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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭石通 | zh_TW |
dc.contributor.advisor | Shih-Tong Jeng | en |
dc.contributor.author | 林奕安 | zh_TW |
dc.contributor.author | I-An Lin | en |
dc.date.accessioned | 2024-02-22T16:33:39Z | - |
dc.date.available | 2024-02-23 | - |
dc.date.copyright | 2024-02-22 | - |
dc.date.issued | 2024 | - |
dc.date.submitted | 2024-02-03 | - |
dc.identifier.citation | 謝誌紘. (2020). 利用生物資訊方法探討在水稻中受過氧化氫調控的小核醣核酸.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/91752 | - |
dc.description.abstract | 當植物面臨許許多逆境時,會造成氧化逆境,釋放過氧化氫,開啟下游防禦基因來抵抗逆境造成的傷害。過氧化氫可以藉由調控微小核糖核酸 (miRNA) 來改變部分基因的表達,參與多種調控網路,儘管目前已經找到許多miRNA調節目標基因的案例,但是在氧化逆境下miRNA所建構的調控網路尚未建立完整。本研究中我們針對水稻 (Oryza sativa L., cv. Tainung 67) 中24核苷酸長度的miRNA與其目標基因進行研究,觀察DNA甲基化的調控情形。以14天大的水稻小苗處理20 mM過氧化氫後,發現有一個24核苷酸長度的miRNA名為nv021的表現會在處理1小時後會被抑制,同時nv021被報導會和AGO4蛋白質共同沉澱下來,說明nv021可能具有DNA甲基化的能力。藉由psRNATarget預測出nv021的可能目標基因為OsSDR110C-MS3,是一種會受到昆蟲啃咬植物時所釋放的二萜類化合物之生成酵素。當水稻受過氧化氫處理,nv021表現量下降,且OsSDR110C-MS3表現量如預期地呈現相反的上升趨勢。又以McrBC-qPCR與亞硫酸鹽定序 (Bisulfite sequencing) 來觀測OsSDR110C-MS3的甲基化變動情況,最後也以McrBC-qPCR分析nv021基因的自身甲基化狀態。為後人提供一個確認miRNA甲基化能力可行的方法。 | zh_TW |
dc.description.abstract | When plants face stresses that cause oxidative stress, they release hydrogen peroxide and turn on downstream defense genes to resist the damage triggered by the biotic and abiotic stresses. Hydrogen peroxide can change the gene expression by regulating microRNA (miRNA) and participates in various regulatory networks. However, the regulatory network regulated by miRNA under oxidative stress is not yet fully established. In this study, we studied the 24-nucleotide long miRNA and its target genes to investigate methylation regulation in rice (Oryza sativa L., cv. Tainung 67). After 14-day-old rice seedlings were treated with 20 mM hydrogen peroxide, it was found that the expression of a 24-nucleotide long miRNA named nv021 was suppressed after 1 hour of treatment. At the same time, miRNA nv021 was reported to co-precipitate with the AGO4 protein, which is involved in DNA methylation by miRNA. These results may indicate that miRNA nv021 may be able to methylate DNA. The putative target gene of miRNA nv021 was predicted by psRNATarget to be OsSDR110C-MS3, which encodes an enzyme that produces diterpenoids after insect biting. The expression of miRNA nv021 decreased, and the expression of OsSDR110C-MS3 increased as expected in rice after hydrogen peroxide treatment. McrBC-qPCR and Bisulfite sequencing were used to observe the methylation changes of OsSDR110C-MS3 in rice under H2O2 treatment. Finally, McrBC-qPCR was used to analyze the self-methylation status of the nv021 gene. This study may provide feasible methods to confirm the methylation ability of miRNA. | en |
dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-02-22T16:33:39Z No. of bitstreams: 0 | en |
dc.description.provenance | Made available in DSpace on 2024-02-22T16:33:39Z (GMT). No. of bitstreams: 0 | en |
dc.description.tableofcontents | 致謝 i
摘要 ii Abstract iii 目 次 iv 圖 次 viii 表 次 x 壹、文獻探討 1 貳、材料及方法 9 A. 研究材料 9 水稻Rice (Oryza sativa) 9 B. 實驗方法 10 一、水耕液製備 10 二、水稻過氧化氫處理 11 三、甲基化程度測定 11 1. DNA 萃取 11 2. McrBC處理標準流程 12 3. 聚合酶連鎖反應(Polymerase chain reaction, PCR) 12 4. DNA膠體電泳 13 5. 即時定量PCR (Quantitative Real-Time PCR, qPCR) 13 四、亞硫酸鹽定序 (Bisulfite sequencing) 14 1. DNA 萃取 14 2. 亞硫酸鹽處理DNA 15 3. 亞硫酸鹽專一性引子設計 16 4. 膠體回收DNA片段 (Gel elution) 17 5. DNA黏合反應 (DNA ligation) 17 6. 大腸桿菌轉型 17 7. DNA定序 18 五、反轉錄聚合酶連鎖反應 (reverse transcription-PCR, RT-PCR) 18 1. RNA 萃取 18 1. 反轉錄反應 (Reverse transcription, RT) 19 2.1 mRNA反轉錄 19 2.2 Mature miRNA反轉錄 19 六、質體構築 20 1. 膠體回收DNA片段 (Gel elution) 20 2. DNA黏合反應 (DNA ligation) 20 3. 大腸桿菌轉型與轉型菌株挑選 20 4. 小量質體抽取 (plasmid DNA Mini-extraction) 21 5. DNA定序 21 6. 限制酶剪切作用與質體構築 22 七、亞細胞定位 (Subcellular Localization) 22 1. 中量質體抽取 (plasmid DNA Midi-extraction) 22 溶液配置 23 2. 原生質體分離 (Protoplast isolation) 25 3. 原生質體轉型 (Protoplast transfection) 27 4. 螢光顯微鏡觀察 28 5. 共軛焦顯微鏡觀察 28 C. 生物資訊分析 29 一、 分析次世代定序資料 29 二、 比較受過氧化氫影響且與AGO4 family有結合之24 nt微小核糖核酸定序資料 30 三、 使用Kismeth觀察亞硫酸鹽定序檢測之甲基化情形 30 四、 由DeepLoc-1.0分析OsSDR110C-MS3之可能細胞定位 30 五、透過mfold預測pri-nv021構型 31 參、結果 32 ㄧ、nv021受過氧化氫誘導 32 二、nv021目標基因預測為OsSDR110C-MS3 33 三、nv021與OsSDR110C-MS3皆受到過氧化氫影響 33 四、nv021對OsSDR110C-MS3造成之甲基化影響 34 五、OsSDR110C-MS3蛋白在水稻中定位 36 肆、討論 38 一、生物資訊分析可能與過氧化氫相關之具有甲基化功能的miRNAs 38 二、新發現的nv021與其可能的目標基因關係探討 38 三、有甲基化功能的miRNAs與目標基因組合之找尋困境 40 四、結論 41 伍、圖表 42 陸、補充資料 56 柒、參考文獻 74 | - |
dc.language.iso | zh_TW | - |
dc.title | 在過氧化氫逆境下水稻24核苷酸長度的微小核糖核酸nv021的甲基化能力分析 | zh_TW |
dc.title | Methylation ability of 24-nucleotide microRNA nv021 in rice under H2O2 stress | en |
dc.type | Thesis | - |
dc.date.schoolyear | 112-1 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 林振祥;靳宗洛;楊淑怡 | zh_TW |
dc.contributor.oralexamcommittee | Jeng-Shane Lin;Tsung-Luo Jinn;Shu-Yi Yang | en |
dc.subject.keyword | 水稻,過氧化氫,微小核糖核酸,甲基化,nv021,SDR基因家族, | zh_TW |
dc.subject.keyword | nv021,SDR gene family,rice,hydrogen peroxide,microRNA,methylation, | en |
dc.relation.page | 79 | - |
dc.identifier.doi | 10.6342/NTU202400422 | - |
dc.rights.note | 同意授權(全球公開) | - |
dc.date.accepted | 2024-02-04 | - |
dc.contributor.author-college | 生命科學院 | - |
dc.contributor.author-dept | 植物科學研究所 | - |
顯示於系所單位: | 植物科學研究所 |
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