciser la réponse immunitaire innée suite à l'exposition ex vivo aux H5-VLP produites dans N. benthamiana. Nous avons démontré les propriétés mitogéniques des H5-VLP sur les PBMC ainsi qu'une activation des lymphocytes B, des cellules NK et de certaines sous populations de lymphocytes T. L'analyse des cytokines sécrétées dans le surnageant des PBMC exposés ex vivo aux VLP suggère qu'une réponse pro-inflammatoire prédomine 48h après exposition et semble résulter essentiellement d'une activation des monocytes CD14+. Notre étude démontre que les VLP produites à partir de la plante de tabac génèrent une réponse immunitaire innée dans les PBMC provenant de patients naïfs, nous permettant ainsi de mieux comprendre les propriétés immunostimulantes de ce nouveau type de vaccin.
De, Figueiredo Pinto Gomes Pera Francisco. "Design and production of a candidate universal influenza A vaccine in Nicotiana benthamiana plants." Master's thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/27063.
Повний текст джерелаVeerapen, Varusha Pillay. "Novel expression and production of Foot-and-mouth disease virus vaccine candidates in Nicotiana benthamiana." Master's thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/27240.
Повний текст джерелаAgüero, González Jesús. "Desarrollo de vectores virales basados en el virus del manchado foliar de los cítricos (CLBV)." Doctoral thesis, Universitat Politècnica de València, 2013. http://hdl.handle.net/10251/34342.
Повний текст джерелаAgüero González, J. (2013). Desarrollo de vectores virales basados en el virus del manchado foliar de los cítricos (CLBV) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34342
TESIS
Verbeek, Matthew James Robert. "The Expression of Shuni Virus Nucleocapsid Protein in Nicotiana benthamiana for Use as a Diagnostic Reagent." Master's thesis, Faculty of Science, 2019. http://hdl.handle.net/11427/31017.
Повний текст джерелаBruckner, Fernanda Prieto. "Aspectos da interação entre a proteína TCTP e o potyvírus PepYMV na infecção de tomateiro e Nicotiana benthamiana." Universidade Federal de Viçosa, 2012. http://locus.ufv.br/handle/123456789/5355.
Повний текст джерелаCoordenação de Aperfeiçoamento de Pessoal de Nível Superior
Viruses are organisms with small genomes of simple organization, which coding about 3 to encode 10 viral proteins. The success of the infection depends on the manipulation of the cell by the virus, by means of complex interactions occurring between viral factors and host factors. The induced changes by virus infection include cell morphology changes, cell cycle changes and alterations in gene expression, among others. Understanding the processes that favor viral infection necessarily involves the study of virus-host interactions. In order to better understand the processes related to infection by tomato potyvirus Pepper yellow mosaic virus (PepYMV) a subtractive library was built 72 hours after infection. Several genes were identified as induced or repressed by viral infection. Among the induced genes, is the gene encoding the Translationally controlled tumor protein (TCTP). TCTP protein is highly conserved in all eukaryotes. Its functions are related to growth control and cell cycle, anti-apoptotic activity, and response to different biotic and abiotic stresses. The involvement of this protein in infection PepYMV has not been established, but studies in a strain of transgenic tomato plants silenced for TCTP showed that the silenced plants have a lower accumulation of PepYMV, indicating that TCTP promotes viral infection. In this study, we sought to advance the understanding of mechanisms involving TCTP in the process of infection by PepYMV. N. benthamiana plants silenced by VIGS TCTP were used to study the effect of silencing in viral infection, and the silenced plants accumulate fewer viruses in early stages of virus infection. Individual expression of viral proteins in N. benthamiana identified P3 and CP as capable of inducing TCTP expression at similar levels to those induced during PepYMV infection, and expression of NIb reduced expression of TCTP. The verification of direct interactions occurrence between viral proteins and TCTP by double-hybrid assay showed that TCTP not interact separately with any of the proteins of viral origin. Purification of proteins of health and infected N. benthamiana plants by affinity with TCTP identified several proteins that putativaly interacts with TCTP. As in two hybrid assay, interactions involving PepYMV proteins were not detected. These results sugests that TCTP actuation must involve the formation of protein complexes involving viral and plant proteins or contribute indirectly to PepYMV infection, without involving direct interactions between TCTP and viral proteins.
Os vírus são organismos com genomas pequenos, de organização simples, que codificam em média 3 a 10 proteínas. O sucesso da infecção depende da manipulação da célula pelo vírus, por meio de interações complexas que ocorrem entre fatores virais e fatores do hospedeiro. As modificações induzidas na célula incluem alterações morfológicas, alteração do ciclo celular e na expressão gênica, entre outras. A compreensão dos processos que favorecem a infecção viral passa necessariamente pelo estudo de interações vírus-hospedeiro. No intuito de compreender melhor os processos relacionados à infecção de tomateiros pelo potyvírus Pepper yellow mosaic virus (PepYMV) uma biblioteca subtrativa foi construída 72 horas após a infecção. Diversos genes cuja expressão foi alterada pela infecção foram identificados. Dentre os genes induzidos, se encontra o gene que codifica a Translationally controlled tumor protein (TCTP). A proteína TCTP é altamente conservada em todos os eucariotos. Suas funções estão relacionadas a controle do crescimento e ciclo celular, atividade anti-apoptótica, e resposta a diferentes tipos de estresses abióticos e bióticos. O envolvimento desta proteína na infecção pelo PepYMV ainda não foi estabelecido, porém estudos em uma linhagem de tomateiro transgênica silenciadas para a TCTP, mostraram que as plantas silenciadas apresentam um menor acúmulo de PepYMV, indicando que a TCTP favorece a infecção por este vírus. Neste trabalho, buscou-se avançar na compreensão dos mecanismos que envolvem a TCTP no processo de infecção pelo PepYMV. Plantas de Nicotiana benthamiana silenciadas para TCTP por VIGS (Virus Induced Gene Silence) foram utilizadas para estudar o efeito do silenciamento na infecção viral, sendo que as plantas silenciadas acumularam menos vírus no início da infecção. A expressão individual das proteínas de origem viral em N. benthamiana identificou a P3 e a CP como capazes de induzir a expressão de TCTP em níveis semelhantes aos observados durante a infecção pelo PepYMV, sendo que a expressão da proteína NIb reduziu a expressão de TCTP. A verificação da ocorrência de interações diretas entre a TCTP e as proteínas virais, por ensaio de duplo híbrido, mostrou que a TCTP não interage separadamente com as proteínas de origem viral. A purificação de proteínas de plantas de N. benthamiana, sadias e infectadas, por afinidade com a TCTP identificou diversas proteínas que possivelmente 7 interagem com a TCTP. Assim como no ensaio de duplo híbrido, a interação com proteínas virais não foi detectada. Estes resultados sugerem que o papel da TCTP deve envolver a formação de complexos proteicos entre proteínas virais e da planta, ou favorecer a infecção de forma indireta.
Stephan, Dirk. "Molekulare Charakterisierung von beet mild yellowing virus (BMYV) und beet chlorosis virus (BChV) sowie Selektion von BMYV Amplicon-transgenen Nicotiana benthamiana." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974988146.
Повний текст джерелаGhoshal, Basudev. "Symptom recovery in Tomato ringspot virus infected Nicotiana benthamiana plants : investigation into the role of plant RNA silencing mechanisms." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/49984.
