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Identification of crucial residues or domain(s) of rice stripe virus NS3 protein required for self-interaction and for silencing suppressor activity
DC Field | Value | Language |
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dc.contributor.advisor | 김국형 | - |
dc.contributor.author | 김한길 | - |
dc.date.accessioned | 2017-07-14T06:45:48Z | - |
dc.date.available | 2017-07-14T06:45:48Z | - |
dc.date.issued | 2016-02 | - |
dc.identifier.other | 000000132612 | - |
dc.identifier.uri | https://hdl.handle.net/10371/125933 | - |
dc.description | 학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부 식물미생물학전공, 2016. 2. 김국형. | - |
dc.description.abstract | Rice stripe virus (RSV), a member of the genus Tenuivirus, is one of the most harmful virus in rice cultivation. The genome of RSV consists of four single-stranded RNAs and encodes seven viral proteins. NS3, which is encoded from the RNA segment 3, was previously reported as a gene silencing suppressor for RNA silencing and self-interaction of NS3 is necessary for maintaining suppressor activity. To identify the crucial amino acid residues or domains(s) required for self-interaction of NS3, I used protein structure prediction program and constructed eleven NS3 mutant clones including four alpha-helix deletion and substitution mutants. With these mutated clones, yeast-two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were conducted for interaction study. Y2H and BiFC results showed that the N-terminal region of NS3 is essential for self-interaction. All of alpha-helix deletion mutants and substitution mutants lost its self-interaction ability in Y2H and BiFC assays. To identify the relationship between NS3 self-interaction and silencing suppressor activity, we used GFP silencing system in Nicotiana benthamiana with Agrobacterium-mediated transient overexpression of each mutated NS3 protein. All the deletion and four alpha-helix substitution mutations resulted in the loss of the silencing suppressor ability except lysine77 substitution mutation which maintained self-interaction capacity. Altogether, these results suggest that the NS3-NS3 self-interaction might be necessary for maintaining suppressor activity as a counter defense to RNA silencing system of the host plant. | - |
dc.description.tableofcontents | INTRODUCTION 1
MATERIALS AND METHODS 4 1. NS3 three-dimensional (3D) structure prediction 4 2. Construction of RSV NS3 mutants 4 3. Vector construction and cloning 9 4. Yeast two hybrid (Y2H) analysis 9 5. Bimolecular fluorescence complementation (BiFC) assay 10 6. RNA silencing suppression analysis 10 RESULTS 12 1. 3D structure prediction of RSV NS3 protein 12 2. Generation of NS3-mutants 12 3. Y2H analysis for identification of NS3 self-interaction site 17 4. BiFC assay for localization of NS3 self-interaction in planta 20 5. Identification of crucial motif for NS3 self-interaction in Y2H 22 6. Identification of crucial motifs for NS3 self-interaction in planta 26 7. RNA silencing suppression analysis 29 DISCUSSION 34 LITERATURE CITED 38 ABSTRACT IN KOREAN 43 | - |
dc.format | application/pdf | - |
dc.format.extent | 2337532 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Rice stripe virus | - |
dc.subject | Protein 3D structure prediction | - |
dc.subject | RNA silencing | - |
dc.subject | Viral Suppressor of RNA silencing (VSR) | - |
dc.subject | Protein-protein interaction (PPI) | - |
dc.subject | GFP silencing | - |
dc.subject.ddc | 630 | - |
dc.title | Identification of crucial residues or domain(s) of rice stripe virus NS3 protein required for self-interaction and for silencing suppressor activity | - |
dc.type | Thesis | - |
dc.description.degree | Master | - |
dc.citation.pages | vi, 44 | - |
dc.contributor.affiliation | 농업생명과학대학 농생명공학부 | - |
dc.date.awarded | 2016-02 | - |
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