水稻CCR4基因的功能分析-
繁衍大量表現和靜默表現的基因轉殖水稻

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姓名

何浩恩(Haw-En Ho) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

水稻CCR4基因的功能分析- 繁衍大量表現和靜默表現的基因轉殖水稻
(Functional analysis of CCR4 genesin rice-generation of overexpression and RNAi knock down transgenic rice plant )

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摘要(中)

基因表現的調控可以是在轉錄階層或是後轉錄修飾階層，甚至兩者都可影響。在真核中mRNA主要的降解機制是由尾端poly (A)的降解開始，此步驟是各樣mRNA降解的速率決定步驟，並在各樣的生物種間都可以發現這樣的情形。在酵母菌中， CCR4-NOT (carbohydrate catabolism repression 4-not)的複合體中的CCR4蛋白質，被認為扮演著降解 mRNA deadenylase的關鍵角色。然而，在植物中CCR4的功能還是非常不清楚。因此本實驗利用單子葉植物，水稻，來分析植物中，CCR4對mRNA 降解的影響。本實驗利用大量表現OsCCR4-1和 OsCCR4-2和RNAi knock down的轉殖水稻，來分析植物中CCR4對mRNA降解的影響。我們成功得到T1代的靜默植株和T2代的大量表現轉殖植株。分析癒傷組織 (callus)的OsCCR4-1和 OsCCR4-2表現量，初步結果認為沒有參予在糖調控基因α-amylase表現的pathway上。進一步，之後可找到T2代轉殖植株和T3 homologous 的植物，將來可以分析OsCCR4在水稻中的生化功能。

摘要(英)

In general, gene expression is regulated at either a transcriptional level or a post-transcriptional level, or even both. In eukaryotes, the major mechanism of mRNA degradation involves a poly A tail deadenylation. The deadenylation is the rate-limiting step in many mRNA degradation events in a wide-range of organisms. In yeast, the CCR4 (carbohydrate catabolism repression 4) protein, a part of the CCR4-NOT complex, have been proposed that play a prominent role as deadenylase in mRNA degradation. However, the function of CCR4 in plants is not clear yet. In this study we use the monocot model plant, rice, to further investigate the general role of plant CCR4 in mRNA degradation. The OsCCR4-1 and OsCCR4-2 overexpression and RNAi knock down transgenic rice were generated, and they will be analyzed for the functions of OsCCR4-1 and OsCCR4-2 in rice subsequently. The T1 RNAi plant and T2 overexpression rice plant have been identified. Continuously we will identified the T2 transgenic plant and T3 homologous transgenic rice plant.The function of OsCCR4-1 and OsCCR4-2.will be analyzed in the future.

關鍵字(中)

★ 水稻
★ 基因轉殖
★ 降解
★ 降解腺呤

關鍵字(英)

★ deadenylation
★ decay
★ turnover
★ mRNA
★ transgenic rice

論文目次

中文摘要...................................................................................................................... III
Abstract .......................................................................................................................IV
縮寫表........................................................................................................................... V
目錄..............................................................................................................................VI
壹，研究動機與目的............................................................................................. 1
貳，序論................................................................................................................ 2
一、基因表現與調控...................................................................................... 2
二、 mRNA 降解的生物意義......................................................................... 2
三、降解的路徑............................................................................................. 3
四、降解腺呤的酵素 deadenylating Enzymes............................................. 3
六、 CCR4 ...................................................................................................... 5
七、水稻......................................................................................................... 6
八、 RNAi 的作用機制................................................................................... 6
九、水稻的ccr4 家族.................................................................................... 7
十、水稻中CCR4-1 和CCR4-2 的表現........................................................ 8
參，實驗材料方法................................................................................................. 9
第一部建構載體......................................................................................... 9
1. 選殖載體................................................................................................. 9
2. 接合反應............................................................................................... 10
3 細菌的轉型作用.................................................................................... 11
4 DNA 序列分析...................................................................................... 13
第二部水稻基因轉殖....................................................................................... 13
5 水稻未成熟胚(或成熟胚)之癒傷組織(callus)誘導............................... 13
6 水稻細胞利用農桿菌(Agrobacterium)轉型 (EHA105 strain)............. 14
第三部水稻轉殖株分析..................................................................................... 15
7 轉殖株GUS assay ................................................................................ 15
8 水稻基因組DNA 的分析..................................................................... 16
9 水稻RNA 分析..................................................................................... 17
10 糖反應分析.................................................................................... 18
肆，實驗結果....................................................................................................... 19
1. OsCCR4 基因的比對.................................................................................... 19
2. OsCCR4-1 及OsCCR4-2 RNAi 基因轉殖水稻的建立................................. 19
2.1. 構築RNAi 位置............................................................................ 19
2.2. 構築載體RNAi 轉殖入水稻中.................................................... 19
2.3. 基因轉植水稻癒傷組織的建立與確認分析................................. 19
2.4. 繁衍表達OsCCR4-1及OsCCR4-2 RNAi基因轉殖水稻植株的分析
20
3. 繁衍OsCCR4-1 及OsCCR4-2 大量表現轉殖水稻...................................... 21
3.1. 癒傷組織的建立與分析................................................................ 21
3.2. 大量表現OsCCR4-1 及OsCCR4-2 基因的T2 水稻轉殖植株分析
22
4. 分析水稻中OsCCR4 基因的表現模式........................................................ 22
4.1. OsCCR4 基因在含糖與缺糖環境下表現情形.............................. 22
伍，討論.............................................................................................................. 23
1. OsCCR4 基因的比對.................................................................................... 23
2. 分析OsCCR4-1 與OsCCR4-2 在水稻內的功能-OsCCR4-1 及OsCCR4-2
RNAi 基因轉殖水稻的建立................................................................................. 23
3. 過量表達OsCCR4-1 及OsCCR4-2 轉殖水稻的分析.................................. 24
4. OsCCR4-1 及OsCCR4-2 和糖調控基因表現的探討................................ 24
5. OsCCR4-1 及OsCCR4-2 的蛋白表達........................................................ 25
陸，文獻參考....................................................................................................... 45
附錄I(藥品配方)........................................................................................................ 50
附錄II (載體構築)....................................................................................................... 61

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指導教授

陸重安(Chung-An Lu)

審核日期

2009-1-20

推文