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研究生:曾勇祥
研究生(外文):Yung-Hsiang Tseng
論文名稱:綠竹不同生長階段之基因差異性表現分析
論文名稱(外文):Analysis of gene expression in different growth stages of green bamboo Bambusa oldhamii
指導教授:王愛玉
指導教授(外文):Ai-Yu Wang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:76
中文關鍵詞:差異性顯示法綠竹變性聚丙烯醯胺膠體電泳
外文關鍵詞:reverse Northern analysisgreen bamboodenaturing polyacrylamide gel electrophoresisdifferential display
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由於竹類植物兼具快速生長與獨特的生理、生化特性,在探討醣類代謝與快速生長關係的議題上,是相當好的研究材料。為瞭解調控綠竹筍快速生長之機制,本論文採用差異性顯示法 (differential display) 針對綠竹 (Bambusa oldhamii)不同生長階段之基因表現進行分析。我們首先由 8 種竹筍樣品抽取 total RNA,並以不同的 arbitrary primes 與 two - base anchored primers 進行 reverse transcription - polymerase chain reactions (RT - PCR); 接著在 174 組 PCR 反應中,挑選出 349 個具有差異性表現的片段,進行選殖與定序,共得到172條基因序列。BLAST 分析結果,未知基因有 68 條,具有相對應蛋白質的序列共有 57 條。針對差異性片段進行 reverse Northern hybridization 分析,由結果可觀察到選殖基因在竹筍各組織的表現情形。

Bamboo possesses fast growth, remarkable physiological and biochemical characteristics, and thus are regarded as an excellent research material for investigating the relationships between carbohydrate metabolism and fast growth. To gain insight into the mechanisms regulating the fast growth of bamboo shoots, we employed differential display to analyze gene expression in different developmental stages of shoots of Bambusa oldhamii. Total RNAs were isolated from 8 bamboo sample’s and reverse transcription - polymerase chain reactions were performed using different arbitrary primers and two-base anchored oligo (dT) primers. 349 differentially expressed fragments were selected from 174 sets of PCR reactions. After cloning and sequencing, 172 gene sequences were obtained. According to the result of BLAST analysis, 68 cDNAs are unknown genes, and 57 cDNAs encode specific protein sequences. By employing reverse Northern hybridization analysis, we were capable of observing the expression patterns of cloned genes in various tissues of bamboos.

目錄

目錄 …………………………………………………………………… I
縮寫表 ………………………………………………………………… V
摘要 ………………………………………………………………… VI
Abstract …………………………………………………………… VII

第一章 序論 ………………………………………………………… 1
第一節 研究材料與研究目的 ………………………………………………… 1
1.1 竹 1
1.2 以竹作為實驗材料的意義與重要性 2
1.2.1 綠竹筍經濟價值高,是台灣獨具特色的農產品 2
1.2.2 竹具獨特的生理現象 2
1.3本論文之目的 3

第二節 分析基因差異性表現的方法 ……………………………… 4
2.1 刪減雜合法 4
2.2 抑制性刪減雜合法 5
2.3 代表性差異分析法 6
2.4 基因表現系列分析法 6
2.5 差異表現分析法 7
2.6 DNA 微陣列晶片 8

第三節 論文採用之基因差異性表現分析方法及研究重點………… 9
第二章 材料與方法 ………………………………………………… 10
第一節 實驗材料 …………………………………………………… 10
1.1 植物材料 10
1.1.1 綠竹樣品採集與處理 10
1.2 菌株 10
1.2.1 大腸桿菌菌株 10
1.3 載體 11
1.4 核酸引子 11
1.4.1 Oligo(dT) 引子 11
1.4.1.1 One-base anchor primers 11
1.4.1.2 Two-base anchor primers 11
1.4.2 H-AP primers 11

第二節 實驗藥品與儀器 ………………………………………… 12
2.1 實驗藥品 12
2.1.1 一般化學試劑 12
2.1.2 分生試劑、限制�◆P核酸修飾酵素 12
2.1.3 培養基 13
2.1.4 放射性藥品 13
2.2 實驗儀器 13
2.2.1 核酸電泳設備 13
2.2.2 離心機 13
2.2.3 其他儀器 13

