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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭秋萍(Chiu-Ping Cheng) | |
| dc.contributor.author | Yu Wu | en |
| dc.contributor.author | 吳宇 | zh_TW |
| dc.date.accessioned | 2021-06-15T16:08:24Z | - |
| dc.date.available | 2019-08-25 | |
| dc.date.copyright | 2015-08-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52129 | - |
| dc.description.abstract | 病毒需要藉助其宿主的蛋白進行複製，然而目前所知參與病毒複製的宿主蛋白仍相當有限。本研究室利用strepto tag方法選取了一些與馬鈴薯X病毒屬的單股正極RNA病毒──竹嵌紋病毒(Bamboo mosaic virus, BaMV)──及其衛星核酸(satBaMV)之5’或是3’端非轉譯區結合的宿主候選蛋白。本研究測試作用於糖解作用的兩種蛋白質──烯醇化酶(enolase, ENL)與磷酸甘油酸變位酶(phosphoglycerate mutase, PGM)對BaMV複製的影響。將這兩種蛋白的部分內生基因以菸草脆裂病毒(Tobacco rattle virus)為載體系統分別靜默圓葉菸草(Nicotiana benthamiana)，發現BaMV在ENL靜默的植物中累積量下降至對照組之10%，而在PGM靜默的植物中卻與對照組沒有顯著差異。相反地，當ENL在菸草中過量表現，BaMV的累積量便會上升至150%，而在ENL靜默的菸草中過量表現ENL，則可回復BaMV的累積量。而BaMV在原生質體的累積量並不會受到ENL抑制劑影響。另外測試另一種馬鈴薯X病毒屬病毒──馬鈴薯病毒X (Potato virus X, PVX)──也有相同的結果，其累積量在ENL靜默的植物中下降至約30%。除此之外，ENL與PGM在菸草中的表現量並不會受到BaMV感染的影響。整體而言，ENL對於BaMV、satBaMV及PVX在圓葉菸草的累積量有正向調控的影響。 | zh_TW |
| dc.description.abstract | Viruses cannot replicate without recruitment of host proteins. However, the information of host proteins that are involved in virus replication is quite limited. Previously, some host factor candidates were found to be bound with the 5’ and 3’ untranslated regions of Bamboo mosaic virus (BaMV), a positive-sense RNA virus, and the satellite of BaMV (satBaMV) by strepto tag method. This study aims to examine the effects of enolase (ENL) and phosphoglycerate mutase (PGM), two enzymes from glycolysis pathway, on BaMV replication. We first knocked down these genes by the Tobacco rattle virus-based virus-induced gene silencing (VIGS) system and found that the accumulation of BaMV decreased to 10% of the mock-treated control when ENL was knocked down in Nicotiana benthamiana, while knocking down of PGM did not significantly change BaMV accumulation. In contrast, BaMV accumulation increased to 150% when ENL was overexpressed. The overexpression of ENL recovered the accumulation of BaMV in ENL knocked down N. benthamiana. These results suggest that ENL positively regulates the accumulation of BaMV in N. benthamiana but no difference in BaMV accumulation was observed when protoplasts of N. benthamiana were treated with ENL inhibitor, NaF. Similarly, the accumulation of satBaMV decreased to about 40% and accumulation of another Potexvirus, Potato virus X (PVX), decreased to about 30% in ENL knocked down N. benthamiana compared with that in mock-treated control. In addition, the expression level of ENL and PGM was not significantly changed during BaMV infection. In summary, ENL positively regulates the accumulation of BaMV, satBaMV and PVX in N. benthamiana. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T16:08:24Z (GMT). No. of bitstreams: 1 ntu-104-R02b42025-1.pdf: 4098026 bytes, checksum: 30d7c1119c453103f741c42eb7f2659b (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 口試委員審定書…………………….…………………………………………………i
致謝……………………………………………………………………..…….……….ii Abstract (Chinese)………………………….………………………………………...iii Abstract (English)….………………………………………………………………....iv Contents……………………………………………………………..…………….......vi Contents of Tables………………………………………………………………….…ix Contents of Figures……………………………………………..…………...……...…x Introduction…………………………………………………………………………....1 1. Host factors of virus replocation..……………………………………………..1 2. Bamboo mosaic virus and the associated satRNA………….…………………3 3. The host factors involved in replication of BaMV…………………...….…….5 4. Unusual functions of enolase and phosphoglycerate mutase…..…….………..6 Materials and Methods………………………………………………………...………8 1. Virus isolates…………………………………………………………………8 2. Plasmid construction…………………………………………………………8 3. VIGS, overexpression and virus inoculation………………………………..11 4. Protoplasts isolation and cultured with NaF……………………....….…….12 5. Total RNA extraction……………………………….………..……………..13 6. RNA analysis by qPCR and northern blot analysis…………………………14 7. Total protein extraction and western blot analysis………………………….15 Results…………………………….………………………………..………………...17 1. The sequence of ENL and PGM in N. benthamiana are similar to these in N. tabacum…………………………………………………………………………….17 2. The accumulation of BaMV decreases in ENL silencing N. benthamiana, while knocking down of PGM does not change the accumulation of BaMV…...