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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭石通 | |
dc.contributor.author | Chun-Wei Huang | en |
dc.contributor.author | 黃俊瑋 | zh_TW |
dc.date.accessioned | 2021-06-08T03:48:04Z | - |
dc.date.copyright | 2019-01-22 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-01-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21815 | - |
dc.description.abstract | 過氧化氫為活性氧化物質的一種,在濃度適中的狀況下在植物當中扮演得訊息傳遞分子的角色。但當植物面臨傷害、蟲害、乾旱、炎熱等逆境的時候,會產生過量的ROS進而導致氧化逆境而使得植物的生長發育受到影響。微小核糖核酸(microRNA)在植物當中屬於非編碼小核糖核酸中的一員(non-coding small RNA),雖然僅具有21-24個核苷酸,卻在植物基因調控路徑中扮演的相當重要的角色,這些微小核糖核酸會藉由降解核醣核酸或是抑制傳譯的方式來達到基因調控的目的。在近年來許多研究發現微小核糖核酸不只參與在植物生長發育過程當中,更發現它們與植物面對逆境的機制息息相關,但對於氧化逆境中微小核糖核酸的功能的研究仍然不多,因此,我們實驗室利用次世代微小核酸定序以及降解組定序的方式在水稻中預測出了51種已知的微小核糖核酸可能參與在氧化逆境下植物的基因調控之中。本篇研究更近一步驗證次世代定序的結果並且發現了miR535在氧化逆境之下其表現量會受到抑制,同時藉由生物資訊方式預測下游調控之目標基因OsSPL2以及OsFLS2,可能具有調控水稻基因表現量的功能,經由菸草短暫表達實驗驗證,發現所預測之miR535前驅物確實會產生具有功能的成熟miR535,但經由實驗結果驗證後發現,miR535未如預測結果調控OsSPL2,推測其調控反應應為miR156作用,本研究雖未對miR535功能有所定論,未來可利用轉殖方式來去確認其調控功能與機制。 | zh_TW |
dc.description.abstract | Hydrogen peroxide (H2O2), one of reactive oxygen species (ROS), is a natural by-product of cellular metabolism, and acts as a signal molecule in response to both biotic and abiotic stresses in plants. MicroRNAs (miRNAs), non-coding small RNAs, play critical roles in regulating gene expression through either degradation of miRNA target genes or inhibiting the process of gene translation. To date, only few studies have concentrated on miRNA-mediated gene regulation in response to oxidative stress in plants. In this study, small RNA sequencing has revealed that 51 conserved miRNAs were regulated during the short period of H2O2 treatment in rice seedling roots. The osa-miR535 with novel functions was identified for further study. The expression of osa-miR535 was decreased under short-term oxidative stress. Through the anaylyses of bioinformatics and degradome sequencing two potential target genes of osa-miR535 were OsSPL2 and OsFLS2. Surprisely, the interaction between osa-miR535 and OsSPL2 could not be confirmed by agroinfiltration. However, according to previous reserches and reanalysis of bioinformatics we speculated that the gene regulation of OsSPL2 might be caused not by osa-miR535 but by osa-miR156. Taken together, our results demonstrated that 51 conserved miRNA were regulated under oxidative stress and osa-miR535 play a role in oxidative stress while the function of osa-miR535 still need further investigation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:48:04Z (GMT). No. of bitstreams: 1 ntu-108-R05b42027-1.pdf: 2428817 bytes, checksum: aeefa9cd2f07d55ec63e8ab1296dfedb (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 致謝 I
