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系統識別號 U0026-1701201115021700
論文名稱(中文) 蝴蝶蘭miR156及miR172功能性之研究
論文名稱(英文) The functional study of miR156 and miR172 in Phalaenopsis aphrodite subsp. formosana
校院名稱 成功大學
系所名稱(中) 熱帶植物科學研究所
系所名稱(英) Institute of Tropical Plant Sciences
學年度 99
學期 1
出版年 100
研究生(中文) 蕭順榮
研究生(英文) Shuan- Rung Hsiao
學號 z3697103
學位類別 碩士
語文別 中文
論文頁數 80頁
口試委員 指導教授-詹明才
口試委員-黃浩仁
口試委員-吳素幸
口試委員-鄭秋萍
中文關鍵字 生長時期轉換  開花時期調控 
英文關鍵字 microRNAs  miR156  miR172  phase transitions 
學科別分類
中文摘要 MicroRNAs (miRNAs) 是一類長約21個核苷酸的單鏈小分子RNA,lin-4是最早於線蟲 (Caenorhabditis elegans) 中首次發現的miRNA,目前miRNAs在許多生物中已被發現。miRNAs和目標基因結合後經由轉錄抑制或mRNA降解等方式調控基因表現。植物體中,miRNAs主要參與生長發育、荷爾蒙和逆境等調控。阿拉伯芥中已知miR156和miR172兩個 miRNAs參與在生長時期轉換和開花時期調控,從營養生長進入生殖生長時期,miR156表現逐漸下降,其目標基因SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL)因而增加表現,SPL進而促進miR172表現,其目標基因APETALA2 (AP2) 和AP2-like 基因, TARGET OF EAT 1 (TOE1), TOE2受到抑制因而誘導開花。本實驗以白花蝴蝶蘭 (Phalaenopsis aphrodite subsp. formosana) 為材料,白花蝴蝶蘭為台灣重要原生種具有多花色、獨特花型且具高觀賞價值,但因其生長週期長,所以本篇藉由探討microRNAs (miR156和miR172) 在白花蝴蝶蘭開花調控所扮演的角色,未來希望能縮短蝴蝶蘭開花所需時間。我們利用小分子RNA定序技術並配合生物資訊分析和其他物種已知miRNAs比對後得知蝴蝶蘭有22個miRNA families,當中包含miR156 和miR172。並利用小分子RNA北方點墨法證實 miR156 和miR172確實存在蝴蝶蘭,且miR156在葉片表現高於梗和花苞,miR172則是梗和花苞表現較高。進一步由實驗室所建立的蝴蝶蘭transcriptome databaes中找尋可能的目標基因,PaSPL和PaAP2 分別具有可被miR156 和miR172所結合之位置且皆會被cleavage。其中PaSPL於RNA層次上在不同組織的表現和 miR156 互相互補,推測miR156具有調控PaSPL的功能 ; PaAP2在RNA層次之表現上並沒有因miR172表現低而增加。本實驗結果顯示蝴蝶蘭具有植物間高保守性的miRNAs且存在經由miRNAs所調控的開花路徑。
英文摘要 MicroRNAs (miRNAs) are ~21-nucleotide noncoding RNAs that have been identified in various organisms. In plants, miRNAs are mostly involved in the regulation of important growth and developmental processes such as leaf and root development, phase transitions, and flowering. Recent studies explore two temporally expressed miRNAs in Arabidopsis, miR156 and miR172 play important roles in regulation of vegetative phase change and flowering time regulation. From juvenile-to-adult transition, the transcription of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) accumulation depend on the decrease of miR156 with time, and SPL genes promote miR172 transcript which then repress the APETALA2 (AP2), and AP2-like genes、TARGET OF EAT 1 (TOE1)、TOE2 to induce flowering. In this study, we used Phalaenopsis aphrodite subsp. formosana as material. To exam the possibility of controlling miR156 and miR172 expression in this long life cycle plant to accelerate the flowering time, we perform small RNA sequencing analysis and combining bioinformatics, we discovered 22 conserved miRNA families, and identified miR156 and miR172 othologs from Phalaenopsis orchid. Also, we discovered miR156 expresses higher in leaves than in stalks and in flower buds; however miR172 mainly expressed in stalks. These patterns are similar with those in Arabidopsis, imply us that we have possibility to overexpress miR172 to facilitate flowering in orchid the same way as Arabidopsis. Further, we identified the target genes of miR156 and miR172 from our orchid transcriptome databaes, PaSPL and PaAP2 which contain a miR156 and miR172 complementary site. The expression of PaSPL is abundant in stalks and flower buds that is complementary with expression of miR156. This suggests that miRNA-mediated flowering pathway exist in Phalaenopsis orchid.