…………………………………………………………………..17 3. The accumulation of BaMV increases in ENL overexpressed N. benthamiana………………………………………………………………………...19 4. BaMV accumulation recovery in ENL silencing N. benthamiana after ENL overexpression………………………………………………………………………19 5. The accumulation of BaMV was not changed in N. benthamiana protoplasts cultured with ENL inhibitor, NaF, but reduced in NaF treated N. benthamiana plants……………………..……………………………….….21 6. The accumulation of PVX was reduced in ENL knocked down N. benthamiana………………………………………………...................................22 7. The accumulation of satBaMV was reduced in ENL knocked down N. benthamiana………………………………………………………………………...23 8. The satBaMV mediated down regulation of BaMV replication is not affected by silencing of ENL…………………………………………………………24 9. The expression of ENL or PGM is not significantly changed during BaMV infection………………………………………………...……………….......25 Discussion……………………………………………………………………………26 1. Knocking down of enolase may cause serious effects to plant……………..26 2. The effect of ENL on the accumulation of BaMV, PVX and satBaMV…....26 3. The infection of BaMV does not cause Warburg effect on ENL and PGM in N. benthamiana……………………………………..…………………………...…27 4. The possible functions of ENL to BaMV accumulation…………….……...28 Conclusion……………………………………………………………………………31 References……………………………………………………………………………62 Contents of Tables Table S1. Abbreviations used in this study………………………………………...….…..58 Table S2. Plasmids list. ………………………………………………...…………………....60 Contents of Figures Fig. 1. Alignment of ENL RNA and protein sequence in N. benthamiana with these in N. tabacum…………..………………………………………………………………...….…..32 Fig. 2. Alignment of PGM RNA and protein sequence in N. benthamiana with these in N. tabacum.…........................................................................................................…..36 Fig. 3. The accumulation of BaMV in ENL or PGM knocked down N. benthamiana……………………………………………….………………………….40 Fig. 4. The accumulation of BaMV in ENL overexpressed N. benthamiana………….42 Fig. 5. The accumulation of BaMV in GFP or ENL knocked down and overexpressed N. benthamiana……………………………………..…………………..……………..44 Fig. 6. The accumulation of BaMV in N. benthamiana protoplasts incubated with various concentration of ENL inhibitor.……………………………………….46 Fig. 7. The accumulation of BaMV in pKB and NaF coinfiltrated N. benthamiana…47 Fig. 8. The accumulation of BaMV in NaF treated N. benthamiana…………………..49 Fig. 9. The accumulation of PVX in ENL or PGM knocked down N. benthamiana…51 Fig. 10. The accumulation of satBaMV in ENL knocked down transgenic N. benthamiana ORF1…………………………………………………………….53 Fig. 11. BaMV and satBaMV accumulation in ENL knocked down N. benthamiana……………………………………………………………………………55 Fig. 12. The expression of ENL and PGM in BaMV inoculated N. benthamiana…...57 Fig. S1. Glycolysis pathway…………………………………………………………………61 | |
| dc.language.iso | en | |
| dc.subject | 病毒誘導基因靜默 | zh_TW |
| dc.subject | 竹嵌紋病毒 | zh_TW |
| dc.subject | 竹嵌紋病毒衛星核酸 | zh_TW |
| dc.subject | 圓葉菸草 | zh_TW |
| dc.subject | 宿主蛋白 | zh_TW |
| dc.subject | 烯醇化? | zh_TW |
| dc.subject | 磷酸甘油酸變位? | zh_TW |
| dc.subject | satBaMV | en |
| dc.subject | virus-induced gene silencing | en |
| dc.subject | phosphoglycerate mutase | en |
| dc.subject | enolase | en |
| dc.subject | host factor | en |
| dc.subject | Nicotiana benthamiana | en |
| dc.subject | Bamboo mosaic virus | en |
| dc.title | 烯醇化酶與磷酸甘油酸變位酶在圓葉菸草中對竹嵌紋病毒累積量所扮演之角色 | zh_TW |
| dc.title | The roles of host enolase and phosphoglycerate mutase in BaMV titer in Nicotiana benthamiana | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 林納生(Na-Sheng Lin) | |
| dc.contributor.oralexamcommittee | 徐堯煇(Yau-Heiu Hsu) | |
| dc.subject.keyword | 竹嵌紋病毒,竹嵌紋病毒衛星核酸,圓葉菸草,宿主蛋白,烯醇化?,磷酸甘油酸變位?,病毒誘導基因靜默, | zh_TW |
| dc.subject.keyword | Bamboo mosaic virus,satBaMV,Nicotiana benthamiana,host factor,enolase,phosphoglycerate mutase,virus-induced gene silencing, | en |
| dc.relation.page | 74 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-08-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-104-1.pdf 未授權公開取用 | 4 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。