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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭石通 | zh_TW |
| dc.contributor.advisor | Shih-Tong Jeng | en |
| dc.contributor.author | 林鈺玲 | zh_TW |
| dc.contributor.author | Yu-Ling Lin | en |
| dc.date.accessioned | 2021-07-10T21:52:42Z | - |
| dc.date.available | 2024-08-15 | - |
| dc.date.copyright | 2019-08-19 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77249 | - |
| dc.description.abstract | 植物在外界環境下，會受到生物及非生物等逆境。其中，傷害逆境對植物影響最為嚴重，妨礙了植物的生長及發育。而植物也已經演化出許多防禦機制來因應傷害逆境，包含修復受傷組織，及活化系統性防禦機制，使尚未受到傷害之部位產生防禦，因應即將到來之逆境。植物的microRNA (miRNA)由21至24個核苷酸所組成的非編碼小片段分子，藉由降解目標基因之mRNA或抑制轉譯的方式，來調控目標基因。然而，目前對於參與在甘藷系統性防禦之miRNA了解甚少。本篇研究藉由次世代定序技術建構傷害處理組以及對照組的甘藷系統葉small RNA定序(small RNA sequencing)資料庫，和未經處理的甘藷系統葉轉錄體資料庫(transcriptome)，交互比對資料庫後，預測會受傷害影響的一群miRNA candidates，以及這些miRNA candidates所調控的目標基因。分析發現，有319個miRNA可能參與系統葉防禦反應，其中131個為已知的miRNA，188個為新穎的miRNA。我們挑選了長度為21個核苷酸的新穎miRNA，tag6568。利用甘藷系統葉轉錄體資料庫(transcriptome)預測出tag6568的目標基因可能為LRR receptor-like protein kinase 1 (RPK1) 和CDPK-related kinase 1 (CRK1)。經即時定量PCR分析後primary form tag6568和mature form tag6568在系統葉的表現量會受傷害誘導，而其目標基因RPK1和CRK1表現量會受傷害抑制。然而在菸草短暫基因表現實驗中，結果顯示RPK1和CRK1表現量不受tag6568調控，這些結果表明RPK1和CRK1是由生物資訊預測出tag6568的目標基因，然而他們的相互作用和生理功能將進一步研究。 | zh_TW |
| dc.description.abstract | Wounding stress is the most serious impact on plants, which affects plant growth and development. Plants have evolved many defense mechanisms to deal with damage, including repairing injured tissues and activating systemic defense mechanisms. Plant microRNAs (miRNAs), a group of 21-24 nucleotides small non-coding RNA, direct messenger RNA cleavage or inhibition translation to regulate gene expression. Previous studies show that miRNAs may involve in local wounding. However, little is known about the miRNAs involved in the systematic defense of sweet potatoes. In this study, next generation sequencing (NGS) was used to generate the transcriptome and two small RNA sequence databases of sweet potato (Ipomoea batatas cv. Tainung 57) systemic leaves with or without wounding treatment. These databases were comparatively analyzed to predict the small RNAs affected by systematic wounding, and target genes regulated by small RNAs. Here we find 319 miRNA candidates involve in the wounding of the sweet potato systemic leaves, 131 of them are conserved miRNAs, and 188 of them are novel miRNAs. We focus on a wounding-induced 21-nutleotide-long novel miRNA named tag6568. Target gene search based on sweet potato transcriptome database, we find that LRR receptor-like protein kinase 1(RPK1) and CDPK-related kinase 1 (CRK1)may be the putative target. Verified by quantitative-RT PCR, expression of primary form tag6568 and mature form tag6568 are both increased after wounding. Conversely, the expression ofRPK1and CRK1 were decreased. These result indicated that RPK1 and CRK1 may regulate by tag6568. However, the results show that RPK1 and CRK1aren’t targrted by tag6568 inTransient expression assay by agro-infiltration.These results indicate that RPK1 and CRK1 are predicted by bioinformatics to target genes of tag6568, however their interactions and physiological functions will be further studied. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:52:42Z (GMT). No. of bitstreams: 1 ntu-108-R06b42022-1.pdf: 1991256 bytes, checksum: c8a754891afe50fa42047a8e62cf1bdf (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 致謝 I