Повний текст джерелаScience, Faculty of
Botany, Department of
Graduate
Naude, Jason Christopher Delville. "The Expression of Chikungunya Virus Envelope 2 Glycoprotein Variants in Nicotiana benthamiana for the Development of a Diagnostic Reagent." Master's thesis, Faculty of Science, 2021. http://hdl.handle.net/11427/32942.
Повний текст джерелаVarennes-Jutras, Philippe. "Protection des protéines recombinantes sécrétées chez l'hôte d'expression "Nicotiana benthamiana" par expression hétérologue du canal ionique M2 du virus de l'Influenza." Doctoral thesis, Université Laval, 2017. http://hdl.handle.net/20.500.11794/28235.
Повний текст джерелаPlant expression systems are commonly used for the heterologous production of complex recombinant proteins. However, biochemical conditions in the plant cell secretory pathway, such as the presence of poorly-specific proteases or pH variations from one organelle to another, impair the expression of several potentially useful proteins. Approaches have been developed to improve the cellular environment of the host plant in such a way as to increase the quality and yield of secreted recombinant proteins. In this project, we assessed the impact of pH homoeostasis in the leaf cell secretory pathway of wild tobacco Nicotiana benthamiana on the expression and stability of recombinant proteins. We demonstrate the potential of Influenza virus proton channel M2 as a new tool to increase pH in acidic compartments of the cell secretory pathway, eventually useful to stabilize acid-labile recombinant proteins. In line with the well-established influence of pH on cell protease activities, we then show that pH modification induced by the expression of the M2 channel influences the degradation profile of fusion proteins susceptible to proteolysis, thus confirming the impact of pH on protease activities in plant cells and highlighting the potential of M2 as an accessory protein to increase the stability and yield of recombinant proteins in planta. Finally, we describe the impact of the M2 proton channel on the expression of endogenous proteins at the cellular scale. We show that pH alteration in the secretion system upon M2 expression has cell-wide effects on the leaf proteome, affecting the content of proteins found in various cell compartments including the chloroplast, the cytosol and the vacuole. We report the establishment of a ‘hybrid proteome’ in leaf cells expressing the proton channel, composed of protein clusters characteristic of both control, non-infected plants and agroinfected plants actively expressing defense-related proteins. Overall, our data highlight the central role of pH homeostasis on the proteome of plant cells and the strong impact of pH gradient in the cell secretory pathway on the stability and yield of acidic pH-labile and protease-susceptible recombinant proteins.
Charlesworth, Steven Roy. "Investigation into resistance strategies against geminiviruses by understanding and adapting RNA interference." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/121482/2/Steven%20Roy%20Charlesworth%20Thesis.pdf.
Повний текст джерелаXavier, André da Silva. "Efeito do silenciamento dos genes DnaJ e TCTP na infecção de tomateiro e Nicotiana benthamiana pelo potyvírus Pepper yellow mosaic virus (PepYMV)." Universidade Federal de Viçosa, 2012. http://locus.ufv.br/handle/123456789/4413.
Повний текст джерелаConselho Nacional de Desenvolvimento Científico e Tecnológico
During coevolution, plant viruses have developed the ability to modulate the expression of several host genes, or to alter the function of cognate protein to succeed in their multiplication and perpetuation. These virus-induced changes might lead to a high level of dependency, creating an indissoluble link between virus and host. The objective of this study was to investigate the contribution of a DnaJ of Nicotiana benthamiana (homologous of the tomato protein) and of the TCTP protein from tomato during PepYMV infection. These two genes were identified as differentially expressed in a cDNA library constructed from tomato plants infected by PepYMV. Kinetic studies of DnaJ expression in PepYMV-infected N. benthamiana demonstrated that the induction occurs at both 72 hours post-inoculation (hpi) and 14 days post-inoculation (dpi). Plants of N. benthamiana silenced for DnaJ by means of VIGS and tomato plants cv. Moneymaker silenced by transgenesis for TCTP were obtained and mechanically inoculated with PepYMV. Viral infection was confirmed by ELISA and viral load determined by qRT-PCR. Silencing of the DnaJ gene in N. benthamiana interfered with the early stages of viral infection (72 hpi) but its effect on established infections (14dpi) was inconclusive. Non-transformed tomato plants showed severe symptoms of the disease, while TCTP-silenced transgenic plants showed greatly attenuated symptoms or remained asymptomatic. Viral load was dramatically reduced in silenced plants. The subcellular localization of a TCTP-GFP fusion protein in healthy or PepYMV-infected N. benthamiana plants was analyzed by confocal microscopy. In healthy plants TCTP was nuclear and cytoplasmic, while in infected plants at 14 dpi, its subcellular localization was exclusively cytoplasmic. Together, these results suggest that both TCTP and DnaJ are proteins which positively regulate the infection cycle of PepYMV, being required for disease development in the case of TCTP or for the rapid establishment of viral infection in the case of DnaJ. Further studies should be conducted in order to unravel the mechanisms by which these host factors are used to benefit the viral infection.
Durante a coevolução, os vírus de plantas desenvolveram a capacidade de modular a expressão de alguns genes do hospedeiro, ou alterar a função cognata de proteínas para obter sucesso em sua multiplicação e perpetuação. Essas alterações induzidas pelos vírus podem culminar em elevados níveis de especialização, tornando o vínculo com seus hospedeiros indissociável. O objetivo deste trabalho foi investigar a contribuição de uma DnaJ de Nicotiana benthamiana homóloga de tomateiro e da proteína TCTP de tomateiro durante a infecção pelo potyvírus PepYMV. Esses dois genes foram identificados como diferencialmente expressos em uma biblioteca de cDNA construída a partir de tomateiro infectado pelo PepYMV. Estudos de cinética de expressão do gene DnaJ em N. benthamiana infectadas pelo PepYMV demonstraram que a indução do gene ocorre 72 horas pós-inoculação (hpi) e aos 14 dias pós-inoculação (dpi). Plantas de N. benthamiana silenciadas para DnaJ por meio de VIGS e de tomateiro cv. Moneymaker silenciadas por transgenia para o gene TCTP foram obtidas e inoculadas mecanicamente com o PepYMV. A infecção viral foi confirmada por ELISA e a carga viral determinada por qRT-PCR. O silenciamento do gene DnaJ em N. benthamiana interferiu nos estágios iniciais da infecção viral (72 hpi), porém seu efeito em infecções já estabelecidas (14dpi) foi inconclusivo. Plantas não-transformadas de tomateiro exibiram sintomas severos da doença, enquanto as plantas transgênicas silenciadas para o gene TCTP apresentaram sintomas muito atenuados ou permaneceram assintomáticas. Nas plantas silenciadas a carga viral foi drasticamente reduzida. A localização subcelular de TCTP fusionada à proteína GFP em plantas de N. benthamiana sadias ou infectadas pelo PepYMV foi analisada por microscopia confocal. Em plantas sadias a localização de TCTP foi nuclear e citoplasmática, porém em plantas infectadas aos 14dpi, a localização de TCTP foi exclusivamente citoplasmática. Os resultados obtidos sugerem que tanto TCTP quanto DnaJ são proteínas que regulam positivamente o ciclo de infecção do PepYMV, sendo necessárias para o desenvolvimento da doença no caso de TCTP, ou para o rápido estabelecimento da infecção no caso de DnaJ. Estudos posteriores devem ser conduzidos afim de descobrir o mecanismo pelo qual esses fatores do hospedeiro são utilizados em benefício da infecção viral.
