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研究生:陳瑩瑾
研究生(外文):Ying-Chin Chen
論文名稱:利用T-DNA插入突變水稻基因庫找出種子組織特異性的啟動子
論文名稱(外文):Screeing of Seed-specific Promoters from T-DNA Insertional Transgenic Rice Plants
指導教授:賀端華
指導教授(外文):Tuan-Hua Ho
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:74
中文關鍵詞:水稻種子啟動子
外文關鍵詞:riceseedpromoter
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在調控基因表現的過程中,啟動子 ( promoter ) 扮演著重要的角色。啟動子可控制該基因表現之強度、組織特異性、時期性,以及對環境的反應。在受到不同環境的誘導 (如缺水、高鹽、抗低溫),啟動子決定了基因活化的不同條件。我們可根據啟動子具有專一調控基因表現的分子機制特性,用來導入外來基因,了解與研究基因的功能。組織特異性或可誘發的啟動子可用於比較表現與不表現外源基因對細胞所造成的差異,更可避免持續表現具外源蛋白質所引起的副作用,因此善加利用啟動子的特性,挑選構築表現量高、調控機制健全又不影響植物生理的啟動子成為了實驗設計必需考量的第一步。目前在雙子葉中常被使用的啟動子有:CaMV35S (持續性表現)、CruA (種子專一性)、RubS (葉子專一性)、Pat (球莖專一性)、Spo (塊根專一性);單子葉常使用的則有: Act、Ubi (持續性表現)、Glu (種子專一性)、αAmy (種子發芽專一性)。
利用報導基因隨機插入基因體來尋找基因及啟動子的研究策略已經成功應用在許多不同物種上,包括了細菌、果蠅、老鼠、和植物等。本篇研究利用中央研究院余淑美及邢禹依教授建立之台灣水稻T–DNA插入突變基因庫 ( T–DNA insertional rice mutant library) 中的基因突變水稻作為研究材料。藉由對突變水稻組織之 GUS染色分析,我們得到了四個只有在種子才會有GUS蛋白活性的突變株。經過T–DNA 插入點兩旁序列比對及RT-PCR的實驗分析,我們進一步確認了這些標定基因之種子組織特異性。將這些標定基因的啟動子序列 (長度約2 kb) 與報導基因 GUS 構築表現載體,經轉殖技術 (基因槍暫時表現及農桿菌媒介法) 將之送入野生型水稻中進行表現。基因槍暫時表現實驗結果我們証實M36928A及M29866兩個水稻突變品系為種子組織特異性的,待收到經農桿菌轉殖的轉基因水稻T1種子後,便可確認該啟動子在種子表現的再現性。未來透過對啟動子序列組成的分析,將有助於了解該基因的功能。
中文摘要……………………………………………… i
英文摘要…………………………………………… ii
縮寫字對照表………………………………………… iv
前言…………………………………………………… 1
前人研究……………………………………………… 2
一、水稻功能性基因體研究………………………… 2
二、啟動子的重要性及基因調控…………………… 5
三、尋找啟動子的方法……………………………… 7
四、啟動子的應用…………………………………… 7
五、啟動子應用的例子……………………………… 8
六、利用T-DNA插入突變體庫找尋具種子組織特異性之啟動子 9
材料與方法…………………………………………… 11
一、基因突變水稻…………………………………… 11
二、基因突變水稻種子篩選與分析………………… 11
1.GUS 活性篩選……………………………………… 11
2.Franking sequence 分析………………………… 11
3.Starch/hormone plate assay 澱粉/荷爾蒙培養基分析……13
三、基因表現分析…………………………………… 14
1.Total RNA 之萃取………………………………… 14
2.反轉錄聚合酶連鎖反應…………………………… 14
3.南方墨點分析 ………………………………… 16
四、表現載體構築方法……………………………… 17
1.宿主勝任細胞 ( competent cells ) 的製備…… 18
2.少量質體 DNA 之萃取……………………………… 18
3.大量質體 DNA 之萃取 ……………………… 19
4.聚合酶連鎖反應…………………………………… 20
5.自洋菜瓊脂膠體回收 DNA 片段 ………… 21
6. DNA 片段與載體之黏接作用……………………… 21
7.質體轉型作用-電穿孔法………………………… 21
五、植物基因轉殖及後續分析…………………… 21
1.基因轉殖植物材料……………………………… 21
2.農桿菌媒介基因轉殖法…………………………… 22
3.基因槍短暫性表現………………………………… 24
結果…………………………………………………… 26
討論…………………………………………………… 60
參考文獻……………………………………………… 65
附錄…………………………………………………… 70
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