水稻CAF1基因在水稻懸浮培養細胞之研究

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方振丞(Jhen-Cheng Fang) 查詢紙本館藏

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生命科學系

論文名稱

水稻CAF1基因在水稻懸浮培養細胞之研究
(Functional Analysis of OsCAF1 Genes in Rice Suspension Cells)

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摘要(中)

mRNA降解作用為一個重要的基因表現調控機制，參與調控基因的表現，改變胞內狀態，以因應不同外在環境的變化。Poly(A) tail deadenylation為mRNA降解的速率決定步驟，而CCR4-associated factor 1s（CAF1s）在真核生物中催化deadenylation的一個主要酵素。水稻中含有四個不同的CAF1，分別為OsCAF1A、OsCAF1B、OsCAF1G、OsCAF1H。由北方墨點法與RT-PCR的分析顯示，OsCAF1s分別受到糖及不同的非生物逆境所調控。針對rOsCAF1A與rOsCAF1B重組蛋白進行胞外活性分析，結果指出其確實具有deadenylase的活性。先前的研究指出水稻受糖調控之α-澱粉降解酶的基因，αAmy3，其mRNA的半衰期會受到糖的調控。分析有糖及缺糖培養情況下之水稻細胞αAmy3 mRNA poly(A) tail長度，結果顯示在缺糖培養時其poly(A) tail長度較有糖培養長。有趣地是，在大量表現OsCAF1A轉殖水稻懸浮細胞中，發現其參與在αAmy3 mRNA的累積。由先前研究指出，OsMYBS1與OsMYBS2被證實參與αAmy3基因的表現，分別作為正向與反向的轉錄調控因子。由此分析大量表現OsCAF1A轉殖水稻懸浮細胞OsMYBS1與OsMYBS2的表現情況，結果顯示OsMYBS1表現會大量提升而OsMYBS2表現量會因此而下降。以上結果顯示OsCAF1A參與在OsMYBS1與OsMYBS2 deadenylation過程中，進而調控αAmy3 mRNA的累積。

摘要(英)

One of central mechanisms to determine the expression level of genes for adjustment cellular status in response to various signals is messenger RNA (mRNA) degradation. Poly(A) tail deadenylation is a rate-limiting step of mRNA degradation. CCR4-associated factor 1s (CAF1s) is one of major enzymes to catalyze mRNA deadenylation in eukaryotes. Four members of CAF1 family, designated OsCAF1A, B, G and H, were analyzed and characterized in rice (Oryza sativa). Northern experiments and RT-PCR revealed diverse transcription patterns in response to sugar and various abiotic stresses among the four genes. Two recombinant proteins, rOsCAF1A and rOsCAF1B, had a deadenylation activity in vitro. It is known that mRNA half-life of αAmy3, which encodes a α-amylase, is regulated by sugar. Poly(A) tail analysis indicated that the length of poly(A) tail of αAmy3 mRNA was longer in the sugar-starved cells than in sugar-feasted cells. Interestingly, ectopic expression approach indicated that OsCAF1A was involved in αAmy3 mRNA accumulation. Previously, the OsMYBS1 and OsMYBS2 have been demonstrated to play a positive and negative regulator for αAmy3 expression, respectively. RT-PCR analysis indicated that ectopic expressed OsCAF1A caused the expression level of OsMYBS2 decreasing and the OsMYBS1 increasing. These results suggested that OsCAF1A may play a crucial role in mRNA deadenylation of OsMYBS1 and OsMYBS2 to regulate αAmy3 mRNA accumulation in rice.

關鍵字(中)

★ 糖
★ mRNA的降解
★ α-澱粉水解酶

關鍵字(英)

★ OsMYBS2
★ OsMYBS1
★ α-amylase
★ CCR4-associated factor 1s
★ mRNA deadenylation
★ mRNA degradation
★ sugar

論文目次

中文摘要.............................. ....................I
Abstract.............................. ...................II
本文目錄.............................. ..................III
圖目錄....................................................V
表目錄..................................................VII
本文目錄
壹、緒論.............................. ....................1
一、mRNA的降解機制：......................................1
二、去腺嘌呤酶（deadenylase）.............................3
三、CCR4-Not complex......................................3
四、CAF1的酵素特性及生理功能：............................4
五、CAF1在植物中的研究：..................................6
六、α-澱粉水解酶（α-amylase）..........................6
七、賀爾蒙對於α-澱粉水解酶基因的調控.....................7
八、OsMYBS1、OsMYBS2、OsMYBS3對α-澱粉水解酶基因的調控....8
貳、研究目的.............................................10
參、材料與方法...........................................11
Rice cell culture and sugar treatment....................11
Plasmid..................................................11
Reverse transcription-polymerase chain reaction (RT-PCR).11
Real-time quantitative RT-PCR............................11
Poly(A) tail (PAT) assay.................................12
Plasmid construction.....................................12
Rice Embryo Transient Expression Assay................... 12
Luciferase and GUS Activity Assay........................13
Rice Transformation ......................................14
肆、實驗結果.............................................15
1. 分析水稻α-amylase基因................................15
1.1 αAmy3基因在有糖及缺糖的表現模式.....................15
1.2 比較αAmy3基因於有糖及缺糖下Poly(A) tail長度.........15
1.3 分析αAmy3基因其3’ UTR是否會受到糖的調控............15
2. 分析水稻中CAF1基因的表現..............................16
2.1 CAF1在不同物種間蛋白質序列分析比對...................16
2.2 CAF1在不同物種間的演化樹關係圖.......................16
2.3 OsCAF1s在有糖缺糖環境下表現模式......................17
2.4 OsCAF1s在各種逆境下的表現模式........................17
3. 分析OsCAF1A大量表現和靜默表現轉植水稻.................18
3.1 確認OsCAF1A大量表現與靜默表現轉殖水稻懸浮培養細胞....18
3.2 OsCAF1A轉殖懸浮培養細胞中αAmy3基因表現量............19
3.3 OsCAF1A轉殖懸浮培養細胞中OsMYBS1與OsMYBS2基因表現量..19
4. 建立OsCA1B大量表現與靜默表現轉殖水稻..................20
4.1 載體的構築...........................................20
4.2 確認OsCAF1B靜默表現轉殖成功之T0水稻癒傷組織..........20
伍、討論.................................................21
Table 1..................................................25
陸、參考文獻.............................................39
柒、附錄.................................................43

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指導教授

陸重安(Chung-An Lu)

審核日期

2011-7-29

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