Solofoharivelo, Marie Chrystine. "Molecular Characterizations of Transgenic Nicotiana Benthamiana Plants Resistant to Red Clover Necrotic Mosaic Virus and Effects of Mixed Infections with Potato Virus Y on RNAi-Mediated Resistance." Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/194799.
Повний текст джерелаYu, Ming. "Characterization of cDNAs from Nicotiana benthamiana that encode proteins which interact with tomato golden mosaic virus AL2 protein in the yeast two-hybrid system." NCSU, 2003. http://www.lib.ncsu.edu/theses/available/etd-07182003-150714/.
Повний текст джерелаO'Connor, Steven Patrick. "The production of foot-and-mouth disease virus-like particles in the plant Nicotiana benthamiana: a potential candidate vaccine for foot-and-mouth disease." Master's thesis, University of Cape Town, 2018. http://hdl.handle.net/11427/29378.
Повний текст джерелаGil, Capitán María Teresa. "Inmunomodulación de la infección de Plum pox virus mediante la expresión estable y transitoria de anticuerpos recombinantes contra la NIb replicasa viral en plantas de Nicotiana benthamiana." Doctoral thesis, Universitat Politècnica de València, 2013. http://hdl.handle.net/10251/34204.
Повний текст джерелаGil Capitán, MT. (2010). Inmunomodulación de la infección de Plum pox virus mediante la expresión estable y transitoria de anticuerpos recombinantes contra la NIb replicasa viral en plantas de Nicotiana benthamiana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34204
Palancia
Martínez, Priego Lucía. "Actividad antiviral de pequeños RNAs endógenos y supresión de silenciamiento génico por la proteína 16K del virus del cascabeleo del tabaco (TRV)." Doctoral thesis, Universitat Politècnica de València, 2016. http://hdl.handle.net/10251/61464.
Повний текст джерела[ES] En las infecciones por virus, el desenlace del proceso infectivo debe entenderse como el resultado neto de las interacciones compatibles y de defensa entre el virus y la planta hospedadora. Cuando un virus entra en una célula eucariota debe lidiar con la activación de diferentes mecanismos de defensa del huésped. El silenciamiento génico mediado por RNA constituye una primera línea de defensa innata de la planta, siendo los propios virus inductores, dianas y supresores de este sistema de defensa. Las plantas a través del silenciamiento génico son capaces de limitar la proliferación viral en las células infectadas permitiendo un delicado equilibrio entre la multiplicación del virus y la integridad celular. Sobre este equilibrio se fundamenta la relación de compatibilidad en la interacción planta-virus. En este escenario, los virus utilizan sus proteínas supresoras de silenciamiento (VSR) para modular los efectos antivirales del silenciamiento y reprogramar la expresión génica del huésped proporcionando un entorno favorable para el desarrollo de la infección compatible Con este trabajo hemos abordado el modo en que los virus interaccionan con el silenciamiento génico en el contexto de una infección compatible. Empleando el virus del cascabeleo del tabaco (TRV) como sistema viral y Nicotiana. benthamiana y Arabidopsis thaliana como modelos de huésped, hemos indagado en el potencial de los pequeños RNAs (sRNAs) endógenos para guiar procesos de silenciamiento sobre secuencias virales. Nuestros resultados suLa manera en que TRV interacciona con la ruta de silenciamiento está condicionada gieren la posibilidad de interacciones funcionales entre microRNAs (miRNAs) y secuencias complementarias en el genoma del virus, si bien su relevancia como mecanismo de control de la proliferación viral no se ha estudiado en este trabajo. por el efecto supresor de la proteína 16K. Esta proteína impide, al menos parcialmente, el ensamblaje de los complejos efectores de silenciamiento y puede por tanto comprometer el efecto del silenciamiento antiviral dependiente de sRNAs tanto virales (vsiRNAs) como endógenos. El efecto supresor de TRV no parece perturbar globalmente el contenido, composición relativa y actividad de los miRNAs, si bien no es descartable que induzca alteraciones en el metabolismo de especies concretas.
[CAT] En les infeccions per virus, el desenllaç del procés infectiu ha d'entendre's com el resultat net de les interaccions compatibles i de defensa entre el virus i la planta hoste. Quan un virus entra a una cèl·lula eucariota ha de lluitar amb l'activació de diferents mecanisme de defensa de l'hoste. El silenciament gènic per RNA constitueix una primera línia de defensa innata de la planta, i els propis virus son inductors, dianes i supressors d'aquest sistema de defensa. Les plantes a través d'aquest silenciament, són capaces de limitar la proliferació viral a les cèl·lules infectades, permetent un delicat equilibri entre la multiplicació del virus i la integritat cel·lular. En aquest equilibri es fonamenta la relació de compatibilitat existent a la interacció planta-virus. En aquest escenari, els virus utilitzen les seues proteïnes supressores de silenciament (VSR) per tal de modular els efectes antivirals del silenciament i reprogramar l'expressió gènica de l'hoste, proporcionant un entorn favorable per al desenvolupament de la infecció compatible. Amb aquest treball hem abordat la manera en la que els virus interaccionen amb el silenciament gènic en el context d'una infecció compatible. Emprant el virus del cascavelleig del tabac (TRV) com a sistema viral i Nicotiana. benthamiana i Arabidopsis thaliana com a hostes models, hem indagat en el potencial dels RNAs endògens curts de doble cadena (sRNAs) per guiar processos de silenciament sobre seqüències virals. Els nostres resultats suggereixen la possibilitat de interaccions funcionals entre microRNAs (miRNAs) i seqüències complementàries al genoma del virus, tot i que la seua rellevància com a mecanisme de control de la proliferació viral no ha estat tractat en aquest treball. La manera en la que TRV interacciona amb les rutes de silenciament es troba condicionada per l'efecte supressor de la proteïna 16K. Aquesta proteïna impedeix, al menys parcialment, l'acoblament dels complexos efectors del silenciament i pot llavors comprometre l'efecte del silenciament antiviral depenent de sRNAs, tant virals (vsiRNAs) com endògens. L'efecte supressor de TRV no sembla pertorbar globalment el contingut, composició relativa i activitat dels miRNAs, tot i que no es pot descartar que induïsca alteracions en el metabolisme d'espècies concretes.
Martínez Priego, L. (2016). Actividad antiviral de pequeños RNAs endógenos y supresión de silenciamiento génico por la proteína 16K del virus del cascabeleo del tabaco (TRV) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61464
TESIS
Hoffmann, Laurent. "Etude du métabolisme des phénylpropanoïdes; analyse de l'interaction de la caféoyl-coenzyme A 3-O-méthyltransférase (CCoAOMT) avec son substrat et caractérisation fonctionnelle d'une nouvelle acyltransférase, l'HydroxyCinnamoyl-CoA : shikimate/quinate hydroxycinnamoyl Transférase (HCT)." Phd thesis, Université Louis Pasteur - Strasbourg I, 2003. http://tel.archives-ouvertes.fr/tel-00003598.
Повний текст джерелаBusto, Jennifer Lee. "Transcriptional changes in Nicotiana benthamiana induced by tobamoviral transfection." Thesis, 2005. http://proquest.umi.com/pqdweb?index=1&did=913527421&SrchMode=1&sid=1&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1235524057&clientId=23440.
