RNA全長定序技術分析水稻於冷逆境中的剪接變體

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凌郁婷(Yu-Ting Ling) 查詢紙本館藏

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生命科學系

論文名稱

RNA全長定序技術分析水稻於冷逆境中的剪接變體
(Splicing variants analysis in rice upon cold stress by isoform sequencing)

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至系統瀏覽論文 (2026-10-16以後開放)

摘要(中)

選擇性剪接 (Alternative splicing, AS)為真核生物的後轉錄修飾機制 (post-transcription modification)裡重要的一環。在植物中，選擇性剪接不僅能產生多樣化的剪接變體 (splicing variants)，更能於外部環境刺激下（如：非生物脅迫性逆境）做出反應，適度調整使植物產生抗性。
低溫對水稻 (Oryza sativa L.)產量的影響巨大，因為其過度穩定水稻內不正確的RNA結構，導致RNA分子失去原本的活性與功能。為了研究受低溫影響的初級mRNA轉錄物 (pre-mRNA transcripts)剪接和選擇性剪接的參與情況，本論文使用PacBio同源異構體定序 (Isoform sequencing, Iso-seq)來克服短RNA定序技術的限制；而先前有研究指出水稻中DEAD-box RNA Helicase42 (OsRH42)基因是低溫下mRNA成熟過程中有效剪接的必要條件，因此樣本中使用OsRH42 RNAi轉基因植株與野生型水稻進行了比較。結果顯示，與水稻基因組注釋IRGSP-1.0相比，轉錄物的種類和數量更多，並且透過RT-PCR的方式證明篩選出來的資料，不僅顯示了AS和新轉錄物的多樣化表達，同時證實所獲得的Iso-seq數據的可靠性。
轉錄因子 (Transcription factors, TFs)和剪接因子 (Splicing factors, SFs)是受AS調節的典型因子，而在整理了不同的TF家族和SF分類後，發現低溫逆境和OsRH42基因的剔除對基因表現數量及其剪接變體皆有影響。進一步分析發現，WRKY轉錄因子家族中，被證實作為調節器參與水稻冷逆境的調控機制的兩個TF基因——OsWRKY71和OsWRKY76，受低溫影響而增加其表現量；於我們的數據中發現除了和註釋相同外，亦有其他種類的剪接變體組成，如：內含子保留 (intron retention, IR)與外顯子跳躍 (exon skipping, ES)等，而該結果可以作為往後研究個別轉錄因子、剪接因子與其splicing variants 參與水稻冷逆境中的作用與功能之重要依據。

摘要(英)

Alternative splicing (AS) plays important roles in the post-transcriptional modification of eukaryotes, including plants. Not only does AS generate a diverse range of splicing variants, but it also allows in response to external environmental stimuli, such as abiotic stresses, which can be fine-tuned for the stress responses. The impact of low temperature stress is significant for rice (Oryza sativa L.) yield because it leads to over-stabilization of incorrectly RNA structure and therefore inactivation of RNA molecules. To investigate the pre-mRNA splicing affected by the low temperature and the involvement of AS, we used PacBio isoform sequencing (Iso-seq) to overcome the limitations of short RNA sequencing technologies. Since the DEAD-box RNA Helicase42 (OsRH42) is known to be necessary to support effective splicing of pre-mRNA during mRNA maturation at low temperature, we used the OsRH42 RNAi transgenic lines which was compared with wild type . Our results present more diverse and abundant transcript isoforms, in comparison to rice genome annotation IRGSP-1.0. We also demonstrated the screened data by RT-PCR which showed the diverse expression in both AS and novel transcript, further confirming the confidence of the obtained Iso-seq data. Transcription factors (TFs) and splicing factors (SFs) are typical factors that regulate by the AS. After collating the different TF families and SF classifications, we found that cold stress and OsRH42-Ri lines had an impact on TF number and their splicing variant. Our data suggested that the TF families, OsWRKY71 and OsWRKY76, known as cold stress regulator in rice, which increase by cold, composed of annotated and AS variants. The results can therefore be further developed as a way to study the role of AS in different TFs after exposure to the cold.

關鍵字(中)

★ RNA全長定序
★ 選擇性剪接
★ OsRH42
★ 水稻
★ 冷逆境

關鍵字(英)

論文目次

摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VI
表目錄 VII
附錄 VII
縮寫檢索表 VIII
壹、序論 1
1. pre-mRNA剪接機制 1
2. 選擇性剪接 (Alternative splicing) 3
3. 低溫逆境與其對於水稻的影響 5
4. DEAD-box RNA helicase簡介與生理影響 7
5. 水稻DEAD-box RNA helicase 42 (OsRH42)簡介與功能 9
6. PacBio Isoform Sequencing (Iso-seq) 10
貳、實驗目的與研究動機 13
參、材料與方法 14
1. 植株樣品之逆境處理 14
1.1 植株培養 14
1.2 低溫 (4℃)逆境處理 14
2. PacBio Isoform Sequencing 14
2.1 萃取樣品之total RNA 14
2.2 樣品定序 15
2.3 樣品分析與水稻註釋序列對照 15
3. RT-PCR驗證逆境下差異表現之isoform 16
3.1 去除染色體DNA汙染 16
3.2 電泳RNA瓊脂膠體 16
3.3 合成cDNA 17
3.4 設計專一引子 17
肆、實驗結果 18
1. Iso‑seq提高水稻全長cDNA的鑑定 18
2. Iso‑seq揭示的水稻新transcripts 19
3. 根據Iso-seq結果的AS綜合分析 21
4. 四個樣品中IR與ES的個別分析 23
5. 水稻中響應冷逆境反應的轉錄因子 25
6. 水稻中響應冷逆境反應的剪接因子 27
伍、討論 31
1. Iso-seq數據與水稻基因庫選擇 31
2. AS分析中的剪切位點影響 31
3. TF與SF受OsRH42之影響 32
陸、參考資料 34
柒、圖表 39
捌、附錄 60

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指導教授

陸重安(Chung-An Lu)

審核日期

2023-10-16

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