博碩士論文 93224019 詳細資訊




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姓名 黃偉哲(Wei-Che Huang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 水稻CCR4基因之功能分析- 水稻CCR4基因的選殖、定性及表現
(Functional Analysis of The CCR4 Genes in Oryza sativa-Cloning, Characterization and Expression of the Rice CCR4 )
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摘要(中) 從酵母菌到人類都存在著CCR4-NOT複合體,這複合體對於基因表達是個整體的調節者。複合體中的各個蛋白質對於不同層面的mRNA 代謝都有關聯,其中包括transcription initiation、elongation和mRNA degradation。其中一種影響基因表現的調控是控制mRNA 的半衰期。大部分的情況下,調控真核細胞mRNA半衰期的主要路徑是先將poly(A) tail縮短。在酵母菌的研究得知,CCR4蛋白質是CCR4-NOT 複合體中的一部分,目前已經證實在細胞質中具有deadenylation的作用。然而CCR4基因在植物系統中的功能並不清楚,所以本篇論文將進一步探討CCR4基因在水稻中的功能。
首先,透過資料庫比對分析發現有5個和酵母菌CCR4同源性較高的OsCCR4蛋白質,同時也發現皆屬於Mg2+-dependent exonuclease蛋白質家族。並發現在缺糖條件下,5個OsCCR4基因表現量較含糖條件下高,對於含糖條件下的相關基因可能有調控的作用。OsCCR4基因在水稻中分別有其組織特異性,其中OsCCR4-2則對於所有組織都有其重要性。藉由GFP融合OsCCR4-1、OsCCR4-2,發現OsCCR4-1、OsCCR4-2在細胞核及細胞質中皆有表現,所以可能參與了細胞核及細胞質中mRNA的deadenylation。並且成功建立過量表達GFP融合OsCCR4-1及OsCCR4-2的轉殖水稻,之後可進一步分析OsCCR4-1與OsCCR4-2在水稻內的功能。在E. Coli 表達系統中,也成功的表達出soluble的OsCCR4-1、OsCCR4-2重組蛋白質,之後將透過In vitro deadenylase activity分析,確認OsCCR4蛋白質的生化功能。
摘要(英) The CCR4-NOT complex is a global regulator of gene expression that is conserved from yeast to human. The proteins of the complex are involved in several aspects of mRNA metabolism, including transcription initiation and elongation and mRNA degradation. One of the regulations of gene expression is influenced by the control of mRNA turnover. In most cases, the major pathway of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. In yeast, the CCR4 (carbohydrate catabolism repression 4) protein, as part of the CCR4-NOT complex, has been show to be responsible for cytoplasmic deadenylation. However, the function of CCR4 in plants is not clear yet. In this research, we will investigate the functions of CCR4 in rice. First, there are five OsCCR4 proteins have high homology with yeast CCR4 which compared with the database. And all belongs to Mg2+-dependent exonuclease protein family. The five OsCCR4 genes have higher expression under sugar starvation, so that they have the possibility to regulate the sugar-related genes. The OsCCR4 genes have their tissue specificity in rice. Besides, OsCCR4-2 plays a important role for all tissues in rice. Through the GFP fused with OsCCR4-1 and OsCCR4-2 separately, we observed that OsCCR4-1 and OsCCR4-2 express both in nucleus and cytoplasm. So the OsCCR4-1 and OsCCR4-2 may participate the mRNA deadenylation in nucleus and cytoplasm. The transgenic rice which overexpressed the GFP fused with OsCCR4-1 and OsCCR4-2 separately were successful established, and will be analyzed for the functions of OsCCR4-1 and OsCCR4-2 in rice subsequently. The soluble recombinant proteins of OsCCR4-1 and OsCCR4-2 were expressed in E. Coli. In vitro assay of the deadenylase activity will be performed to ensure the biochemical function of OsCCR4-1 and OsCCR4-2.