Повний текст джерелаHuang, Wei-Pin, and 黃薇頻. "Tracing Bamboo Mosaic Virus RNA Molecules in Nicotiana benthamiana Protoplasts." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/14346358446864419971.
Повний текст джерела國立中興大學
生物科技學研究所
96
Bamboo mosaic virus (BaMV), a potexvirus, is a single-strand, positive-sense RNA virus which contains five open reading frames (ORFs) which encode proteins for replication, movement and structure and a caped 5''UTR and a 3''UTR with a poly A tail. There is good amount of information on the replication of potexviruses but information on the sub-cellular localization of the viral RNA and the site of replication is lacking. Though there are different methods to detect RNA localization by optical microscopy, most of them are limited to fixed samples. We employ a technique which exploits the ability of phage MS2 coat protein to bind specifically to only its cognate RNA. In this strategy, the MS2 RNA sequence is inserted into target RNA (BaMV genomic RNA) and MS2 coat protein is fused with green fluorescent protein (GFP) which contains a nuclear localization signal (NLS). When the modified target RNA and the GFP-NLS-CPMS2 construct are co-transformed into protoplasts the GFP-NLS-CPMS2 fusion protein binds to the target RNA and the green fluorescence signal indicates the localization of the target RNA. The unbound GFP-NLS-CPMS2 is retained in the nucleus due to the NLS thereby reducing noise. This method can be used for four dimensional study of RNA localization in live cells. First, the MS2 RNA sequence had to be inserted into BaMV RNA genome. According previous study, the PVX 3''UTR insertion construct and only subgnomic RNAs could accumulate normally. Simultaneously, the CPMS2-GFP fusion protein was constructed in pBI 121 vector. As BaMV.S.(MS2)6 was transfected in GFP-NLS-CPMS2 fusion transiently expressed protoplasts1 dpi, observation of GFP localization indicated the BaMV RNA subcellular localization. GFP signal was co-localized and moved with mobile mitochondria stained with Mitotracker OrangeCMTMRos. Employing this novel method we observed BaMV RNA localizing at the mitochondria and that a large proportion of the RNA would most probably be the subgenomic RNAs which also contains the MS2-hps since the mutant RNAs are defective in genomic RNA accumulation.
"Expression of Recombinant Zika Virus-Like Particles in Nicotiana benthamiana." Master's thesis, 2018. http://hdl.handle.net/2286/R.I.50512.
Повний текст джерелаDissertation/Thesis
Masters Thesis Molecular and Cellular Biology 2018
Guo, Shang-Ming, and 郭尚明. "Interactions between Cymbidium mosaic virus and Odontoglossum ringspot virus in Nicotiana benthamiana." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/e3x9c3.
Повний текст джерела國立臺灣大學
植物病理與微生物學研究所
105
Mixed infection of plant viruses usually leads to intrahost virus-virus interactions. Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CymMV) commonly co-infect orchid plants and cause more severe symptoms, which is defined as synergistic effect. Recently, we found that the synergistic effect between ORSV and CymMV did exist on Nicotiana benthamiana protoplasts. This interaction seems to be regulated by the silencing suppression activity of ORSV p126. In this study, we continued to explore the interactions between ORSV and CymMV on N. benthamiana. In addition to p126, transiently expressed ORSV capsid protein (CP) facilitated CymMV accumulation on the inoculated leaves of N. benthamiana, but ORSV movement protein did not. The mechanism under this phenomenon remains unknown. Individual domains of ORSV p126 were proved without RNA silencing suppression ability and could not improve CymMV accumulation. In this study, we constructed five different domain combination of p126 and found that all four domains are necessary for RNA silencing suppression. Surprisingly, viral RNA and CP accumulation of both ORSV and CymMV had no significant difference between singly and doubly inoculated leaves of N. benthamiana plants through agroinoculation. However, by means of sap inoculation, more severe symptoms on both inoculated and systemic leaves of doubly infected plants were observed compared to singly infected ones. Next, we detected the viruses in systemic leaves of ORSV and CymMV doubly infected plants by indirect-ELISA, and found that the systemic movement-deficient CymMV could systemically infect N. benthamiana. These results suggested that although mixed infection of ORSV and CymMV did not exhibit synergistic interaction on inoculated leaves, ORSV still facilitated CymMV in other mechanism, probably on movement. Interestingly, facilitation on CymMV systemic movement disappeared when CymMV was co-inoculated with systemic movement-deficient ORSV (ORSVE100), which suggested the systemic movement of CymMV may rely on ORSV CP or ORSV infection processes. To understand the specificity of ORSV-CymMV synergism, we co-expressed CymMV with some well-known RNA silencing suppressors (RSSs), e.g. Turnip mosaic virus (TuMV) HCPro, Cucumber mosaic virus 2b, Potato virus X (PVX) p25 and Tomato bushy stunt virus p19. Except for PVX p25, all RSSs could significantly increase the accumulation of CymMV, which indicated that p126-mediated enhancement of CymMV accumulation probably can be replaced by other RSSs. Furthermore, we were curious about whether CymMV can systemically infect N. benthamiana with the aid of other ORSV-related or ORSV-unrelated viruses. For mixed infection of TuMV+CymMV, and PVX+CymMV, about 57% and 50% infected plants showed systemic CymMV infection. Co-infection of CymMV and Tomato mild green mosaic virus (TMGMV), a tobamovirus, facilitated systemic movement of CymMV on all tested plants. Finally, we constructed three eGFP-expressing CymMV clones and used one of them to confirm the experimental results.
McLachlan, Juanita. "A Study of Nicotiana Benthamiana Protein Interactions with Tomato Bushy Stunt Virus." Thesis, 2013. http://hdl.handle.net/1969.1/149490.
Повний текст джерелаLi, Jin-Guei, and 黎金桂. "Establishment of Transgenic Nicotiana benthamiana Plants Expressing the Bamboo Mosaic Virus Replicons." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/66779453434479418175.
Повний текст джерела國立中興大學
農業生物科技學研究所
89
Bamboo mosaic virus (BaMV) is a flexuous rod-shaped plant virus with a positive-sense RNA, about 6366 nucleotides in length (excluding the poly(A) tail). BaMV have five conserved open reading frames (ORF). The three overlapping ORFs (ORF 2, 3, and 4), known as the triple gene block (TGB), which encode proteins of 28, 13, 6 kDa, respectively. ORF 5 encodes 25 kDa viral capsid protein (CP). The TGB proteins and the coat protein are thought to play roles in virus movement between plant cells. Some BaMV isolates contain a satellite RNA (satBaMV) which is 836 nucleotide long (excluding poly(A)) and contains an ORF for a protein of 20 kDa (P20). Whether the satBaMV uses the same movement machinery as BaMV in plant is not known yet. Coinoculation of BaMV defective in TGB or CP with satBaMV into Nicotiana benthamiana protoplasts had found that both TGB- and CP-defective BaMV could support satBaMV replication. The purpose of this study is to produce the transgenic Nicotiana benthamiana that express the replicative BaMV RNA defective in TGB or CP. Complementary DNAs of defective BaMV RNA that has deletion of TGB and CP gene were constructed in a plant expression vector pKyLx7 and transferred to N. benthamiana by Agrobacterium-mediated transformation. Transgenic plants were selected in kanamycin-containing medium and those expressing defective BaMV RNAs were further identified by the PCR, Southern, Northern and Western blot analyses. The stability of transgene maintained in the F1 progenies was screened in kanamycin-containing medium. The ratio of kanamycin resistance genotype of F1 progenies was 3/4, which followed the Mendelian law. We had selected 15 homozygous transgenic N. benthamiana lines expressing the BaMV dT and 3 homozygous transgenic N. benthamiana lines expressing the BaMV dC. After inoculation with satBaMV, we found that transgenic Nicotiana benthamiana plant expressing the defective BaMV RNA that has deletion of TGBps could not support the movement of satBaMV. This result indicates that BaMV movement protein genes are required for satBaMV movement.
