水稻熱誘導OsCAF1H去腺苷化酶之功能分析

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梁凱茵(Kai-Yin LIANG) 查詢紙本館藏

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生命科學系

論文名稱

水稻熱誘導OsCAF1H去腺苷化酶之功能分析
(Characterization of a heat responsive OsCAF1H deadenylase in rice)

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至系統瀏覽論文 (2024-8-1以後開放)

摘要(中)

植物由於不能移動，在面對環境逆境時，會藉由快速的基因調控影響後續的蛋白質表現，進而改變生理狀態以達到適應環境逆境的目的。mRNA是基因表現的重要一環，其數量多寡是由轉錄階層與後轉錄階層共同調控。在真核生物中，mRNA deadenylation是大部分mRNA降解的初始步驟，也是速率決定步驟，作用方式為縮短poly(A) tail的長度，降低mRNA的穩定度，使其容易被胞內其他酵素降解或降低轉譯的效率，最終影響了蛋白質含量。CCR4-NOT複合體在mRNA deadenylation中扮演重要角色，它乃利用carbon catabolite repressor 4 (CCR4)和CAF1 (CCR4-associated factor 1)這兩個deadenylase參與在降解mRNA的機制中。水稻含4個CAF1基因，其中OsCAF1H在水稻受高熱時，表現量會大幅度提升，因此本篇論文將進一步探討水稻CAF1H基因在水稻受高熱下的功能。
透過啟動子序列比對分析發現水稻CAF1H包含了非典型的heat shock element (HSE)序列，並在GUS活性分析和組織化學染色法分析實驗中證實，水稻CAF1H基因是一個受熱誘導表現的基因。生理學實驗的部分，相較野生型(WT)、靜默表現(RNA interference) 和先前建立的基因剔除(knock out)轉植株，大量表現(overexpression) 水稻CAF1H基因的轉植株對高熱會有較好的耐受性，表現在水稻種子受熱處理後的發芽率和水稻植株受熱處理後的存活率。根據RNA-seq分析的結果，我們發現Histone 2A基因在野生型水稻受熱後表現量會顯著下降，但在OsCAF1H 基因剔除的轉植株卻沒有明顯的差異，推測Histone 2A可能為OsCAF1H的下游目標基因之一。

摘要(英)

Plants cannot move under environmental stress so they use subsequent protein expression through rapid gene regulation, thereby changing their physiological state to achieve the purpose of adapting to environmental stress. mRNA is an important part of gene expression, and its quantity is regulated by the transcriptional and post-transcriptional level. In eukaryotes, mRNA deadenylation is the first step in the degradation of most mRNAs, and it is also a rate-determining step. In this step, deadenylase will short the length of poly(A) tail to make the mRNA unstable and easily to be degraded and then reduce the efficiency of translation, which ultimately affects protein content. The CCR4-NOT complex plays an important role in mRNA deadenylation, which utilizes two deadenylases, carbon catabolite repressor 4 (CCR4) and CAF1 (CCR4-associated factor 1), to participate in the mechanism of mRNA degradation. Rice contains 4 CAF1 genes, among which OsCAF1H will greatly increase the expression when rice is exposed to heat stress. Therefore, this study will further explore the function of rice CAF1H under heat stress in rice.
Through GUS activity analysis and histochemical staining experiments, we knew that the rice CAF1H gene is a heat-inducible gene, and according to the promoter sequence alignment analysis, we found that rice CAF1H contained an atypical heat shock element (HSE) sequence. According to the germination rate test and the survival rate test, the CAF1H overexpression lines show better tolerance to heat stress compare with WT and CAF1H knock out lines. According to the results of RNA-seq analysis, we found that the expression of Histone 2A was significantly decreased in wild-type after heat stress, but there was no significant difference in the OsCAF1H knockout transgenic lines. It is speculated that Histone 2A may be one of the downstream target genes of OsCAF1H.

關鍵字(中)

★ 水稻
★ 熱逆境
★ OsCAF1H
★ 去腺苷化酶

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 VI
壹、序論 1
1. mRNA降解 1
2. Deadenylase 3
3. CCR4-NOT complex 4
4. CAF1 5
5. CRISPR/Cas9 6
6. 熱逆境 8
貳、材料與方法 10
1. cDNA製備 10
1.1 製備DEPC treated ddH2O (RNase-free solution) 10
1.2 萃取total RNA 10
1.3 去除染色體DNA汙染 10
1.4 合成cDNA 11
2. CRISPR-Cas9 轉植株 11
2.1 設計引子 11
2.2 Spacer序列接合至sgRNA質體中 11
2.3 sgRNA與Cas9質體進行LR 12
3. CRISPR-Cas9 轉植株突變點分析 12
3.1 瓊脂膠體回收DNA 12
4. GUS activity assay 13
4.1 萃取植物總蛋白質 13
4.2 蛋白質含量測定 13
5. GUS assay 14
6. GUS staining assay 14
7. 在逆境下OsCAF1H基因表現分析 15
7.1 水稻植株培養 15
7.2 逆境處理 15
7.3 基因表現分析 15
參、實驗結果 17
1. OsCAF1H為熱誘導基因 17
2. CRISPR/Cas9 OsCAF1H knockout轉植株建立 18
3. OsCAF1H參與水稻耐熱機制調控 19
4. OsCAF1H下游目標基因分析 21
肆、討論 23
1. OsCAF1H為熱誘導基因 23
2. CRISPR/Cas9 OsCAF1H knockout轉植株建立 24
3. OsCAF1H參與水稻耐熱機制調控 25
4. OsCAF1H下游目標基因分析 26
伍、參考資料 27
陸、圖表 31
柒、附錄 56

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指導教授

陸重安

審核日期

2022-1-28

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