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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 葉信宏(Hsin-Hung Yeh) | |
| dc.contributor.author | Chao-Jung Wu | en |
| dc.contributor.author | 吳昭蓉 | zh_TW |
| dc.date.accessioned | 2021-06-15T02:23:07Z | - |
| dc.date.available | 2016-08-22 | |
| dc.date.copyright | 2011-08-22 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-16 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43542 | - |
| dc.description.abstract | 病毒本身基因體極小，包含之遺傳訊息不多，因此必須倚靠寄主系統完成其複製及增殖的過程；然而對於病毒透過何種方式調控寄主基因，目前所知仍然有限。先前實驗室研究，為探究這些寄主基因，利用菸草嵌紋病毒(Tobacco mosaic virus, TMV)感染阿拉伯芥原生質體後，收取0.5-24小時抽取原生質體RNA進行microarray試驗，篩選影響病毒複製早期的基因(Chen, 2010)。其中寄主基因PAP85，在感染後0.5-24小時表現量皆上升。而PAP85無論在轉基因植物或在原生質體進行PAP85 knock-down實驗，病毒累積量皆降低，可知PAP85影響病毒累積。本研究的目的，在於探討TMV調控寄主基因表現的可能方式。首先以快速增殖DNA尾端法(Rapid amplified of cDNA ends, RACE)找出PAP85轉錄起始點約在轉譯起始點上游24個核苷酸處，同時也利用電腦比較分析PAP85找出CIACADIANLELHC, NODCON1GM, OSE1ROOTNODULE, TATABOX4, TATAPVTRNALEU, TBOXATGAPB. CIACADIANLELHC可能為參與病毒誘導之cis-acting elements。接著嘗試建立原生質體過渡性短暫試驗(transient assay)系統，但因核酸轉染效率低，得到不一致之結果。為克服此一困難，以長度不一(107, 1227 及2000 bps) 之PAP85啟動子區域聯結GUS報導基因進行基因轉殖，以便進行病毒感染後PAP85啟動子功能性分析。目前已有轉基因植物可進行試驗，且在pCambia1301-PAP85-P2000與-P1227之轉基因植物中發現其GUS報導基因表現形式與內生性PAP85一致。另外，並分析四個在TMV感染前期類轉錄因子基因(transcription factor-like genes)的knock-out突變株，發現在At5g41140突變株中，病毒累積量減少，顯示At5g41140可能參與病毒累積的過程。 | zh_TW |
| dc.description.abstract | The genomes of plant viruses are small and encode limited genetic products. To complete their life cycles, plant viruses highly require assistance from host factors. In previous research, to study these host factors, Arabidopsis protoplasts infected by Tobacco mosaic virus (TMV) were used to screen the host genes responsive to TMV replication at initial stage of infection through microarray analysis (Chen, 2010). It is found that host gene PAP85 is continuously up-regulated during 0.5-24 hours post inoculation, and virus accumulation decreased both in PAP85 transient knock-down protoplasts and pap85-RNAi transgenic plants. In this thesis, we started to study how TMV induces the PAP85 in the initial stage during virus infection. Rapid amplification of cDNA ends (RACE) was conducted to determine the transcription start site of PAP85 and found that it is located at 24 bps upstream to translational start site. Computer assisted comparison analysis was conducted to identify the possible cis-acting elements, and CIACADIANLELHC, NODCON1GM, OSE1ROOTNODULE, TATABOX4, TATAPVTRNALEU, TBOXATGAPB. CIACADIANLELHC were identified that may be involved in regulation of PAP85. PAP85 promoter assay systems were initially established on transiently-expressed protoplasts; however, inconsistent results were obtained due to low transfection rates. The transgenic plants with different lengths (107, 1227 and 2000 bps) of PAP85 promoter fused GUS reporter gene were obtained. In plants transformed with pCambia1301-PAP85-P2000 and pCambia1301- PAP85-P1227, the GUS expression pattern is similar to the reported endogenous PAP85 expression. Besides, the knock-out mutants of 4 transcription factor-like genes (At1g09530, At2g38970, At4g29530, At5g41140) upregulated during TMV initial infection were obtained from the Arabidopsis Biological Resource Center (ABRC). After TMV inoculation, the virus accumulation decreased only in At5g41140 knockout plants, suggesting that At5g41140 was involved in virus accumulation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T02:23:07Z (GMT). No. of bitstreams: 1 ntu-100-R97633004-1.pdf: 2611612 bytes, checksum: 30d30584afedddc96f4a98037597baad (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 致謝 i