Hsueh, Chia-Hsin, and 薛家欣. "The effect of NbRbohB on Bamboo mosaic virus replication in Nicotiana benthamiana." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/05121950829248358246.
Повний текст джерела國立中興大學
生物科技學研究所
104
Respiratory burst oxidase homologs (Rboh), alias NADPH oxidase, plays a critical role in reactive oxygen species (ROS) generation. In Nicotiana benthamiana, there are two kinds of Rboh, NbRbohA and NbRbohB. NbRbohB, induced by specific pathogen signals, participates in mechanisms for plant disease resistance. Bamboo mosaic virus (BaMV), a member of the Potexvirus genus, contains a single-stranded positive-sense RNA genome. A previous proteomic approach identified a potential interaction between NbRbohB and BaMV replicase. To explore the relationship between NbRbohB and BaMV replication, NbRbohB was down-regulated by the Tobacco rattle virus (TRV)-induced gene silencing method and the silencing plant was subsequently inoculated with the recombinant BaMV virion that carries green fluorescent protein gene (GFP) in this study. Western blot analysis, revealed that the accumulations of GFP and BaMV CP were reduced significantly in NbRbohB-silenced plants. Northern blot and RT-PCR also demonstrated that the accumulation of BaMV genomic RNA was decreased when NbRbohB was silenced. The same VIGS method was used to analyze the effects of NbRbohB on Foxtail mosaic virus (FoMV) and Potato virus X (PVX) replication. Silencing NbRbohB reduced the accumulation of FoMV CP but not PVX CP. According to public EST databases, the full-length cDNAs of NbRbohB1 and NbRbohB2 were acquired, and they were inserted into the expression vector pBI221. Overexpression of NbRbohB1 in BaMV-infected protoplasts significantly increased the accumulation of BaMV CP. In conclusion, NbRbohB1 plays a significant role in promoting BaMV and FoMV replication.
Odokonyero, Denis 1984. "Identification of ARGONAUTES Involved in Antiviral RNA Silencing in Nicotiana benthamiana." Thesis, 2012. http://hdl.handle.net/1969.1/148228.
Повний текст джерелаAi, Wei-Ping, and 艾瑋苹. "The effect of isocitrate dehydrogenase on Bamboo mosaic virus replication in Nicotiana benthamiana." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/04832184388759104405.
Повний текст джерела國立中興大學
生物科技學研究所
103
Viruses recruit many of the host cell factors to complete infection cycle. Recently, many studies have committed to explore the relationship between the virus and its host factors. Bamboo mosaic virus ( (BaMV)) , belonging to the genus Potexvirus, is a single-stranded 6.4-kb positive sense RNA virus.. In our lab, BaMV relpicase complex was prepared from Nicotiana benthamiana that had been Agroinfiltrated agroinfiltrated with BaMV replicase-expression cassette, followed by partial purification using immunoprecipitation. Several speculative host factors were subsequently identified by LC-MS/MS, including AtClpC, pleiotropic drug resistance like protein, scarecrow-like protein 5, isocitrate dehydrogenase ( (NbICDH)) and Calcium-transporting ATPase 4. It was found that when NbICDH was down-regulated by virus-induced gene silence silencing ( (VIGS)) , the accumulation of BaMV coat protein was increased. NbICDH Overexpression overexpression NbICDH in protoplasts, decreased the accumulation of BaMV coat protein was decreased. The result suggests that NbICDH may have antiviral function in N. benthamiana. The catalytic site residue Y205 of NbICDH were was replaced by alanine by mutagenesis to examine the involvement of the enzyme’s catalytic activity in BaMV replication. Overexpression of the mutant proteins in protoplasts showed the same inhibition effect on the accumulation of BaMV coat protein as the wild-type NbICDH. This result suggests that the catalytic activity of NbICDH is not required for decreasing the viral replication. In the future, the regulation of BaMV replication by NbICDH should be demonstrated.
Chuang, Chi-Mau, and 莊棨貿. "Effects of a putative methyltransferase from Nicotiana benthamiana on Bamboo mosaic virus replication." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/94415236392530050905.
Повний текст джерела國立中興大學
生物科技學研究所
96
After entering host cells, virus requires host factors for replication and hosts might generate defensive mechanisms. Bamboo mosaic virus (BaMV) is a flexuous-rod positive-sense RNA virus belonging to the genus Potexvirus of the family Flexiviridae. BaMV genome contains 5 open reading frames (ORFs). ORF1 encodes a 155-kDa viral replicase, which possesses an RNA-dependent RNA polymerase (RdRp) activity at the C terminus. In previous study, a putative Nicotiana benthamiana methyltransferase (NbMts) was found to interact with BaMV RdRp domain in a yeast two-hybrid screening against a leaf cDNA library of N. benthamiana. According to predictions on websites, NbMts has a signal peptide at N terminus for membrane targeting and motifs for AdoMet binding. In this study, we used protoplast transformation to study the relation of NbMts to the viral replication. To express NbMts under the control of 35S promoter, the corresponding cDNA was inserted in pBI221 to become pBI-NbMts. Protoplasts were transformed with BaMV infectious cDNA (pCBG) and pBI221 (or pBI-NbMts). After cultivation for indicated periods of time, accumulations of the viral coat protein were analyzed by Western blot. The results indicate that NbMts overexpression inhibits BaMV replication in protoplasts, and the BaMV-inhibition effect was dosage dependent. The BaMV-inhibition effect was most obvious at 16~24 h post cotransfection of pCBG and pBI-NbMts. Deletion of the signal peptide or mutations at the putative AdoMet-binding motifs abolish the BaMV-inhibition effects. Addition of AdoMet in the incubation medium enhanced the repression. NbMts overexpression also inhibited the replication of Foxtail mosaic virus, a member of Potexvirus, but not Tobacco mosaic virus in protoplasts.
Wu, Li-Chin, and 吳麗琴. "Proteomic Analysis of Differential Protein Expression of Bamboo mosaic virus Infected Nicotiana benthamiana." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/89582669429601137127.
