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研究生:林明岩
研究生(外文):Ming-Yean Lin
論文名稱:斑馬魚轉錄因子oct-1基因的選殖與表現
論文名稱(外文):Cloning and expression of zebrafish transcription factor oct-1 gene
指導教授:宋永義張繼堯張繼堯引用關係
指導教授(外文):Yung-Yi SungChi-Yao Chang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:畜產學研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:88
中文關鍵詞:斑馬魚oct-1基因
外文關鍵詞:zebrafishoct-1 gene
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摘 要
本研究主旨為探討斑馬魚轉錄因子oct-1基因的選殖與表現。藉由已發表的鯉魚含有POU domain的pit-1基因DNA片段為探針,來篩選斑馬魚成魚腦部DNA基因庫,以篩選到斑馬魚POU轉錄因子家族的基因。由此基因庫篩選出選殖體3-1,全長為1152 bp。此段基因與斑馬魚的轉錄因子oct-1基因部份序列相同。利用此段DNA當做DNA探針去篩選斑馬魚5’- Stretch plus DNA基因庫,篩選出含有1058 bp和2800 bp的oct-1的基因片段,將之命名為11-2 ( 靠近5’端 )與8-1 ( 靠近3’端 )。以PCR的方式將11-2與8-1連接成完整的oct-1 DNA,再進行次選殖至載體PBK-CMV中。 利用11-2 DNA當探針,進行整體原位雜化,以偵測oct-1在不同時期胚胎表現的情形。研究結果顯示,在1個細胞期的細胞中最早偵測到oct-1 mRNA,之後在2細胞期、4細胞期、8細胞期、16細胞期、32細胞期、64細胞期、128細胞期、1 K細胞期、2K細胞期、High 、oblong 、Sphere 、Epiboly和Shield等時期的細胞區, 都有偵測到。在Prim 5 ( 24小時 ) 時期,於腦部、神經管、脊索、體節部位,都有很強的oct-1 mRNA表現。在Prim 11 ( 30小時 ) 時期 腦部、神經管仍然有很強的表現,但是在脊索、體節部位表現量卻明顯降低。而在Protruding-month ( 72小時 ) 及Upright ( 96小時 ) 時期的脊索、體節都偵測不到oct-1 mRNA,只有在腦部和神經管持續有很強的表現。為了要在大腸桿菌系統中表現Oct-1蛋白質,以確定Oct-1蛋白質的分子量,將oct-1 cDNA轉譯區域次選殖至pET-20b(+)後,以IPTG誘發Oct-1蛋白質,發現IPTG在3 mM、16小時可誘發的效果最好,Oct-1蛋白質的分子量約為66 kDa,且為不可溶性蛋白質。由斑馬魚oct-1 選殖體 11-2,經限制酵素EcoRI 和SacII 剪切後,分離出靠近5’端的0.2 Kb的片段 ,利用此片段當作DNA探針,以篩選斑馬魚啟動子的區域。最後得到一個oct-1基因組DNA選殖體2-1,接入片段約為9Kb。目前已完成定序的Oct-1起始密碼ATG之前的上游區域為3212bp,在序列上有14個TAAT與1個CCAAT序列。
Abstract
In this study, we used a carp pit-1 gene DNA fragment containg POU domain as probe to screen zebrafish POU family gene. We successfully obtained a POU-domain containg gene, oct-1, named clone 3-1 from zebrafish brain DNA library. Computer comparison of the sequence indicated that this sequence was identical to zebrafish oct-1 gene. By using this partcial oct-1 DNA fragment as probe to screen the zebrafish 5''-stretch plus cDNA library, we got two clone 11-2 (5''flanking ) and 8-1 (3''flanking) containing 1058bp and 2800bp, respectively.
We used PCR mediated ligation method to obtain full-length oct-1 cDNA and detected the oct-1 expression patterns in different zebrafishembryonic stages by whole-mount in situ hybridization. The results suggest that oct-1 presents in one-cell stage, through 2-cell, 4-cell, 8-cell, 16-cell, 32-cell,64-cell,128-cell, 1 k cell, 2 k cell, High, Oblong, sphere, Epiboly and Shield stage . The oct-1 gene strongly expressed in brain、neural tube、notochord and somite at prime 5 ( 24 hr )stage. The oct-1 gene still strongly expressed in brain、neural tube, but weakly in notochord and somite at prime 11 ( 30 hr )stage. In Protruding-month ( 72 hr )and upright ( 96 hr )stages, we can’t detect oct-1 mRNA in notochord and somite, but the oct-1 gene still expressed in brain and neural tube.
To express recombinant Oct-1 protein and to confirm the Oct-1 molecular weight, we subclone the oct-1 gene into pET-20 b(+) overexpressed in E.coli by IPTG induction. The recombinant Oct-1 protein can be expressed by 3 mM IPTG induction for 16hr , and the molecular weight of the insoluble recombinant Oct-1 protein is approximately 66 kDa.
With the probe prepared from the oct-1 cDNA clone 11-2 by EcoRI and SacII digestion, we identified a 9Kb oct-1 upstream DNA fragment named 2-1. Now,we have sequenced 3212 bp in the oct-1 upstream region. There are 14 TAAT sites and 1 CCAAT site in this sequence.
目 次
I. 摘要…………………………………………1
II. 緒言…………………………………………2
III.文獻檢討……………………………………3
IV. 材料與方法…………………………………10
V. 結果與討論…………………………………38
VI. 結論…………………………………………71
VII.附錄…………………………………………72
VIII.英文摘要.…………………………………75
IX. 參考文獻……………………………………77
X. 簡歷…………………………………………88
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