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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃楓婷(Feng-Ting Huang),楊健志(Chien-Chih Yang) | |
| dc.contributor.author | Chia-Lin Wu | en |
| dc.contributor.author | 吳佳霖 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:54:54Z | - |
| dc.date.copyright | 2018-08-19 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-16 | |
| dc.identifier.citation | References
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21955 | - |
| dc.description.abstract | R-loop是由DNA-RNA雜合體 (hybrid) 及一未配對單股DNA所構成之〝三股核酸結構〞，R-loop已在許多物種中被發現參與轉錄層次的調控。然而植物中關於R-loop之研究仍剛起步。本實驗室先前研究顯示，在阿拉伯芥側根發育中扮演重要角色的生長素相關基因LBD18 (LATERAL ORGAN BOUNDARIES DOMAIN18)，會在體外實驗中 (in vitro) 形成R-loop。本研究利用吲哚-3-乙酸 (IAA) 與可促進R-loop累積之DNA拓樸異構酶抑制劑喜樹鹼 (CPT) 處理，探討在阿拉伯芥中R-loop對生長素相關基因之調控。研究結果顯示CPT處理會抑制側根的生長，且針對在生長素訊息傳遞路徑中具代表性的十二個基因分析其mRNA表現量，結果指出CPT處理會降低LAX3、PIN1、IAA14及LBD16的表現量約30-50%，暗示了此四基因較有可能形成R-loop。為探討上述結果確實是由R-loop所造成，進一步使用可辨認R-loop之DNA-RNA雜合體 (hybrid) 的S9.6單株抗體，進行DNA-RNA免疫沉澱法接續qPCR (DRIP-qPCR) 測量各基因之R-loop多寡，結果顯示在未處理CPT或IAA的情況下，PIN1、IAA14及LBD18之R-loop就比預期不受R-loop調控之ARF19高出約4-6倍，此外CPT處理後PIN1之R-loop增加約40-50%，且其mRNA表現量減少50%，PIN1為生長素的運輸者，在側根發育前期扮演建立生長素最大濃度的角色，綜合上述結果，R-loop可能藉由參與PIN1之調控，進而影響阿拉伯芥的側根發育。 | zh_TW |
| dc.description.abstract | R-loop is a “three-stranded nucleic acids complex” including a DNA-RNA hybrid and an unpaired single-stranded DNA. R-loop has been identified to participate in transcriptional regulation in various organisms. However, the R-loop research in plants is still limited. Previous studies in our laboratory suggested that LBD18 (LATERAL ORGAN BOUNDARIES DOMAIN18), one of auxin-related genes, could form R-loop in vitro. Indole-3-acetic acid (IAA) and camptothecin (CPT, a DNA topoisomerase 1 inhibitor) were used in this study to investigate the roles of R-loop in the regulation of auxin-related genes in Arabidopsis. It is expected that CPT treatment will lead to R-loop accumulation. Phenotypic analysis showed that the lateral root growth was inhibited in CPT-treated Arabidopsis. Twelve representative auxin-related genes were selected from the auxin signaling pathway to analyze their mRNA expression level. The results showed that the expression of LAX3, PIN1, IAA14 and LBD16 decreased about 30-50% after the CPT treatment. It implied that these four genes had higher R-loop forming potential than that in other genes. To further investigate if above results were actually caused by R-loops, S9.6 monoclonal antibody was employed to recognize DNA-RNA hybrids of R-loops to conduct DRIP-qPCR (DNA-RNA immunoprecipitation followed by qPCR) to quantify R-loop enrichment of individual gene. The results showed that R-loops at PIN1, IAA14 and LBD18 were 4-6 fold higher than ARF19 which was expected not to be regulated by R-loops under the condition of no IAA or CPT treatment. In addition, R-loops at PIN1 increased about 40-50% while its mRNA expression decreased 50% after the CPT treatment. PIN1 functions as an auxin transporter that establishes of an auxin maximum in the early stage of lateral root development. In summary, R-loops might modulate lateral root development via participating in the regulation of PIN1 in Arabidopsis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T03:54:54Z (GMT). No. of bitstreams: 1 ntu-107-R05b22042-1.pdf: 3827281 bytes, checksum: 463421b0eff254de6dbb55dd90f410de (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | Table of content
Table of content 1 Abbreviation List 3 摘要 5 Abstract 6 Chapter 1 Introduction 7 1.1 The presence of R-loop is common to the genomes from various organisms 7 1.2 Known biological functions of R-loop 8 1.3 RNase H and DNA topoisomerase 1 affect the stability of R-loops in vivo 11 1.4 Auxin-related genes modulate lateral root development 12 1.5 Motivation 14 Chapter 2 Materials and Methods 17 2.1 Plant materials and growth condition 17 2.2 Phenotype observation 18 2.3 Quantitative RT-PCR 18 2.4 DNA-RNA immunoprecipitation, followed by qPCR (DRIP-qPCR) 21 2.5 R-loop dot-blot 26 Chapter 3 Results 28 3.1 Lateral root growth was affected by CPT and IAA treatment 28 3.2 The mRNA expression of auxin-related genes were differentially regulated by CPT and IAA treatment 29 3.3 The appropriate conditions of nucleic acid preparation and the CPT treatment 32 3.4 Modified DRIP-qPCR seemed to be with a higher specificity than the original one 34 3.5 R-loops at PIN1, IAA14 and LBD18 were promoted in CPT-treated Arabidopsis 35 Chapter 4 Discussion 38 4.1 The effects of IAA and CPT on Arabidopsis growth 38 4.2 The roles of R-loop in the auxin signaling pathway 39 4.3 Possible reasons why modified DRIP-qPCR led to be with a higher specificity 40 4.4 Time period of R-loop formation and its physiological function in Arabidopsis 40 4.5 Conclusions 42 4.6 Future prospects 44 References 45 Figures 52 Tables 65 Appendixes 73 問答集 84 | |
| dc.language.iso | en | |
| dc.title | 阿拉伯芥生長素相關基因之R-loop對其基因表現量之影響 | zh_TW |
| dc.title | Roles of R-loop in the regulation of auxin-related genes in Arabidopsis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 靳宗洛(Tsung Luo Jinn),鄭秋萍(Chiu Ping Cheng) | |
| dc.subject.keyword | R-loop,阿拉伯芥,側根發育,生長素,喜樹鹼, | zh_TW |
| dc.subject.keyword | R-loop,Arabidopsis,lateral root development,auxin,camptothecin (CPT), | en |
| dc.relation.page | 88 | |
| dc.identifier.doi | 10.6342/NTU201803555 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2018-08-16 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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