水稻OsDEADl-1基因的功能性探討

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黃俊凱(Chun-Kai Huang) 查詢紙本館藏

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生命科學系

論文名稱

水稻OsDEADl-1基因的功能性探討
(Functional analysis of OsDEADl-1 gene in rice)

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摘要(中)

在此研究中，我們得到了一株水稻的轉殖品系 Osdeadl-1 (Osdead box protein like-1) 的轉殖株，而此轉殖株並沒有同型合子的突變株後代。 OsDEADl-1比對分析結果是屬於DEAD box RNA helicase的一員，在生理功能上RNA helicase解開雙股RNA，確保RNA形成正確的型式以執行其正確的功能。 OsDEADl-1 cDNA包含1506去氧核醣核酸。藉由RT-PCR分析中OsDEADl-1在不同水稻組織中皆有表現。藉由綠螢光蛋白 (GFP) 標定在OsDEADl-1上，在洋蔥表皮細胞及水稻癒傷組織的原生質體中清楚觀測GFPOsDEADl-1只有在細胞核中表現。同時我們在阿拉伯芥中找到AtDEADl-1， AtDEADl-1與OsDEADl-1有77%相似度，在 Atdeadl-1突變株的T3種子亦無法得到同型合子的突變株，顯示AtDEADl-1及OsDEADl-1突變後將造成阿拉伯芥及水稻致死的外表型。並藉由RT-PCR分析結果AtDEADl-1在各個不同的組織中也皆有表現。在Osdeadl-1及Atdeadl-1突變株子代的基因型分析時，發現野生型植株比上異型合子的植株比例為1:1，最後於AtDEADl-1 異型合子突變株的配子與野生型阿拉伯芥 (Len.)正反交的實驗中，顯示Atdeadl-1異型合子突變株的雄配子體可能在其受精或雄配子發育上有缺陷，所以造成無法產生同型合子的突變株後代。

摘要(英)

In this study, we had a mutant line Osdeadl-1 (Osdead box protein like-1). The Osdeadl-1 mutant didn’t find any homozygous mutant progeny. In blast analysis, OsDEADl-1 is a member of the DEAD box RNA helicase. RNA helicase has biological function to unwind double strand RNA to assure the correct foldings of RNA molecules to execute the correct biological functions. OsDEADl-1 cDNA includes 1506 nucleotides. The OsDEADl-1 expresses in all tissues in Oryza sativa by RT-PCR analysis. By green fluorescent proteins fused with OsDEADl-1, we clear observe GFPOsDEADl-1 expressed in the nucleus in the onion epidermal cell and rice callus protoplast. At the same time, we found AtDEADl-1 in Arabidopsis thaliana. AtDEADl-1 and OsDEADl-1 have 77% similarity. In the population of the T3 seeds of Atdaedl-1 also can’t find homozygous mutant. The data reveals the AtDEADl-1 and OsDEADl-1 mutant is lethal phenotype in Arabidopsis thaliana and Oryza sativa. And by RT-PCR result, AtDEADl-1 gene expresses in different tissue in Arabidopsis thaliana. In genotype analysis of the progeny of the Osdeadl-1 and Atdeadl-1 were obtained a ratio of 1:1, as heterozygote to the wild type. Last, in the gametophytes of Atdeadl-1 heterozygous mutant to cross with the wild type (Len.) suggests male gametophyte may have defect in its gametophyte development or its insemination. This is the reason we can’t get homozygous mutant.