第三節 實驗流程與方法 ………………………………………… 15
3.1 RNA之抽取、檢定與分析 …………………………………… 16
3.1.1 RNA 使用溶液與器具之前處理 16
3.1.2 RNA 之抽取 16
3.1.3 RNA 樣品之定量 17
3.1.4 甲醛洋菜膠體電泳 17
3.2 差異表現分析法 ……………………………………………… 17
3.2.1 RNA樣品處理 17
3.2.2 RNA 樣品稀釋 18
3.2.3 反轉錄反應 18
3.2.4 聚合�○s鎖反應 18
3.2.5 DNA 標準分子量之放射線標示 19
3.2.6 變性聚丙烯醯胺膠體電泳 19
3.2.6.1 膠體製備 19
3.2.6.2 電泳 19
3.2.7 差異性表現 DNA 片段的選取、回收與增殖放大 20
3.2.7.1 差異性表現 DNA 片段之選取與回收 20
3.2.7.2 DNA片段增殖放大 20
3.2.8 PCR產物之純化 21
3.3 重組質體之建構與轉形 ……………………………………… 21
3.3.1 重組質體之建構 21
3.3.1.1 Blunt-end ligation 21
3.3.1.2 T-A cloning 22
3.3.2 質體之轉形 22
3.3.2.1 Competent cell 之製備 22
3.3.2.2 轉形 23
3.3.3 轉形株質體快速檢定法 23
3.3.4 菌種保存 23
3.4 DNA之抽取、檢定與分析 ……………………………………… 24
3.4.1質體 DNA 之小量分離法 24
3.4.2 DNA之定量 24
3.4.3 DNA之限制�﹞尷R 24
3.4.4 DNA 洋菜膠體電泳 25
3.4.5 膠體中 DNA 片段之回收與純化 25
3.5 放射性探針之製備與純化 …………………………………… 26
3.5.1放射性探針之製備 26
3.5.2 放射性探針之純化 26
3.5.3 放射性探針之檢測 26
3.5.3.1 薄層色層分析法 26
3.5.3.2 鹼性洋菜膠體電泳 27
3.5.3.3探針放射性強度之定量 27
3.6 Reverse Northern 分析 …………………………………… 27
3.6.1 膜之製備 27
3.6.2 雜合反應 28
3.6.3 放射性探針之去除 28
第三章 結果與討論 ……………………………………………… 29
第一節 差異表現分析法反應條件之探討 ……………………… 29
1.1 One-base anchor primers 與 two-base anchor primers 之比較 29
1.2 不同膠體濃度之電泳分析結果對於差異性片段選取的影響 30
1.3 不同放射線核�˙譯q對於電泳分析結果之影響 30
1.4 T14MT primers 對於實驗結果影響之探討 30
1.5 不同年份竹筍樣品之比較 31
1.6 最佳反應條件之決定 31
1.7 DNA 標準分子量的選取以及標示放射線核種的決定 31
1.8 進行 DD negative control 以確定 RNA 樣品中無 DNA 殘留 32

第二節 差異性片段的選取與增殖放大情形 …………………… 32
2.1 差異性片段的挑選原則 32
2.2 差異性片段選取統計 33
2.3 差異性片段 PCR 增殖反應之結果 33

第三節 重組質體之檢定 ………………………………………… 34

第四節 具差異性 DNA 片段定序結果與序列比對 ……………… 34

第五節 差異性 DNA 片段組織特異性之探討 …………………… 35
5.1 實驗 positive control 之選取 35
5.2 放射性探針合成片段長度之探討 35
5.3 Reverse Northern 分析結果 36
5.4 造成 DD-PCR 與 reverse Northern 結果不同之可能原因探討 36

第六節 選殖基因的分類 ………………………………………… 37
第四章 結論與未來研究方向 …………………………………… 38
第一節 結論 …………………………………………………… 38

第二節 未來研究方向 …………………………………………… 39
2.1 比較樣品單純化 39
2.2 Reverse Northern hybridization 及 Northern hybridization 進行確認 39
2.3 選殖全長 cDNA 片段 40


參考文獻 …………………………………………………………… 41


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