Abstract II 中文摘要 III 目錄 IV 第一章 前言 1 一、 植物中的活性氧化物質以及氧化逆境 1 1. 活性氧化物質(Reactive oxygen species, ROS) 1 2. 植物中ROS的生合成 1 3. 植物中ROS的清除 2 4. 氧化逆境(Oxidative stress) 2 5. ROS作為訊息傳遞分子 2 二、 植物中的微小分子RNA(microRNA) 3 1. 植物小分子RNA 3 2. 植物中的微小核糖核酸(miRNA)生合成 3 3. 植物miRNA調節目標基因量表現機制 4 4. 植物生長發育與miRNA 5 5. 植物逆境與miRNA 5 三、 miRNA535 6 四、 SQUAMOSA promoter-binding protein-like transcription factors (SPL) 6 1. SPL 家族 6 2. miRNA與SPL 7 五、 研究目的與方向 9 第二章、材料與方法 10 一、 植物材料 10 1. 水稻 10 2. 菸草 10 二、 實驗處理方式 11 1. 過氧化氫處理 11 三、 RNA樣品處理流程 11 1. Total RNA萃取 (Total RNA extraction) 11 2. RNA膠體電泳 (RNA gel electrophoresis) 12 3. 使用TURBO DNA-free™ Kit進行DNAase處理 12 4. miRNA目標基因表現測定之反轉錄反應 13 5. Mature form miRNA表現測定之反轉錄反應 13 四、 植株基因組(Genomic DNA)抽取 13 1. Genomic DNA抽取 13 五、 基因表現量測定以及基因全長釣取 14 1. 引子設計 14 2. 聚合酶連鎖反應 (Polymerase chain reaction,PCR) 14 3. 即時定量PCR (Real-Time PCR) 15 4. DNA膠體電泳 (DNA gel electrophoresis) 15 5. miRNA目標基因miRNA調控位置釣取 15 六、 質體構築 16 1. 由膠體回收DNA片段 16 2. DNA 連接反應 (DNA Ligation) 16 3. 大腸桿菌轉型作用以及篩選 17 4. 質體DNA(Plasmid DNA)小量抽取 17 5. DNA定序 18 6. 質體構築和限制酶酵素剪切 18 七、 菸草基因短暫表達與水稻轉殖 18 1. 菸草基因短暫表達試驗LBA4404勝任細胞製備 18 2. LBA4404轉型作用以及篩選 19 3. 菸草基因短暫表達(Transient expression assay by ago-infiltraction) 19 第三章 結果 20 一、 Small RNA Sequencing分析 20 二、 過氧化氫處理putative miRNA分析以及分類 22 三、 保守性miRNA-miR535特性分析 23 四、 miR535的表現量測定 24 五、 miR535的目標基因預測分析 25 六、 miR535目標基因表現量測定 26 七、 pre-miR535產生mature form miR535 26 八、 miR535目標基因的驗證 27 第四章 討論 29 一、 次世代定序分析短時間氧化逆境調控之miRNA 29 二、 過氧化氫調控miR535 30 三、 miR535序列分析 32 四、 miR535可能調控目標基因預測與分析 33 五、 miR535可能之目標基因SPL2驗證 35 六、 過氧化氫處理下OsSPL2與OsFLS2功能 36 七、 miR535與miR156和miR529之功能 37 八、 結論 39 第五章 結果圖表 40 表一、使用引子列表 40 表二、經由Small RNA sequencing分析流程所得到保守型miRNA 42 表三、受過氧化氫誘導之osa-miR535目標基因 46 圖一、經由Small RNA sequencing分析流程所得到保守型miRNA分類 47 圖二、osa-miR535之基因資訊分析 48 圖三、osa-miR535-5p 與 miR156、miR529序列比較 49 圖四、野生型水稻台農67號在過氧化氫處理下根系miR535-5p的表現量 50 圖五、野生型水稻台農67號在過氧化氫處理下根系pre-miR535的表現量 51 圖六、野生型水稻台農67號在過氧化氫處理下根系OsFLS2的表現量 52 圖七、野生型水稻台農67號在過氧化氫處理下根系OsSPL2的表現量 53 圖八、以農桿菌短暫表現分析pre-miR535與mature miR535之表現量 54 圖九、以農桿菌短暫表現分析miR535與其目標基因OsSPL2 之結合位之作用關係 55 圖十、比較實驗室Small RNA分析方式與吳明聰學長分析結果差異 56 第六章 附錄 57 附表一、吳明聰學長分析Small RNA sequencing 中受到過氧化氫處理表現量有差異之保守性miRNA 57 附圖一、Small RNA seqeuncing 定序長度分布圖 59 附圖二、Smalll RNA seqenucing 分析流程 60 附圖三、osa-miR535-5p經由過氧化氫處理下在Small RNA sequencing的讀值變化 61 附圖四、利用psRNAtarget預測osa-miR535-5p 62 附圖五、降解組定序中OsSPL2中miR535-5p切位 63 附圖六、降解組定序中OsSPL2中miR156切位 64 附圖七、比較miR535-5p和miR156與OsFLS2序列互補性 65 附圖八、pCAMBIA-1300-35s-pre-miR535質體 66 附圖九、pCAMBIA-2300-35s-35s::OsSPL2-BS-eGFP 67 附圖十、psRNATarget分析osa-miR535-3p之目標基因 68 附圖十一、osa-miR444經由過氧化氫處理下在Small RNA sequencing的讀值變化 69 附圖十二、野生型水稻台農67號在過氧化氫處理下根系miR444的表現量 70 附圖十三、降解組定序中miR444之目標基因OsMADS23預測與切位 71 附圖十四、降解組定序中miR444之目標基因OsMADS27預測與切位 72 附圖十五、野生型水稻台農67號在過氧化氫處理下根系OsMADS23的表現量 73 附圖十六、野生型水稻台農67號在過氧化氫處理下根系OsMADS27的表現量 74 第七章 參考文獻 75 | |
dc.language.iso | zh-TW | |
dc.title | 水稻過氧化氫相關之微小核醣核酸 miR535 研究 | zh_TW |
dc.title | Study of Hydrogen Peroxide Responsive miR535 in Rice
(Oryza sativa L.) | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 洪傳揚,張孟基,林振祥 | |
dc.subject.keyword | 氧化逆境,過氧化氫,微小核糖核酸,定序資料分析,miR535,OsSPL2,OsFLS2, | zh_TW |
dc.subject.keyword | Oxidative stress,H2O2,miRNA,sequencing data analysis,miR535,OsSPL2,OsFLS2, | en |
dc.relation.page | 83 | |
dc.identifier.doi | 10.6342/NTU201803876 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2019-01-21 | |
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 |
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