論文目次 目錄
中文摘要I
英文摘要II
誌謝III
目錄IV
表目錄VII
圖目錄VIII
第一章 序論1
一、前言1
二、影響植物開花之因素2
三、春化作用途徑 (Vernalization pathway)2
四、Small RNA3
五、MicroRNAs的發現4
六、MicroRNAs在植物的生合成途徑4
七、MicroRNAs的特徵5
八、MicroRNAs的命名5
九、搜尋MicroRNAs之策略6
十、MicroRNA 在動植物之功能6
十一、miR156和 miR172與開花時間之調控9
十二、研究目的11
第二章 材料與方法12
一、材料12
二、蝴蝶蘭小分子RNA定序12
1. 白花蝴蝶蘭營養生長和涼溫催花組各個時間點材料收集12
2. 蝴蝶蘭total RNA之純化 (Trizol reagent method)12
3. 小分子RNA定序分析13
三、利用不同的資料庫進行蝴蝶蘭小分子RNA定序之分析比對 13
四、小分子RNA北方點墨法14
1. 置備15% PAGE (8M Urea)14
2. 跑膠 (Running the gel)14
3. 轉印(Transfer) 和Cross-linking15
4. 雜合反應 (hybridization)15
5. 冷光訊號的偵測15
五、利用RACE擴增miRNA目標基因之全長16
1. 3′ RACE16
2. 5′ RACE17
3. 自洋菜膠體電泳回收DNA片段19
4. 質體接合19
5. 製備大腸桿菌 (E. coli) 的勝任細胞 (competent cell)19
6. 細菌的轉型作用 (transformation)20
六、親緣演化分析 (phylogenetic analysis)20
七、miRNAs目標基因表現型態確認21
1. 蝴蝶蘭total RNA之純化 (LiCl method)21
2. 利用反轉錄聚合酶連鎖反應 (RT-PCR) 偵測基因表現21
3. 利用定量PCR (Real-time PCR) 偵測基因表現22
八、利用5′ RACE檢測目標基因經miRNAs切除後所殘餘之片段23
1. Ligating the RNA oligo to truncated mRNA23
2. Reverse transcribing mRNA (SuperScript™ III RT Reaction)23
3. Amplifying cDNA Ends24
第三章 結果25
一、誘導白花蝴蝶蘭開花需於涼溫環境之下25
二、白花蝴蝶蘭small RNA定序結果25
三、利用miRBase資料庫進行白花蝴蝶蘭小分子RNA定序之分析比對26
四、保守性 (conserved) miRNA families於白花蝴蝶蘭不同組織表現情形26
五、預測白花蝴蝶蘭miR156和miR172的目標基因26
六、PaSPL和PaAP2全長序列的分析28
七、白花蝴蝶蘭PaSPL及PaAP2與其他植物間親緣演化分析28
八、白花蝴蝶蘭PaSPL及PaAP2與其他植物間進行多序列比對28
九、白花蝴蝶蘭miR156和miR172和目標基因所結合的位置和序列29
十、利用5′ RACE檢測目標基因經miRNAs切除後所殘餘之片段29
十一、miR156和PaSPL於白花蝴蝶蘭不同組織表現情形29
十二、miR172和PaAP2於白花蝴蝶蘭不同組織表現情形30
十三、PaSPL和PaAP2於白花蝴蝶蘭不同組織表現情形30
第四章 討論31
一、白花蝴蝶蘭小分子RNA高通量定序結果31
二、白花蝴蝶蘭保守性miRNA families31
三、白花蝴蝶蘭miR156和miR172目標基因34
四、白花蝴蝶蘭miR156與目標基因PaSPL表現情形34
五、白花蝴蝶蘭miR172與目標基因PaAP2基因表現情形35
六、結論36
第五章 圖表37
第六章 參考文獻62
附錄70

表目錄
表一、蝴蝶蘭小分子RNA高通量定序結果37
表二、蝴蝶蘭和其他物種中miRNAs比對為perfectly matched的序列和讀值 38
表三、蝴蝶蘭高保守性miRNAs family在各組織之序列分析總值41
表四、蝴蝶蘭miR156和miR172的預測目標基因42

圖目錄
圖一、蝴蝶蘭經涼溫處理後誘導開花之生理變化44
圖二、進行蝴蝶蘭小分子RNA定序之四組組織及其RNA品質45
圖三、統計蝴蝶蘭小分子RNA在不同組織部位的長度分佈統計圖46
圖四、高保守性miRNAs於蝴蝶蘭不同組織表現情形47
圖五、PaAP2進行3′ 和5′ RACE擴增反應48
圖六、PaSPL的RT-PCR分析及胺基酸編碼49
圖七、PaAP2的RT-PCR電泳圖及胺基酸編碼50
圖八、PaSPL與阿拉伯芥、水稻中miR156目標基因的親緣演化分析51
圖九、PaAP2與阿拉伯芥、水稻、玉米中miR172目標基因的親緣演化分析52
圖十、白花蝴蝶蘭PaSPL與阿拉伯芥 (AtSPL2、10、11) 及水稻 (OsSPL3、12)進行多序列比對53
圖十一、白花蝴蝶蘭PaAP2與阿拉伯芥 (AtAP2) 、水稻(Os04g55560)進行多序列比對54
圖十二、蝴蝶蘭miR156和miR172與目標基因結合位置及其它物種序列之比較55
圖十三、利用5′ RACE證實PaSPL被miR156 cleavege的核苷酸位置56
圖十四、利用5′ RACE檢測PaAP2被miR156 cleavege的核苷酸位置57
圖十五、利用5′ RACE證實PaSPL2被miR156 cleavege的核苷酸位置58
圖十六、miR156和PaSPL於蝴蝶蘭不同組織表現情形59
圖十七、miR172和PaAP2於蝴蝶蘭不同組織表現情形60
圖十八、PaSPL和PaAP2於蝴蝶蘭不同組織表現情形61
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