水稻OsVALs基因的功能性分析-
水稻OsVALs基因的選殖、定性及表現

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姓名

陳麗雅(Li-Ya Chen) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

水稻OsVALs基因的功能性分析- 水稻OsVALs基因的選殖、定性及表現
(Functional analysis of OsVAL genes in rice - identification, characterization and expression )

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摘要(中)

高等植物的種子發育可分為胚胎發育期及成熟期。種子成熟後隨即進入休眠，然後於適當的時間萌芽。B3 domain 家族是參與種子發育的重要轉錄因子。以玉米為例，vp1 基因突變後，玉米種子不會經過休眠期，直接在母體的穗上萌發。相似的，阿拉伯芥中玉米VP1的orthology基因，ABI3，在abi3 基因突變後，阿拉伯芥種子也會對離層酸不感知，並產生綠色不成熟的種子。最近，在阿拉伯芥中發現三個VP1/ABI3-Like(VAL)基因，AtVAL1、AtVAL2、及AtVAL3 ，已經知道它們在阿拉伯芥幼苗的生長發育上扮演重要的角色。此外，AtVAL1/HSI2 也是受糖調控Spomin 啟動子的抑制子。
水稻是世界上重要的糧食作物之ㄧ，也是很好的單子葉模式植物。由資料庫的比對，找到在水稻中有兩個和阿拉伯芥AtVALs序列相近OsVALs基因，分別將它們命名為OsVAL1和OsVAL2。本篇論文中，利用建構OsVALs過量表達及基因靜默的轉殖水稻，分析在單子葉植物水稻中，OsVALs 是否參與糖訊息調控的途徑中以及對水稻的生長發育是否具有影響。在初步的實驗中發現，OsVAL1及OsVAL2 在水稻各部位組織皆有表現，並且在水稻種子萌發的過程，第一天就開始表現。此外，在OsVAL1 過量表達轉殖水稻方面，有四個獨立的品系(#19、#41、#59、#61)的葉片上有明顯褐斑的生成；而在OsVALs 基因靜默方面，相較於野生型水稻，則有較早結穗的外表型。除此之外，也發現OsVALs在水稻中是受糖所調控的，在缺糖的情況下比有糖的情況表現量多，並且推測，對於水稻的澱粉水解酶αAmy3 可能是作為一個抑制子的角色。

摘要(英)

The VP1/ABI3, which belongs to B3 domain like transcription factor,plays an importment role during plant seed embryogenesis and maturation. Previous studies have shown that when vp1/abi3 mutate, seeds could not sense endogenous abscisic acid and then direct germinate without dormancy. Recently,three VP1/ABI3-LIKE (VAL) genes, AtVAL1, AtVAL2, and AtVAL3, have been identified from Arabidopsis and played essential roles in seedlings growth. Moreover, AtVAL1/HSI2 has been demonstrated as a transcriptional repressor for a sugar-inducible Spomin promoter.
Rice is one of the most important crops and also a model plant for monocots. In order to study the function of VAL genes in rice seed development and in sugar signal transduction pathway, we identified two AtVAL homologs, OsVAL1 and OsVAL2, and then gain-of-function and loss-of-function analyses were applied. We find that OsVALs are ubiquitously expressed in rice different tissues, and emerge after rice seeds imbibed in water only one day. Moreover, four independent lines of OsVAL1 overexpression transgenic rice exhibit distinct brownish lesions. On the other hand, OsVAL RNAi transgenic rice plants present earlier heading than wild-type. In addtion,OsVALs are sugar-regulated and may play as a repressor for the rice αAmy3 gene.

關鍵字(中)

★ 水稻
★ 糖

關鍵字(英)