Повний текст джерела國立中興大學
生物科技學研究所
95
Bamboo mosaic virus (BaMV), is a single-stranded positive-sense RNA virus with flexuous rod-shaped morphology. A number of studies have been devoted to analyze the replication of plus-stranded RNA viruses; several host proteins are known to be involved in assembling the viral RNA replication complex, activating the complex for RNA synthesis, and other steps. However, the host has the defense system to against viral replication and spreading which are also mediated through proteins. In this study, we have tried to identify the differentially expressed proteins between mock and BaMV inoculated N. benthamiana leaves by proteomics approach. Since positive-sense RNA virus replication is usually associated with rearrangements of membranous organelles, we first isolated the membrane fraction (P30) of the N. benthamiana leaves. Proteins associated with P30 fraction were resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE) and identified by MALDI-TOF MS. Comparison of protein patterns from P30 fractions of mock and BaMV-infected N. benthamiana in 2-D gels revealed several differential expressed proteins. The differential expression of ribosomal protein L25, RubisCO small chain, calmodulin-1 and glycoprotein endopeptidase-like protein. Among these proteins, ribosomal protein L25 was only found in P30 fractions sample from BaMV-infected plants. It had been reported that ribosomal protein L25 is homologous to general stress proteins CTC. We utilized TRV- based virus-induced gene silencing (VIGS) system to generate L25-knockdown plants and to investigate the effect of this protein on BaMV accumulation. Real-time PCR results showed that the levels of the L25 mRNA in the various L25 knock-down plant were reduced to about 25 and 40 % to those of the control plants. Western blots and northern blots were used to analyze the accumulation of viral coat protein and viral RNA at seven day post-inoculation of BaMV virions. Results showed that the accumulation of BaMV coat protein and viral RNA in L25-knockdown plant were reduced. But no interference on the accumulation of FoMV coat protein was observed in all L25-knockdown plants. Together, these data suggest that L25 likely plays an important role in the BaMV accumulation. Keywords:Bamboo mosaic virus / host factors / proteomic
Wang, Ssu-yuan, and 王斯遠. "The effect of NbSCL6 on Bamboo mosaic virus infection cycle in Nicotiana benthamiana." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/67005081453115320570.
Повний текст джерела國立中興大學
生物科技學研究所
102
Bamboo mosaic virus ( BaMV) is a single-stranded positive-sense RNA virus with a 5¢cap and a 3¢ poly(A) tail. BaMV belongs to the genus Potexvirus and the family Flexiviridae. The gene expression profile in Nicotiana benthamiana may be altered after BaMV infection. To identify the possible host genes involving the infection cycle of BaMV, our lab used cDNA-AFLP technique to screen the differentially expressed genes in BaMV-inoculated N. benthamiana plants. ACTC7-1 is an upregulated gene when BaMV infects N. benthamiana. To characterize the function of ACTC7-1 involving in BaMV infection cycle, I used the virus-induced gene silencing (VIGS) technique to known down the expression of ACTC7-1 leveling N. benthamiana plant and then inoculated BaMV onto the knockdown leaves. The accumulation levels of BaMV coat protein was determined by Western blotting analysis. The accumulation of BaMV was enhanced when after the expression of ACTC7-1 was knocked down. To further analyze the effect of ACTC7-1 on BaMV infection is on viral RNA replication or virus movement, I infected BaMV RNA into the ACTC7-1-knockdown protoplasts which were derived from the knockdown plants. The result of the accumulation of BaMV coat protein in N. benthamiana protoplasts was also increased. Overall of these preliminary results suggest that ACTC7-1 is probably involved in the replication of BaMV. Furthermore, I used RACE technique to clone the full-length of ACTC7-1 and blasted the sequence to WORKBENCH. The identity of ACTC7-1 could be the SCARECROW-LIKE transcriotion factor in GRAS family. The future work of this research will focus on the transient expression the full-length of ACTC7-1 fused with Orange fluorescent protein in N. benthamiana to localize this protein and examine the effect on BaMV accumulation
Chien, Wan-Chu, and 簡婉竹. "Generation of transgenic Nicotiana benthamiana plants conferring resistance against Cucurbit chlorotic yellows virus." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/eghd44.
Повний текст джерелаCheng, Chun-Wei, and 程鈞煒. "The effect of Bamboo mosaic virus accumulation by a putative methyltransferase in Nicotiana benthamiana." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/59291778919213580102.
Повний текст джерела國立中興大學
生物科技學研究所
99
Abstract Bamboo mosaic virus (BaMV), a positive-sense RNA virus with the length of 6.4-kb, contains five ORFs. ORF1 of the BaMV encodes a 155-kDa replicase consisting of mRNA capping enzyme domain, helicase-like domain, and RNA-dependent RNA polymerase (RdRp) domain. In the previous study, the interaction between the BaMV RdRp domain and a host putative methyltransferase (PMtsNb1) was identified from a yeast two-hybrid screening against a leaf cDNA library of Nicotiana benthamiana. Over expression of PMtsNb1 in N. benthamiana protoplasts by CaMV 35S promoter significantly decreased the viral coat protein accumulation by 40% and the viral in vitro RdRp activity also by 50%. To assure that these inhibitions were related to the interaction of PMtsNb1 and RdRp, the PNbMts1 fused with GFP and HA-tagged RdRp were co-expressed in protoplast. In this experiment, the HA tagged RdRp could be recognized in the immunoprecipitation of PNbMts1-GFP fusion protein. In addition, mutations at the putative AdoMet-binding motifs abolished the BaMV-inhibition effect. Furthermore, addition of AdoMet in the incubation medium of protoplast enhanced the inhibition effect of PNbMts1. Besides, the accumulation of viral coat protein was less in PNbMts1-overexpressing N. benthamiana than in the wild type plant. In contrast, knock-down of PNbMts1 by virus-induced gene silencing in N. benthamiana increased the accumulation of the viral coat protein about four to six times. In summary, we have identified a novel virus-resistant protein, PNbMts1, which can inhibit the accumulation of BaMV.
Valenzuela, Aguila Sofia [Verfasser]. "Transformation of Nicotiana benthamiana with different BWYV (Beet western yellows virus) sequences to test for virus resistance = Transformation von Nicotiana benthamiana mit verschiedenen Sequenzen des BWYV (Beet western yellows virus) zur Virus-Resistenztestung / von Sofia Valenzuela Aguila." 2000. http://d-nb.info/959528695/34.
Повний текст джерелаChen, Xiang-Yu, and 陳相伃. "The study of ferredoxin-NADP+ oxidoreductase involved in Bamboo mosaic virus replication in Nicotiana benthamiana." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/75888843185615854959.
Повний текст джерела國立中興大學
生物科技學研究所
105
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Alphaflexiviridae. We used cDNA-AFLP technique to isolate the host gene fragment which had differential expression after BaMV infection in tobacco plant (Nicotiana benthamiana). One of downregulated gene fragments, ACAG1, is sharing high identity to the non-specific ferredoxin NADP+ reductase sequence in the database; we then designated this gene NbFNR. It is a flavoenzyme that involved in the process of photosynthesis electron transport chain, catalyzes NADP+ into NADPH reaction. In order to investigate whether NbFNR affects the infection cycle of BaMV, we used virus-induced gene silencing (VIGS) technique to reduce the expression level of NbFNR gene in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and viral RNA was significantly lower than that of the control group. Further experiment of transiently expressed NbFNR fused with T7-tag or Orange fluorescent protein (OFP) in plants that could elevate the accumulation of BaMV coat protein. The localization of NbFNR-OFP was observed at chloroplast as expected by confocal microscopy. Overall of these results suggest that NbFNR localized at the chloroplast plays a positive role in BaMV replication.
Liu, Hsin-Yi, and 劉欣宜. "The study of gibberellic acid insensitive involved in Bamboo mosaic virus replication in Nicotiana benthamiana." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/88268758104731508985.
