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研究生:朱玉菁
研究生(外文):Yu-Jing Zhu
論文名稱:黃果蠅丙酮酸鹽激脢的基因選殖與其基因表現的研究
論文名稱(外文):Gene Cloning and Gene Expression of Pyruvate Kinase from Drosophila melanogaster
指導教授:簡一治
指導教授(外文):Yi-Chih Chien
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:科學教育研究所
學門:教育學門
學類:普通科目教育學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:46
中文關鍵詞:黃果蠅丙酮酸鹽激脢基因選殖基因表現
外文關鍵詞:Drosophila melanogasterpyruvate kinasegene cloninggene expression
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中文摘要
以5’ Rapid Amplification of cDNA End (5’RACE)的方法找出黃果蠅丙酮酸鹽激脢的轉錄起始點,並以黃果蠅丙酮酸鹽激脢基因組DNA當探針篩選λEMBL-3黃果蠅基因組圖書館,分離出一個含黃果蠅丙酮酸鹽激脢基因組DNA 5’ 端的選殖體-λpk61,再以ABI 377核酸自動定序儀定出黃果蠅丙酮酸鹽激脢轉錄起始點上游 734 個鹼基對的序列,將這些序列加以分析找出了許多可能與轉錄有關及可能會與蛋白質結合的核心序列。
由北方墨漬法分析不同發育期、不同性別黃果蠅丙酮酸鹽激脢基因的表現,發現在黃果蠅蛹期丙酮酸鹽激脢的表現明顯弱於黃果蠅幼蟲期及成蟲期,而在黃果蠅成蟲期不論雌或雄成蟲,其丙酮酸鹽激脢的基因表現差異不大,顯示在黃果蠅雄蟲中丙酮酸鹽激脢的基因表現有劑量補償的現象出現。此外,北方墨漬實驗亦顯示在不同發育期、不同性別的黃果蠅中可能只有一種丙酮酸鹽激脢 mRNA 存在。
RT-PCR則是被用來決定黃果蠅成蟲丙酮酸鹽激脢轉錄物的種類,而從結果中每一個聚合脢連鎖反應僅複製出一條複製片斷,顯示在黃果蠅成蟲中可能只有一條丙酮酸鹽激脢轉錄物,綜合北方墨漬及RT-PCR的結果推測在黃果蠅中可能只有一種丙酮酸鹽激脢基因存在。

Abstract
5’ Rapid Amplification of cDNA End (5’RACE) analysis was applied to identify the transcription start point (tsp) of pyruvate kinase gene (PyK) from Drosophila melanogaster. Screening a EMBL-3 genomic library of D.melanogaster using PyK genomic DNA as a probe, a genomic DNA clone (pk61), which contains the 5’ untranscribed region of D. melanogaster PyK gene was isolated. The insert was reconfirmed to contain Pyk gene by Southern blot analyses. A sequence of 734 bp upstream from tsp of Pyk was determined, from which some core sequences that might regulate transcription or might be recognized by proteins were proposed.
The PyK expression of D. melanogaster from different developmental stages and sexes was analyzed by Northern blot analyses. The results that the amounts of PyK transcripts produced in larvae and adults was higher than those in pupae, indicates that Pyk expression is low in pupal stages. The fact that similar level of PyK expression in both male and female adults, suggests that there is a dosage compensation phenomenon in Pyk gene of D. melanogaster male adults. In addition, the results that there is a single band present in all samples of the Northern blot analyses reveals that there is only a single PyK mRNA in different developmental stages and sexes of D. melanogaster.
RT-PCR analysis was performed to determine the number of species of PyK transcript in adults of D. melanogaster. The result that only a single amplicon appeared in each amplication, suggests that there might be a single PyK transcript expressed in adults of D. melanogaster. Furthermore, this might suggest that there is only a single PyK gene in D. melanogaster.

目錄
致謝…………………………………………………………..I
中文摘要………………………………………………….…II
英文摘要……………………………………………………III
目錄………………………………………………………….IV
表、圖目錄…………………………………………………...V
一、前言……………………………………………………….1
二、研究目的…………………………………………………6
三、材料與方法………………………………………………7
四、結果……………………………………………………..16
五、討論…………………………………………………….23
六、未來展望……………………………………………….27
七、參考文獻……………………………………………….28
表1…………………………………………………………...35
圖1-11……………………………………………………….36

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