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RNA silencing suppression activity of the proteins encoded by the brome mosaic virus RNA genome

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dc.contributor.advisor Bujarski, Jozef J. en_US Weber, Philipp Heinrich en_US 2017-08-02T21:16:20Z 2017-08-02T21:16:20Z 2014
dc.description Advisors: Jozef J. Bujarski. en_US
dc.description Committee members: Kenneth Gasser; Gabriel Holbrook. en_US
dc.description.abstract The purpose of this study was to investigate the RNA silencing suppression activity of the proteins expressed by the Brome mosaic virus (BMV). For this purpose a binary, pCambia-based vector containing a GFP (Green Fluorescence Protein) gene was introduced into leaves of Nicotiana benthamiana (N. benthamiana) by agroinfiltration. The GFP gene gets transiently expressed, visualized by fluorescence activity under UV-light, and partially silenced by the RNA silencing activity (RNAi) of N. benthamiana, as described before. The RNAi-based silencing in turn can be suppressed by a co-infiltrated and also transiently expressed vector, which carries a RNA silencing suppressor-gene. The suppression of RNA silencing can be visualized by a higher intensity of fluorescence under UV-light. Two pROK2-based binary vectors, expressing the open reading frames (ORF) of either only the movement protein (MP) 3a or the coat protein (CP) were constructed. The expression of the genes and therefore the presence of the correspondent proteins in the plant leaves was verified by western blot analysis. The T-DNA constructs expressing the two other BMV proteins 1a (BMV RNA 1) and 2a (BMV RNA 2) were received from Prof. Kao. The vector encoding the GFP-gene, as well as a construct harboring the 2b-gene, used as positive control, was received from Dr. Canto. Every plasmid was introduced separately into Agrobacterium tumefaciens via electroporation. Subsequently, the agrobacteria suspensions were agroinfiltrated into N. benthamiana. The agrobacteria carrying the gene to be tested were always coinfiltrated with agrobacteria carrying the GFP-gene as a reporter gene. After three days, the RNA silencing suppression activity of the transiently expressed gene was determined by the comparison of the intensity of fluorescence under UV-light against a positive respective negative control. All proteins encoded by the BMV RNA genome displayed the same intensity of fluorescence as the negative control. The positive control always showed a much higher intensity of fluorescence than any of the proteins to be tested for RNA silencing suppression activity. The results indicate that none of the BMV proteins has local RNA silencing suppression activity. en_US
dc.format.extent 86 pages en_US
dc.language.iso eng en_US
dc.publisher Northern Illinois University en_US
dc.rights NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors. en_US
dc.subject.lcsh Bromoviridae--Morphology en_US
dc.subject.lcsh RNA viruses--Morphology en_US
dc.subject.lcsh Plant viruses--Morphology en_US
dc.subject.lcsh Viral genomes en_US
dc.subject.lcsh Virology en_US
dc.subject.lcsh Genetics en_US
dc.title RNA silencing suppression activity of the proteins encoded by the brome mosaic virus RNA genome en_US
dc.type Text en_US
dc.contributor.department Department of Biological Sciences en_US M.S. (Master of Science) en_US

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