Повний текст джерела國立中興大學
生物科技學研究所
105
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus, a member of Potexvirus genus of Alphaflexiviridae family. In a previous study, 90 differentially expressed genes were identified from BaMV-inoculated N. benthamiana plants using the technique cDNA-amplified fragment length polymorphism (cDNA-AFLP). One of the differentially expressed genes ACCT7-1 was upregulated in BaMV-inoculated N. benthamiana plants. Furthermore, the full-length cDNA was cloned using rapid amplification of cDNA ends (RACE) and sequence. The identity of ACCT7-1 was revealed as a homolog of GA-INSENSITIVE (GAI), a member of DELLA family, when compared to the databases of National Center for Biology Information (NCBI). Therefore, ACCT7-1 is then designated as NbGAI. NbGAI was characterized by BaMV inoculated on virus-induced gene silencing (VIGS) leaves and protoplasts. The results showed that the accumulation of BaMV coat protein was decreased significantly in both knockdown leaves and protoplasts compared to the controls. These results can be inferred that NbGAI may play a positive role in assisting the virus replication. Furthermore, the fusion protein NbGAI with Orange fluorescent protein (OFP), NbGAI-OFP is expressed in plants and BaMV is then inoculated. The accumulation of BaMV coat protein is increased significantly compared with the control plants with the expression of OFP only. The results indicate that NbGAI play a role in assisting BaMV accumulation. NbGAI is a member of DELLA family and contains a nuclear localization signal (NLS). In this study, I have used confocal microscopy to confirm the nuclear localization of NbGAI in cell. In addition, the NLS of NbGAI is predicted by screening online software: cNLS Mapper. These results indicated that NbGAI could play an assisting role in BaMV replication.
Lin, Jhe-Wei, and 林哲緯. "Investigating the relation between NbXRN4 and replication efficiency of Bamboo mosaic virus in Nicotiana benthamiana." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/55638555805311058677.
Повний текст джерела國立中興大學
生物科技學研究所
103
Bamboo mosaic virus (BaMV) is a 6.4-Kb positive-sense RNA virus. It depends on the host factors for replication and movement. Nicotiana benthamiana infiltrated with Agrobacterium that carries the viral polymerase-coding region was used to produce BaMV replication complex, which was then isolated by Immunoprecipitation using specific antibodies. Twenty-two hypothetical host factors in the complex were identified by LC tandem mass spectrometry. The expression of the potential factors in N. benthamiana were downregulated by the TRV-induced gene silencing method and the silenced plant was subsequently inoculated with the recombinant BaMV virion that carries GFP gene to assess the effects of the factors on BaMV replication. Among the potential factors, silence of NbXRN4, confirmed by the RT-PCR method, significantly reduced the expression of GFP, as evidenced by the green fluorescent spots, on the leaves that had been inoculated with the recombinant BaMV. The expression of GFP in the inoculated leaves was examined by Western blot, and the result confirmed that the accumulation of GFP was decreased in the NbXRN4-silenced plant. Northern blot also indicated that the accumulation of genomic and subgenomic RNAs of BaMV was decreased in NbXRN4-silenced plant. The putative nucleotide sequence of NbXRN4 cDNA was assembled according to the various EST data. In order to obtain the cDNA in full length, specified primers were designed to amplify the gene and put it into pBI221 expression vector. Overexpression of NbXRN4 in N. benthamiana protoplasts significantly increased the viral coat protein accumulation. Next, we expect to make sure the co-localization of the BaMV RdRP and NbXRN4 in plant cell.
Lin, Yan-Cheng, and 林彥丞. "The study of NbGAI involved in the accumulation of Bamboo mosaic virus in Nicotiana benthamiana." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/11635732051930245340.
Повний текст джерела國立中興大學
生物科技學研究所
103
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Flexiviridae. The RNA genome comprises 6366 nts with a 5 cap and a 3 poly (A) tail. In general, the expression profile of host genes could be altered when infected by viral pathogens. These differentially expressed genes might play positive or negative roles in regulating BaMV infection cycle. In a previous study, our lab isolated 90 differentially expressed genes from BaMV-inoculated N. benthamiana plants using cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. One of the upregulated genes post BaMV infection ACCT7-1 was found to involve in BaMV replication since the accumulation of BaMV coat protein was reduced when the expression of ACCT7-1 was knocked down by virus-induced gene silencing (VIGS) in the inoculated leaves and protoplasts. Furthermore, the full-length cDNA was cloned using rapid amplification of cDNA ends technique and sequenced. The identity of ACCT7-1 was revealed as a homolog of GA-INSENSITIVE (GAI), a member of DELLA family, when compared to the databases of National Center for Biology Information (NCBI). Therefore, we then designated this gene as NbGAI. To localize NbGAI in cell, I subcloned NbGAI into pEpyon vector with which can produce the fusion protein of NbGAI with Orange fluorescent protein (OFP) when expressed in plant cells. Under the circumstance of the fusion protein expression, the accumulation of BaMV coat protein is increased compared to the control plants with the expression of OFP only. The results indicate that NbGAI plays a positive role in assisting BaMV accumulation. Since NbGAI is one of the DELLA proteins that regulate plant hormones including gibberellin (GA) and Jasmonate (JA), the involvement of regulating in BaMV replication could be through the JA pathway. In applying JA or GA in plants and followed BaMV inoculation revealed that JA has either no effect or negative on the accumulation of BaMV, whereas the GA plays a positive role on that of BaMV. These results are conflict on the effect of NbGAI in BaMV. Therefore, we conclude that NbGAI, although it is a DELLA protein, may not go through the signaling pathway.
Heish, Cheng-Kai, and 謝丞凱. "The study of NbRTE1 from Nicotiana benthamiana involved in the replication of Bamboo mosaic virus." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/13049716685092917346.
Повний текст джерела國立中興大學
生物科技學研究所
104
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus which belongs to the Potexvirus of alphaflexiviridae. We used cDNA-amplified fragment length polymorphism technique to screen the differentially expressed genes of Nicotiana benthamiana post BaMV inoculation. One of downregulated genes, ACTG7-1, sharing 59% identify to REVERSION-TO-ETHYLENE SENSITIVITY (RTE1) of Arabidopsis was further investigated. We used virus-induced gene silencing (VIGS) technique to knock down the expression of RTE1 and investigate the involvement of NbRTE1 in BaMV infection cycle. The results derived from previous studies revealed that NbRTE1 could negatively regulate the replication of BaMV. My research goals are aiming at the localization of NbRTE1 in cell by using confocal microscopy and clarifying whether the ethylene response pathway is involved in BaMV infection. The RTE1 in Arabidopsis was reported to associate with endoplasmic reticulum (ER), accordingly the localization of NbRTE1 on the ER needs to be confirmed. Because RTE1 is one of the components in the ethylene response pathway and is involved in BaMV replication in N. benthamiana, we would like to examine more components in the pathway and inspect if they are also involved in BaMV infection. NbETR1, the ethylene receptor, specifically interacts with RTE1 to transduce the signaling. The results derived from VIGS indicated that the expression level of ETR1 was decreased to 18% that of the control plants, the accumulation of BaMV coat protein was reduced to 75%. To further inspect if ethylene is involve in the BaMV infection cycle, we have treated the protoplasts with the ethylene inhibitor to block the signaling pathway. The results revealed no significant difference of the BaMV accumulation. Overall, the results suggest that NbRTE1 involved in BaMV replication might not go through the ethylene signaling pathway. The mechanism of NbRTE1 involved in BaMV replication is still need to be clarified
Chiang, Wen-Shan, and 江雯珊. "The study of NbDXR from Nicotiana benthamiana involved in the replication of Bamboo mosaic virus." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/38369194472158216201.
