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研究生:鐘緯璟
研究生(外文):Wei-Ching Chung
論文名稱:利用微矩陣分析百合LLA23在葡萄糖、乾燥與高溫條件下對阿拉伯芥轉殖株的影響
論文名稱(外文):The effect of lily LLA23 on Arabidopsis transgenic plants under glucose, desiccation and high temperature conditions using microarray analysis
指導教授:王國祥
口試委員:王強生鄭萬興
口試日期:2012-12-17
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:101
語文別:中文
論文頁數:127
中文關鍵詞:鐵砲百合ASR乾燥葡萄糖高溫微矩陣即時定量聚合酵素連鎖反應
外文關鍵詞:Lily ASRdroughtglucoseheatmicroarrayQ-PCR
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LLA23為鐵砲百合 (Lilium longiflorum Tumb. cv. Snow Queen) 花粉中屬於ABA-, stress-, and ripening-induced (ASR) 蛋白質的一員,在花粉發育成熟的乾燥時期會大量累積。由於35S::LLA23轉殖株乾燥、高鹽、高溫及低溫環境下展現其耐受性的能力,本研究進一步以微矩陣方法分析葡萄糖及高溫逆境下,比較野生株及35S::LLA23轉殖株整體基因的變化。將2週大的轉殖株與野生株以3 %葡萄糖或在39 ˚C下處理,萃取轉殖株及野生株的總量RNA,以進行微矩陣分析。即時定量聚合酵素連鎖反應分析不同的基因,符合微矩陣所顯示的mRNA層次上之變化。探討30個受逆境影響的基因啟動子,顯示所有基因均受到兩個以上不同的逆境或荷爾蒙調控區的影響,並且發現有尚未被鑑定新的cis-elements存在於啟動子中。本研究由探討在LLA23、葡萄糖、乾燥和高溫下正調控的基因,並描述這些訊息傳遞網絡交流對話和差異性的基因表現,有16個基因對不同的逆境處理都能反應,而高度表現的基因也能在不同的逆境處理找到。即時定量聚合酵素連鎖反應的分析,證實這些基因在各逆境條件下確實被高度誘導。另外,植株在不同的逆境下,也呈現許多負調控或反向調控的基因,更突顯出逆境複雜的訊息傳遞網絡。即時定量聚合酵素連鎖反應分析35S::LLA23轉殖株中離層酸及糖類反應相關的基因,顯示35S::LLA23轉殖株在高糖之下,離層酸生合成、代謝及訊息傳遞相關基因的表現量均比野生株來得少,此結果和35S::LLA23轉殖株為離層酸不敏感型一致。LLA23會影響HXK1依賴型和HXK1不依賴型的離層酸訊息傳遞路徑。綜合以上探討結果,說明LLA23透過複雜的訊息傳遞網路調節基因的表現,以因應多重逆境的影響。

LLA23, a member of abscisic acid-, stress-, and ripening-induced (ASR) family protein previously isolated from lily (Lilium longiflorum) pollen is abundantly accumulated upon desiccation during anther/pollen development. The transgenic plants overexpressing LLA23 exhibit resistance against unfavorably environmental conditions such as drought, salt, heat and cold. In this study, we further used microarray to analyze genome-wide patterns of genes which were altered in the wild-type and 35S::LLA23E plants under 3 % glucose and heat treatments. Two-week-old seedlings of wild-type and 35S::LLA23 plants were treated with 3 % glucose conditions or heated at 39˚C. Total RNA was extracted from wild-type and transgenic seedlings and prepared for microarray analysis. Quantitative real-time PCR (Q-PCR) analyses confirmed the changes in mRNA levels of selected genes observed in microarray. Promoter analysis of 30 define ASR-responsive genes showed that all of these genes had at least two regulatory elements in response to stress or hormones, and new cis-element responsive to a specific stress existed in their promoters. We investigated genes induced by LLA23, 3% glucose, drought and heat stresses, and described the signaling network of cross-talk and differential gene expression. While 16 genes were found to respond to LLA23, glucose, and two different stress conditions, highly-expressed genes existed in each condition confirmed by Q-PCR analysis. In addition, the existence of many down-regulated genes or genes that responded to one condition but adversely responded to another different condition further outstanded the complicated network of signaling transduction. Q-PCR analysis of ABA- and sugar-regulated genes in the 35S::LLA23 plants revealed that all genes had lower expression levels of mRNA in 35S::LLA23 than in wild-type seedlings. It is consistent with ABA insensitive nature of 35S::LLA23 plants. LLA23 affects both HXK- dependent and HXK-independent ABA signaling pathway. In all, LLA23 may regulate various gene expression through complicate signaling transduction network in response to multiple stresses.

中文摘要 1
英文摘要 2
壹、前人研究 3
 一、環境逆境 (stress) 對於植物的影響 3
 (一)乾燥逆境對植物的影響 3
 (二)葡萄糖對植株生長發育之影響 5
 (三)溫度逆境對植物的影響 7
 二、ASR基因序列和其蛋白質之特性分析 8
 三、ASR受環境逆境和發育因子的影響 10
 四、鐵炮百合LLA23的介紹及其相關研究 11
 五、訊息傳遞路徑的對話 (cross-talk) 12
貳、材料與方法 16
 一、植物材料及生長條件 16
 二、總RNA之萃取 16
 三、RNA電泳 17
 四、微矩陣分析 17
 五、反轉錄聚合酶鏈鎖反應(reverse transcription; RT-PCR)和即時定量聚合酶鏈鎖反應(quantitative real-time PCR) 18
 六、DNA電泳 20
叁、結果 21
 一、35S::LLA23轉殖株在正常環境下之微矩陣分析 21
 (一)35S::LLA23轉殖株内受百合ASR調節的基因 21
 (二)受百合ASR正調控的基因啟動子 (promoter) 之分析 24
 二、35S::LLA23轉殖株在不同逆境處理之微矩陣分析 26
 (一)35S::LLA23轉殖株在乾燥逆境處理之微矩陣分析 26
 (二)35S::LLA23轉殖株在3 %葡萄糖處理之微矩陣分析 28
 (三)35S::LLA23轉殖株在高溫處理下的微矩陣分析 29
 三、不同逆境下植物訊息傳遞路徑共同 (convergent) 及差異性 (differential) 正調控基因之表現 31
 (一)於不同逆境下之植物訊息傳遞路徑共有的基因 31
 (二)受單一逆境高度正調控之差異性的基因 35
 (三)在四種條件下皆受到正調控的基因之代謝路徑網絡分析 37
 四、不同逆境下植物訊息傳遞路徑共同及差異性負調控基因之表現 38
 五、不同逆境下造成植物訊息傳遞路徑反向調控之基因表現 39
 六、35S::LLA23轉殖株在4.5 %葡萄糖處理下,離層酸及糖類相關基因之表現40
肆、討論 42
伍、參考文獻 49
陸、圖表 63
 表格(表一~表十三) 63
 圖 (圖一~圖十三) 112
 附表(附表一) 126
 附圖(附圖一) 127


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