中文摘要 iii Abstract iv Table of content vi Chapter I. Introduction 1 1.1 Introduction of TMV 1 1.2 TMV and host factors 3 1.3 Previous studies about a genome-wide screen of host factors related to initial stage of virus replication 5 1.4 Aim of this thesis 8 Chapter II. Material and methods 10 2.1 Sequence acquisition and cis-acting elements analysis 10 2.2 RNA extraction 11 2.3 5’ RACE 12 2.4 Reverse transcriptase-polymerase chain reaction (RT-PCR) 13 2.5 Genomic DNA extraction and polymerase chain reaction (PCR) 14 2.6 Clone construction 15 2.7 Transgenic plants preparation 16 2.8 In vitro transcription 18 2.9 Arabidopsis protoplasts preparation and transfection 19 2.10 Promoter assay using dual luciferase system as reporter gene 20 2.11 Histochemical GUS staining 21 Chapter III. Results 22 3.1 PAP85 transcription start site is at 24 bps upstream to translation start site 22 3.2 Cis-acting elements analysis of PAP85 23 3.3 Clone construction 24 3.4 Promoter assay on protoplasts 25 3.5 Preparation of transgenic plants used in promoter assay 26 3.6 Virus accumulation decreases in At5g41140 knock-out mutant 27 Chapter IV. Discussion 28 References 34 Tables 47 Table 1. Primers used in this study 47 Table 2 The lengths of 5’UTR and correspondent numbers of clones obtained in 5’ RACE 50 Table 3. Common cis-acting elements among more than 10 of the 12 genes 51 Table 4. Common cis-acting elements among the three seed storage protein (At1g03880, At3g22640, At5g44120) 55 Table 5. The cis-acting elements that only PAP85 contained among the three seed storage protein genes (At1g03880, At3g22640, At5g44120) 59 Figures 63 Figure 1 Products of 5’ RACE after polymerase chain reaction amplification 63 Figure 2 Schematic representation of transcription start site of PAP85 and the neighboring region 64 Figure 3 Common cis-acting elements identified from PAP85 and 11 up-regulated genes. 65 Figure 4 Common cis-acting elements between PAP85 and the other two seed storage protein genes, At1g03880 and At5g44120. 66 Figure 5 Cis-acting elements appeared only on PAP85 promoter region 67 Figure 6 The diagram of pGL3-PAP85-P2000 and the restriction enzyme digestion pattern in agarose gel. 69 Figure 7 Luminescence of promoter assay on arabidopsis protoplasts 70 Figure 8 GUS histochemical staining of transgenic plants. 71 Figure 9 Virus accumulation in arabidopsis knock-out mutants 73 Appendix 75 Appendix I pCambia1301-PAP85-P2000 construction. 75 Appendix II The diagrams of T-DNA insertion sites in genomes of the knockout plants 76 Appendix III Genes induced by TMV in microarray data 0.5, 4 and 6 hours post inoculation 77 | |
| dc.language.iso | en | |
| dc.subject | At5g41140 | zh_TW |
| dc.subject | 菸草嵌紋病毒 | zh_TW |
| dc.subject | 阿拉伯芥 | zh_TW |
| dc.subject | 啟動子 | zh_TW |
| dc.subject | 寄主因子 | zh_TW |
| dc.subject | PAP85 | zh_TW |
| dc.subject | Tobacco mosaic virus | en |
| dc.subject | At5g41140 | en |
| dc.subject | PAP85 | en |
| dc.subject | host factor | en |
| dc.subject | promoter | en |
| dc.subject | Arabidopsis thaliana | en |
| dc.title | 與菸草嵌紋病毒複製相關之阿拉伯芥基因PAP85啟動子功能性分析系統之建立 | zh_TW |
| dc.title | Establishment of the promoter assay system of arabidopsis gene PAP85 involved in replication of Tobacco mosaic virus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄭秋萍(Chiu-Ping Cheng),洪挺軒(Ting-Hsuan Hung) | |
| dc.subject.keyword | 菸草嵌紋病毒,阿拉伯芥,啟動子,寄主因子,PAP85,At5g41140, | zh_TW |
| dc.subject.keyword | Tobacco mosaic virus,Arabidopsis thaliana,promoter,host factor,PAP85,At5g41140, | en |
| dc.relation.page | 77 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-08-17 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-100-1.pdf 未授權公開取用 | 2.55 MB | Adobe PDF |
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