Повний текст джерела國立中興大學
生物科技學研究所
104
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus, belonging to the Potexvirus genus of Alphaflexiviridae. To reveal the relationship of host genes involved in BaMV infection cycle, our lab used cDNA-amplified fragment length polymorphism (cDNA-AFLP) to screen the differentially expressed genes from Nicotiana benthamiana after infection. One of the upregulate genes is ACGT8-2, was identified as 1-deoxy-ᴅ-xylulose-5-phosphate reductoisomerase (DXR) in our previous study. The full-length of this gene was cloned and designated as NbDXR. The enzyme is involved in the first committed step of the MEP pathway that converted 1-deoxy-ᴅ-xylulose-5-phosphate (DXP or DOXP) to 2-C-methyl-ᴅ-erythritol 4-phosphate (MEP) for isoprenoid biosynthesis. To further characterize the role of NbDXR in BaMV infection cycle, we used the virus induced gene silencing (VIGS) technology to knock down the expression of DXR in N. benthamiana. The accumulation of BaMV coat protein in both NbDXR-knockdown plants and protoplasts were shown lower than those of the control. These results are implied that NbDXR may play a role in assisting the replication step of BaMV infection cycle. We then further transiently expressed the T7-tag fused NbDXR in plant and shown to have 1.8 folds enhancement of the accumulation of BaMV coat protein compared to that of the control (expressed OFP-T7). Furthermore, the OFP-fused NbDXR was transiently expressed in N. benthamiana and revealed chloroplast localization by confocal microscopy. These results indicated that NbDXR could play an assisting role in BaMV replication. The upstream and downstream genes of DXR in the MEP pathway will be characterized in the future.
Yu, Jong-Ding, and 余中鼎. "The study of Argonautes in Nicotiana benthamiana involved in the infection of Bamboo mosaic virus." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/16142441951239615532.
Повний текст джерела國立中興大學
生物科技學研究所
104
Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Alphaflexiviridae. The genome of BaMV is about 6.4 kb with a 5?cap and a 3?poly (A) tail. In this study we use Nicotiana benthamiana as a model plant to inspect the relationship between Argonautes (AGOs) and BaMV. RNA interference (RNAi) in Eukaryotes is known to play an antiviral defense role especially the RNA viruses. During the replication of viral RNAs, the double-stranded (ds) RNA accumulates and triggers the host silencing process. DICER-LIKE (DCL) proteins cleave the viral dsRNA into short interfering RNAs (siRNAs) of 21~24 nucleotides in length. These siRNAs are loaded into AGOs to form the RNA induced silencing complex (RISC). The RISC contains the specific siRNAs and targets to the viral RNAs. There are 10 AGOs identified in Arabidopsis thaliana. By contrast, only four AGOs (AGO1, AGO2, AGO4, and AGO5) were sequenced in N. benthamiana. In this thesis, I used Tobacco rattle virus (TRV)-based gene silencing technique to knock down the expression of different AGOs in N. benthamiana to reveal the relationship between AGOs and BaMV. The results indicated that reducing the expression of AGO4 did not have any effect on the accumulation of BaMV. The results also imply that AGO4 may not play a key role against BaMV in N. benthamiana. By contrast, the reduction of the expression of AGO1 could result in an increase of the accumulation of BaMV in plants and protoplasts. Furthermore, but the accumulation of BaMV has no significant change in AGO2-silencing plants. Moreover, the expression of AGO2 is increased in AGO1-knockdown plants. The results suggest that AGO2 might be controlled by AGO1 as that revealed in Arabidopsis. The relationship between AGO1 and AGO2 in N. benthamiana needs to be further explored.
Wu, Yi-Jhen, and 吳宜臻. "The effect of MAP kinase phosphatase 1 on Bamboo mosaic virus replication in Nicotiana benthamiana." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/41201104240517043841.
Повний текст джерела國立中興大學
生物科技學研究所
104
MAP kinase phosphatase 1 (MKP1) in Nicotiana benthamiana has 859 amino acid residues. In Arabidopsis thaliana AtMKP1 interacts with MAP kinase 3, 4, and 6 (MPK3, 4, and 6). The interaction compromises the ability of MPK3 and 6 to provoke the pathogen-defending function and salicylic acid production. Recently, NbMKP1 was found to interact with the replication protein of Bamboo mosaic virus (BaMV) through a proteomic approach. In this study, the virus-induced gene silencing (VIGS) method was used to reduce the expression of NbMKP1, and the silencing plant was inoculated with BaMV-GFP virions. The accumulations of BaMV coat protein (CP) and GFP were both enhanced due to the decreased expression of NbMKP1. BaMV genomic RNA was also increased in the same condition. In protoplasts, overexpression of NbMKP1 reduced the accumulation level of BaMV CP. These data suggest that NbMKP1 can inhibit the proliferation of BaMV in N. benthamiana. In addition, the effect of NbMKP1 on accumulations of Foxtail mosaic virus (FoMV) and Potato virus X (PVX) was examined. Decreasing NbMKP1 led to higher expression of FoMV but lower expression of PVX. The inactivated NbMKP1 was expressed in protoplasts to understand if the phosphate-removing activity is critical to the protein’s inhibitory effect on BaMV accumulation. Overexpression of the inactivated NbMKP1 actually accumulated even more BaMV CP than the negative control, suggesting that the catalytic function of NbMKP1 is critical on this matter.
Chiu, Ling-Ying, and 邱齡瑩. "The study of NbLTP1 from Nicotiana benthamiana involved in the replication of Bamboo mosaic virus." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/65508777785579651265.
Повний текст джерела國立中興大學
生物科技學研究所
102
Bamboo mosaic virus (BaMV) is a single-stranded positive sense RNA virus belonging to genus Potexvirus of the family Flexiviridae. The objective of this study is to understand the relationship of the host proteins involved in the replication mechanism of BaMV. The results derived from a previous study, a downregulated gene fragment ACGT12 from Nicotiana benthamiana post BaMV infection identified by cDNA-AFLP technique is further characterized. We used the Tobacco rattle virus (TRV)-based silencing system (virus-induced gene silencing; VIGS) to knock down the expression of ACGT12 and infected the BaMV on the knockdown plants. The results indicate that the accumulation levels of BaMV coat protein in the knockdown plants are lower than that in the control plants. Similar results are observed in the knockdown protoplasts that less coat protein accumulated than that in the control protoplasts. These results suggest that ACGT12 may be involved in the replication process rather than in viral movement. The full-length cDNA of ACGT12 is obtained by rapid amplification of cDNA ends (RACE) technique. The sequence of ACGT12 is blasted to the Biology WorkBench database and matches to that of non-specific lipid transfer protein 1 (nsLTP1) and designated as NbLTP1. Furthermore, the cellular localization of NbLTP1-OFP (fused with Orange fluorescence protein) is mainly associated with the extracellular matrix. However, when the signal peptide is removed, the majority of the expressed mutant protein is associated with chloroplasts. The accumulation of BaMV coat protein is enhanced when NbLTP1 is transiently expressed in plants. Overall, the results indicate that the newly identified host protein NbLTP1 is a positive regulator for the replication of BaMV RNA. In addition to the hydrophobic pocket to accommodate the lipid, NbLTP1 has a conserved calmodulin (CaM)-binding site at the very C-terminus. Therefore, the lipid binding and the CaM-binding properties involving in the replication of BaMV is needed to be further characterized.