中文摘要 II Abstract III 第一章前言 1 一、 植物與逆境 1 二、 植物抵抗傷害逆境的反應 1 三、 植物系統性防禦反應 2 四、 植物小分子 RNA引起基因靜默 3 五、 植物中microRNA生合成路徑 4 六、 植物中miRNA對目標基因調控機制 5 七、 植物microRNA參與的調控 5 八、 LRR receptor-like protein kinase1(RPK1) 6 九、 CDPK-related kinase 1 (CRK1) 7 十、 研究目的與方向 8 第二章材料與方法 9 一、 植物材料 9 1. 甘藷 9 2. 菸草 9 二、 甘藷葉片捏傷處理 10 三、 甘藷葉片處理methyl jasmonate 10 四、 RNA反轉錄cDNA流程 10 1. Total RNA萃取 (Total RNA extraction) 10 2. DNase處理 (DNase treatment) 11 3. 反轉錄反應 (Reverse transcription，RT) 11 五、 表現量測定 12 1. 聚合酶連鎖反應 (Polymerase chain reaction，PCR) 12 2. 即時定量聚合酶連鎖反應 (Quantitative real-time PCR) 12 3. DNA膠體電泳 (DNA gel electrophoresis) 13 六、 質體構築 13 1. 由膠體回收DNA片段 (Gel elution) 13 2. DNA黏合反應 (DNA ligation) 14 3. 大腸桿菌轉型作用與轉型菌株挑選 14 4. 質體DNA小量抽取 14 5. DNA定序 15 6. 限制酶酵素剪切作用與質體構築 15 七、 菸草基因短暫表達與甘藷轉殖 15 1. 農桿菌勝任細胞製備 15 2. 農桿菌轉型作用與轉型菌株挑選 16 3. 菸草基因短暫表達 (Transient expression assay by agro-infiltration) 17 4. 甘藷轉殖 (Agrobacterium-mediated plant transformation) 17 八、 次世代定序與定序資料分析 18 1. Small RNA深度定序 18 2. 次世代定序資料分析 18 第三章結果 22 一、 傷害反應small RNA libraries與transcriptome之資料整理 22 二、 以miRNA生合成特徵進行miRNA candidates預測 22 三、 傷害相關之putative miRNA的保守性分析與歸類分納 25 四、 primary form tag6568可產生novel miRNA tag6568 26 五、 novel miRNA- tag6568與傷害之間的關係 26 六、 預測tag6568所調控的目標基因 27 七、 LRR receptor-like protein kinase 1與傷害之間的關係 28 八、 CDPK-related kinase 1與傷害之間的關係 28 九、 在MeJA處理下，tag6568與目標基因表現量測定 29 十、 驗證tag6568與目標基因之間的關係 29 第四章討論 32 一、 找出甘藷系統葉中傷害相關的miRNA candidates 32 二、 傷害逆境下tag6568的表現 34 三、 甘藷系統葉的選擇 35 四、 tag6568可能調控目標基因預測與分析 36 五、 tag6568可能之目標基因LRR receptor-like protein kinase1(RPK1)調控 37 六、 tag6568可能之目標基因CDPK-related kinase 1 (CRK1)調控 39 七、 結論 42 第五章結果圖表 42 第六章附錄圖表 74 第七章文獻參考 84 表一、傷害相關miRNA candidates分析結果統整 43 表二、野生型甘藷中與傷害相關之conserved miRNA 44 表三、野生型甘藷中與傷害相關之novel miRNA 46 表四、pre-tag6568序列 48 表五、pre-tag6568所產生的small RNA 49 表六、以Mir Score預測tag6568之目標基因 50 表七、使用引子一覽 51 圖一、傷害處理組與對照組之small RNA定序長度分布圖 53 圖二、miRNA candidates預測流程示意圖 56 圖三、預測tag6568的目標基因 57 圖四、預測其他物種的primary form tag6568 58 圖五、pCAMBIA-2300-pre-tag6568質體 60 圖六、pCAMBIA-2300-STTM6568質體 61 圖七、以農桿菌短暫性表現分析pre-tag6568與mature form tag6568之表現量 62 圖八、野生型甘藷在傷害處理下，系統葉之primary form tag6568及mature form tag6568的表現量 63 圖九、野生型甘藷在傷害處理下，本片葉之primary form tag6568及mature form tag6568的表現量 64 圖十、野生型甘藷在傷害處理下，系統葉之LRR receptor-like protein kinase 1及CDPK-related kinase 1的表現量 65 圖十一、野生型甘藷在傷害處理下，本片葉之LRR receptor-like protein kinase 1及CDPK-related kinase 1的表現量 66 圖十二、在JA處理下，tag6568與目標基因的表現量 67 圖十三、以農桿菌短暫性表現分析tag6568和其目標基因RPK1結合位之作用關係 68 圖十四、以農桿菌短暫性表現分析tag6568和其目標基因CRK1結合位之作用關係 69 附錄一、次世代定序資料分析之重點指令、輸入與輸出 70 附表一、以Mir Score預測tag6568之目標基因 74 附表二、以Mir Score預測tag4122之目標基因 76 附表三、以Mir Score預測tag6568及tag4122之共同目標基因 78 附表四、以Mir Score預測可能調控RPK1之miRNA 79 附表五、以Mir Score預測可能調控CRK1之miRNA 80 附圖一、WT unwounding及WT wounding深度定序所得miRNA candidates之單一序列(unique reads)及total reads比較 81 附圖二、野生型甘藷在傷害處理下，系統葉(第二片葉片)之primary form tag6568及mature form tag6568的表現量 82 附圖三、野生型甘藷在傷害處理下，系統葉(第二片葉片)之LRR receptor-like protein kinase 1及CDPK-related kinase 1的表現量 83 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 甘藷 | zh_TW |
| dc.subject | 傷害 | zh_TW |
| dc.subject | 嶄新miRNA | zh_TW |
| dc.subject | 轉錄組 | zh_TW |
| dc.subject | 系統性防禦 | zh_TW |
| dc.subject | transcriptome | en |
| dc.subject | sweet potato | en |
| dc.subject | wounding | en |
| dc.subject | systemic defense | en |
| dc.subject | novel miRNA | en |
| dc.title | 甘藷受傷後系統葉中的嶄新miRNA-tag6568的研究 | zh_TW |
| dc.title | Study of a Novel MicroRNA tag6568 in the Systemic leaves of Sweet Potato upon Wounding | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 黃浩仁;邱子珍;林振祥 | zh_TW |
| dc.contributor.oralexamcommittee | Hao-Jen Huang;Tzyy?Jen Chiou;Jeng-Shane Lin | en |
| dc.subject.keyword | 甘藷,傷害,系統性防禦,轉錄組,嶄新miRNA, | zh_TW |
| dc.subject.keyword | sweet potato,wounding,systemic defense,transcriptome,novel miRNA, | en |
| dc.relation.page | 89 | - |
| dc.identifier.doi | 10.6342/NTU201903222 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-08-14 | - |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-107-2.pdf 未授權公開取用 | 1.94 MB | Adobe PDF |
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