臺灣博碩士論文加值系統

CIA2是一種植物特有的核轉錄因子。根據我們之前的基因微矩陣及生化分析結果，顯示CIA2會藉由促進其下游基因的表現，進而影響到葉綠體的發育及功能。這些CIA2下游基因所轉譯的蛋白質包括參與葉綠體蛋白之運輸、合成及折疊的Toc33、核糖體蛋白質CpRP和CPN10。在阿拉伯芥中，CIL (CIA2-like)是與CIA2胺基酸序列相同度達65%的同源蛋白。CIL和CIA2皆只在綠色植物組織中表現，但是CIL卻不能完全補償CIA2突變所造成的植株外表型或功能缺失，因此推測調節兩種基因表現方式是不同的。我的研究將藉由報導基因的表現程度及生物資訊軟體的分析，嘗試釐清CIA2/CIL基因上游的重要調控序列。目前，透過啟動子序列縮減及以基因槍將含有不同長度啟動子序列的報導基因質體送入阿拉伯芥的葉片中，再分析報告基因活性表現後，發現CIA2基因轉錄起始位點前-1174至-1079的片段及-950至-762片段分別可能會抑制及促進CIA2表現量；而CIL基因轉錄起始位點前-1769至-1668的片段則可能會抑制CIL表現量。此外，以MEME、PLACE、AGRIS生物資訊軟體資料庫尋找到CIA2/CIL序列上之重要順式作用元件，顯示CIA2和CIL可能會受到光線及低溫等環境因子之調節。光及暗處理實驗顯示CIA2的-1174至-1079片段及-950至-762片段可能分別是受光負向及正向調節的序列；CIL的-1769至-1668片段則可能是不受光調節的負向序列。進一步比較這些基因在不同生長時期、溫度的基因表現模式，得知低溫處理下，不同齡植株之CIA2會減少基因表現量；CIL在植株幼期會受到抑制，但隨著年齡增長，低溫會促進其基因表現，且表現量也越高；而高溫處理下，CIA2/CIL在植株幼期均會受到促進。整體來說，因為CIA2及CIL的表現量及對特定環境刺激有不同反應，推論兩基因的表現是由不同的分子機制所調控。

Chloroplast import apparatus 2 (CIA2) is a plant-specific transcription factor. Based on our previous microarray and biochemical results, CIA2 might modulate chloroplast development and function by upregulating the expression of its downstream genes encoding Toc33 (translocon at the outer envelope membrane of chloroplast 33), CpRPs (chloroplast ribosomal proteins) and CPN10 (chaperonin 10), which are participating in chloroplast protein import, synthesis and folding, respectively. There is a CIA2 homolog, named CIL (CIA2-like) in Arabidopsis, sharing 65% sequence identity with CIA2. Both CIL and CIA2 express in green tissues, however, CIL can only compensate cia2 defects partially. Therefore, my research would focus on distinguishing the regulatory mechanism of CIA2/CIL by reporter gene and bioinformatics analyses. So far, the promoter deletion and plant transient assays indicate that CIA2 promoter has a negative and a positive regulatory sequence, located on -1174 to -1079 and -950 to -762, respectively. Also, CIL promoter has a negative regulatory sequence which located on -1769 to -1668. The cis-acting elements on these regulatory sequences also have been explored by homology searching of different bioinformatics databases, including MEME, PLACE, and AGRIS. The results pointed out that both CIA2 and CIL might be affected by light and cold environmental factors. The analysis of CIA2 expression under different light and dark period indicated that the negative and positive regulatory sequence located on-1174 to -1079 and -950 to -762 of CIA2 is resposible for light regulated expression. Also, CIL promoter has a negative regulatory sequence which located on -1769 to -1668 that isn’t resposible for light regulated .We also compared the expression patterns of CIA2/CIL during various developmental stages and under low/high temperatures treatments. The results showed that 4 oC treatment reduced the expression levels of CIA2 in four developmental stages. In contrast, similar treatment results in a lower expression level of CIL in 7-day-old seedling but gradually increased CIL expression acompanying with plant maturation. Fourthermore, 37 oC treatment increased the expression levels of CIA2/CIL in young seedling. In summary, CIA2 and CIL genes are regulated by various molecular mechanisms due to their differential expression yields and responses to various environmental signals.

目錄
中文摘要……………………………………………………………………………………………III
英文摘要……………………………………………………………………………………………IV
緒論………………………………….……………………………………………………………….1
研究材料與方法…………………….……………………………………………………………….3
1. 植物材料…………………………………………………………………………………….3
2. CIA2/CIL的上游調控序列資料取得與分析……………………………………………….3
3. 基因專一性引子對(gene-specific primer)的設計…………………………..………………..3
4. 報導基因質體的選殖…………………………………………………………………………3
5. 轉殖植物葉片樣本準備………………………………………………………………………4
6. 質體殖入阿拉伯芥葉片…………………………………………….………………..……….4
7. 報導基因活性分析……………………………….…..……………………………………….4
8. 總蛋白質萃取……………….……………………………………...........................................5
9. 膠體電泳位移分析……………………………………………………………………………6
10. 基因序列分析…………………………………………………………………………………6
11. 光線環境因子之處理…………...…………………………………………………………….6
12. 溫度環境因子之處理……………………………....................................................................7
13. 核糖核酸(RNA)之萃取……………………………………………………………………….8
14. RNA的定量…..……………………………….………………………………………………8
15. 即時定量反轉錄PCR…………………………………………………………………………8
結果…………………………..……..………………………………………………………..………9
討論…………………………………………………………………………………………………14
參考文獻……………………………………………………………………………………………17
表格與圖片.…………………………………………..………………………….…………………20
表一 專一引子對序列資料…………………………….……………………………….…..……20
表二 利用PLACE資料庫分析CIA2 regulatory sequences 1序列上可能的調節序列……..….21
表三 利用PLACE資料庫分析CIA2 regulatory sequences 2序列上可能的調節序列…….…22
表四 利用PLACE資料庫分析CIA2 regulatory sequences 3序列上可能的調節序列….........23
表五 利用PLACE資料庫分析CIL regulatory sequences 1序列上可能的調節序列…......…..24
表六 利用PLACE資料庫分析CIL regulatory sequences 2序列上可能的調節序列….......….25
表七 利用AGRIS資料庫分析CIA2序列……..…..............................…………………………26
表八 利用AGRIS資料庫分析CIL序列……………………………..…….....………………...27
表九 各序列片段以PLACE資料庫分析之共有調節因子………………..........……....……..28
表十 各實驗中CIA2對CIL基因表現之倍率………………………………………………….29
圖一 CIA2及CIL基因結構………………………………………………….………………….30
圖二 構築CIA2及CIL啟動子系列縮減質體之示意圖…………………......……...…………31
圖三 不同CIA2啟動子片段之活性比較……………………………………………....………32
圖四 不同CIL啟動子片段之活性比較……………………….…………………………..……33
圖五 CIA2/CIL啟動子序列上順式作用元素……………………………………………….….34
圖六 不同光處理對CIA2啟動子片段之活性影響……….………………………………........35
圖七 不同光處理對CIL啟動子片段之活性影響……….……….…………………….............36
圖八 低溫處理對CIA2/CIL基因表現影響…………………………...………………….…….37
圖九 高溫處理對CIA2/CIL基因表現影響…………………………...………………………..38
附錄…………………………………………………………………………………………………39
附錄一 Diurnal資料庫不同實驗條件狀況之列表…………………………………………….40
附錄二 利用Diurnal資料庫分析不同狀況下CIA2/CIL基因表現量…………………………41

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