關鍵字(中) ★ 水稻CCR4基因 關鍵字(英) ★ OsCCR4
論文目次 中文摘要 I
Abstract II
縮寫表 III
本文目錄 V
圖目錄 IX
表目錄 X
本文目錄
壹、研究動機與目的 1
貳、前人研究 2
1.基因表現的調控機制 2
2.mRNA 降解 3
2.1.mRNA 降解的生物意義 3
2.2.mRNA 降解的路徑 3
3.去腺嘌呤酶(deadenylase) 5
4.CCR4-NOT complex 7
5.CCR4 8
6.水稻 11
7.植物基因轉殖系統 12
參、實驗材料與方法 14
第一部分 構築質體 14
1.資料庫序列分析 14
1.1.Database 14
1.2.Analysis Tool 14
2.製備cDNA 14
2.1.製備DEPC-treated ddH2O(RNase-free) 溶液 14
2.2.RNA的純化(TRIZOL® reagent,Invitrogen) 14
2.3.去除RNA中DNA(DNA-freeTM,Ambion) 15
2.4.合成cDNA(The SuperScriptTMIII First-Strand Synthesis System for RT-PCR,Invitrogen) 15
3.以PCR分別合成OsCCR4s基因片段 16
3.1.primer的設計 16
3.2.以PCR合成DNA片段 16
3.3.瓊脂膠體回收限制酵素沏過的DNA(使用 Gel/PCR DNA Fragments Extraction Kit DF300,Geneaid) 17
4.接合反應 18
4.1.線性載體DNA的去磷作用(Dephosphorylation of linearized plasmid DNA) 18
4.2.黏頭端載體DNA與插入DNA片段的接合(Ligation of cohesive-ended DNA fragment) 19
4.3.齊頭端DNA片段的接合(Ligation of blunt-ended DNA fragment) 20
5.細菌的轉殖作用 20
5.1.製備E. coli competent cell(JM109 strain,BL21 strain) 20
5.2.細菌的轉殖(transformation) 21
5.3.小量純化細菌質體DNA法 22
Quick Mini-prep. of plasmid 22
High-Speed plasmid Mini Kit PD300, Geneaid 22
5.4.大量純化細菌質體DNA法(Plasmid Midi Kit,Geneaid) 23
6.DNA序列分析 24
第二部分 水稻基因轉殖 24
7.水稻原生質體轉殖方法 25
7.1.水稻未成熟胚(或成熟胚)之癒傷組織(callus)誘導 25
7.2.水稻懸浮培養細胞的誘導 25
7.3.分離水稻原生質體 25
7.4.水稻原生質體轉殖 26
8.洋蔥表皮細胞(onion epidermal cell)利用微粒子投射法(particle bombardment)的轉殖 26
8.1.洋蔥表皮細胞的準備 26
8.2.基因槍設定 27
8.3.金粒子的製備 27
8.4.DNA附著 27
8.5.轉殖植物樣品處理 28
9.水稻細胞利用農桿菌(Agrobacterium)轉殖的方法 28
9.1.製作Agrobacterium competent cell(EHA105 strain) 28
9.2.農桿菌的轉殖方法 29
9.3.農桿菌的生化檢測(Ketolactose test) 29
9.4.水稻的轉殖作用 30
第三部分 水稻轉殖株分析方法 31
10.轉殖株GUS assay 31
11.轉殖水稻RNA分析 31
11.1.植物RNA的抽取 31
11.2.RT-PCR分析植物RNA 31
12.轉殖水稻蛋白質分析 31
12.1.植物蛋白質的抽取 31
12.2.植物蛋白質分析 32
13.基因組DNA PCR 32
13.1.基因組DNA 的抽取與純化 32
13.2.PCR 33
第四部分 蛋白質表達 33
14.利用pET 載體系統表達目標蛋白質 33
14.1.將插入DNA接入pET 載體 33
14.2.細菌的轉殖 33
14.3.蛋白質表達 33
14.4.細胞蛋白質萃取 34
14.5.蛋白質純化(IMAC sample Kit,Pro-Chem) 34
14.6.蛋白質濃縮 34
14.7.蛋白質分析 35
蛋白質電泳(SDS-PAGE) 35
西方墨點法(Western blot analysis) 35
肆、實驗結果 37
1.OsCCR4基因的選殖 37
1.1.資料庫比對分析 37
1.2.OsCCR4基因特性分析 37
1.3.選殖OsCCR4基因 38
2.分析水稻中OsCCR4基因的表現 38
2.1.OsCCR4基因在含糖/缺糖環境下表現情形 38
2.2.OsCCR4基因在水稻組織的表現模式 39
3.分析OsCCR4-1及OsCCR4-2在細胞中的位置 39
3.1.載體的構築 40
3.2.OsCCR4-1及OsCCR4-2在細胞中的位置 40
4.分析OsCCR4-1與OsCCR4-2在水稻內的功能-OsCCR4-1及OsCCR4-2 overexpression 基因轉殖水稻的建立與分析 41
4.1.過量表達GFP融合OsCCR4-1及OsCCR4-2基因轉殖水稻的建立 41
4.1.1.載體的構築 41
4.1.2.利用GUS活性分析與螢光顯微鏡觀察GFP確認成功之T0轉殖癒傷組織 41
4.2.利用螢光顯微鏡觀察再生成功的轉殖植株根部GFP表現情形 42
4.3.過量表達GFP融合OsCCR4-1及OsCCR4-2基因轉殖水稻的分析 42
4.3.1.pAUG-1轉殖植株分析 42
4.3.2.pAU1-G轉殖植株分析 43
4.3.3.pAUG-2轉殖植株分析 43
4.3.4.pAU2-G轉殖植株分析 44
5.OsCCR4-1、OsCCR4-2重組蛋白的表達 45
伍、討論 46
1.OsCCR4基因的選殖 46
2.水稻中OsCCR4基因的表現 46
3.OsCCR4-1與OsCCR4-2在細胞中的位置 47
4.過量表達GFP融合OsCCR4-1及OsCCR4-2基因轉殖水稻的建立與分析 48
5.OsCCR4-1、OsCCR4-2重組蛋白質的表達 50
陸、參考文獻 86
附錄 I 92
附錄 II 108
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指導教授 陸重安(Chung-An Lu) 審核日期 2006-9-20
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