關鍵字(中)

★ 轉殖
★ 水稻
★ 解旋酶

關鍵字(英)

★ transformation
★ helicase
★ rice

論文目次

中文摘要………………………………………………………………………….…I
Abstract…………………………………………………………………………..….II
本文目錄……………………………………………………………………………III
表目錄………………………………………………………………………………VI
圖目錄………………………………………………………………………………VII
第一章緒論 1
1 水稻簡介 1
2 台灣水稻插入突變資料庫 1
3 OsDEADl-1 1
第二章前人研究 3
1 DEAD-box RNA 解旋酶 3
1.1 RNA 解旋酶的特性 3
1.2 RNA解旋酶在生物體內扮演的角色 4
1.3 DEAD box RNA解旋酶的保留序列的功能 5
1.4 DEAD box RNA解旋酶結構方面的研究 5
1.5 RNA解旋酶可能作用機制的探討 7
1.6 RNA解旋酶在植物學方面研究 8
2 核醣體RNA的生合成路徑 9
2.1 核醣體RNA生合成及處理路徑 9
3 研究動機與目的 11
第三章實驗材料與方法 12
第ㄧ部分構築質體 12
1.以PCR分別合成OsDEADl-1基因全長片段 12
1.1 引子 (primer) 的設計 12
1.2 細胞total RNA的純化 12
1.3 純化不含genomic DNA的RNA 12
1.4 單股cDNA的合成 13
1.5 以PCR合成DNA片段 13
1.6 瓊脂膠體回收限制酵素切過之 DNA 14
2.接合反應 15
2.1 限制酵素作用 15
2.2 線性載體DNA的去磷酸根作用 15
2.3 黏頭端載體DNA與插入DNA片段的接合 15
2.4 齊頭端DNA片段的接合 16
3.細菌的轉殖作用 16
3.1 製備E.coli competent cell (JM109 strain) 16
3.2 細菌的轉殖 16
3.3 小量純化細菌plasmid DNA法 17
3.4 大量純化細菌plasmid DNA 17
4 DNA序列分析 18
第二部份轉植株基因型的分析 18
1.1 訂購轉殖株 18
1.2 種植條件 18
1.3 抽取genomic DNA 19
1.4 PCR確認植株的基因型 19
第三部份水稻基因轉殖 20
1.1 誘導癒傷組織 20
1.2 製備Agrobacterium competent cells (EHA105 strain) 20
1.3 農桿菌的轉殖方法 20
1.4 農桿菌的生化檢測 (Ketolactose test) 21
1.5 水稻的轉殖 21
1.6 水稻懸浮培養細胞的誘導 22
1.7 阿拉伯芥的轉殖 22
第四部份轉殖株分析方法 23
1 GUS的染色分析法 23
1.1 GUS呈色反應溶液 (X-Gluc反應溶液)製備 23
1.2 GUS染色 23
2 南方墨點轉印法(Southern blot analysis) 23
2.1 gDNA的抽取與純化 23
2.2 DNA墨點轉印 23
2.3 探針(probe)的製備 (random primer method) 24
2.3 探針的雜交 24
第四章結果 26
1 轉殖株的初步分析 26
2 克隆OsDEADl-1全長cDNA 26
3 比對分析OsDEADl-1的氨基酸序列 27
4 水稻轉殖株OsDEADl-1後代基因型的分析 27
5 OsDEADl-1在水稻組織的表現模式 27
6 OsDEADl-1在細胞中的位址 28
7 OsDEADl-1重組蛋白的表達 28
8 阿拉伯芥轉殖株AtDEADl-1後代基因型的分析 29
9 AtDEADl-1在阿拉伯芥中表現模式 29
10 AtDEADl-1突變株配子體reciprocal cross 野生型植株的配子體 30
11 利用水稻OsDEADl-1去回復AtDEADl-1突變後所造成的缺失利用水稻 30
12 過量表達OsDEADl-1水稻與RNAi轉殖株的建立 31
第五章討論 32
1 GFP:OsDEAD1-1 在細胞核中的位置 32
2 OsDEADl-1及AtDEADl-1 造成雄配子體的不孕 32
3 AtDEADl-1及OsDEADl-1生理功能的探討 34
第六章參考文獻 36

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指導教授

陸重安(Chun-An Lu)

審核日期

2006-7-24

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