★ HSI2
★ sugar
★ rice
★ B3 domain
★ ABI3
★ VP1
★ VAL

論文目次

中文摘要……………………………………………………………I
Abstract……………………………………………………………II
誌謝…………………………………………………………………III
縮寫表………………………………………………………………IV
本文目錄……………………………………………………………V
附圖目錄……………………………………………………………VIII
圖目錄………………………………………………………………IX
表目錄………………………………………………………………X
本文目錄
壹、緒論 1
一. 種子的發育 1
二. 玉米VP1及阿拉伯芥ABI3基因 1
三. 阿拉伯芥VP1/ABI3-LIKE(VAL)基因 2
四. 阿拉伯芥HSI2基因 3
五. 水稻 7
六. Α-澱粉水解酶 7
七. 水稻種子的萌發與Α-澱粉水解酶基因表現的調控 8
貳、研究動機與目的 9
参、材料與方法 10
第一部分構築質體 10
1.資料庫序列分析 10
1.1. Database 10
1.2. Analysis Tool 10
2.製備cDNA 10
2.1. 製備DEPC-treated ddH2O 10
2.2. RNA的抽取 10
2.3. 去除RNA中DNA 10
2.4. 合成cDNA 11
3.以PCR的方式合成OsVALs的基因片段 11
3.1. primer的設計 11
3.2. 以PCR合成DNA片段 11
3.3. 瓊脂膠體回收限制酵素切過的DNA 12
4.接合反應 13
4.1. 線性載體DNA的去磷作用 13
4.2. 黏頭端載體DNA與插入DNA片段的接合 13
4.3. 齊頭端DNA片段的接合 13
5.以Gateway系統建構表達載體 14
5.1. Topo cloning reaction 14
5.2. LR recombination 14
6.細菌的轉型作用 15
6.1. 製備E. coli competent cell 15
6.2. 細菌的轉型 16
6.3. 小量純化細菌質體DNA法 16
Quick Mini-prep. of plasmid 16
High-Speed plasmid Mini Kit 17
6.4. DNA定序分析 17
第二部分水稻基因轉殖 17
7.水稻細胞利用農桿菌(Agrobacterium)轉殖的方法 17
7.1. 水稻未成熟胚之癒傷組織誘導 17
7.2. 製作Agrobacterium competent cell (EHA105 strain) 18
7.3. 農桿菌的轉殖方法 18
7.4. 農桿菌的生化檢測(Ketolactose test) 19
7.5. 水稻的轉殖作用 19
第三部分水稻轉殖株分析方法 20
8.轉殖株GUS assay 20
9.基因組DNA的PCR分析 20
9.1. 基因組DNA 的抽取 20
9.2. 基因組DNA PCR 20
10.轉殖植物RNA分析 20
10.1. 轉殖植物RNA的抽取 20
10.2. RT-PCR分析轉殖水稻中RNA的表現 21
肆、結果 22
1. OSVALS基因的特性分析 22
1.1. 資料庫比對分析 22
1.2. OsVALs的特性分析 22
2. 建立OSVAL1及OSVAL2過量表達的轉殖水稻 22
2.1. 利用Gateway system構築OsVAL1及OsVAL2的過量表達載體 22
2.2. 將過量表達載體HA-VAL1及HA-VAL2轉殖到水稻中 23
2.3. OsVAL1過量表達轉殖植株的分析 23
2.3.1. OsVAL1過量表達轉殖植株基因組DNA的分析 23
2.3.2. OsVAL1過量表達轉殖植株RNA的分析 23
2.4. OsVAL2過量表達轉殖水稻的分析 24
2.4.1. OsVAL2過量表達轉殖植株基因組DNA的分析 24
2.4.2. OsVAL2過量表達轉殖植株RNA的分析 24
3. 建立靜默表達的OSVAL1及OSVAL2的轉殖水稻 24
3.1. OsVAL1及OsVAL2靜默表達的引子設計 24
3.2. 靜默表達OsVAL1及OsVAL2的載体構築 24
3.3. 將構築好的靜默表達載體pAUVAL1-RNAi及pAUVAL2-RNAi一起
感染(co-transfection)到水稻癒傷組織中 25
3.4. GUS活性分析確認轉OsVALs靜默表達轉殖細胞株 25
3.5. OsVAL1及OsVAL2靜默表達轉殖植株基因組DNA的分析 26
3.6. OsVAL1及OsVAL2靜默表達轉殖植株RNA分析 26
4. 分析OSVALS基因在水稻中的表現情形 26
4.1. OsVALs在水稻不同組織的表現模式 26
4.2. OsVALs在水稻種子萌發的表現情形 27
4.3. OsVALs在有糖及缺糖的表現情形 27
伍、討論 29
1. 水稻OSVALS基因的選殖 29
2. 過量表達OSVAL1及OSVAL2的轉殖植株的建立與分析 29
3. 靜默表達OSVAL1和OSVAL2的轉殖植株的建立及分析 30
4. OSVALS在水稻中的表現情形分析 31
陸、參考文獻 55
附錄I (藥品配製) 58
附錄II (載體構築) 65
附錄III 73

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指導教授

陸重安(Chung-an Lu)

審核日期

2009-1-19

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