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研究生:李惠萍
研究生(外文):Hui-Ping Li
論文名稱:建構含nuclearlocalizationsignal轉位酶以增加轉位子之轉位效率
論文名稱(外文):Construction of the transposase gene containing a nuclear localization signal increase the transposition efficiency of the transposon
指導教授:許宗雄許宗雄引用關係常玉強杜鎮杜鎮引用關係
指導教授(外文):Tzong-Hsiung HseuYuh-Chyang CharngJenn Tu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:71
中文關鍵詞:可誘導啟動子轉位酶轉位子導核訊息
外文關鍵詞:inducible promotertransposasetransposonnuclear localization signal
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Activator (Ac)轉位子最早於1947年被Barbara McClintock在玉米中發現:她發現Ac可自動轉位於基因間並可外作用於(trans-activate)另一組非自動轉位子Dissociation (Ds),趨動Ds之轉位。Ac-Ds在1984年被德國Starlinger選殖並定序(Müller-Neumann et al. 1984),經過進一步研究發現:(1)Ac由4565-bp組成並含有轉位酶(transposase)基因 (2)Ds與Ac之差別僅在於Ds缺少(或突變)了Ac之轉位酶基因 (3)一最小之Ds僅含Ac之兩端各300-bp即可被Ac活化而轉位(Haring et al. 1991)。學者利用其在染色體上跳出原位置,而又插入另一基因位置的特性,釣取一些重要之植物基因,稱基因釣取(gene tagging)。
轉位子有一種「反劑量效應」(inverse dosage effect)現象,乃因轉位酶表現量太高使得Ac轉位頻率降低(Scofield et al. 1992),此現象存在於玉米、菸草、蕃茄,但不存在於阿拉伯芥中(Swinburne et al. 1992)。研究顯示可能是因為過多的轉位酶聚集在核膜上的結果(Heinlein et al. 1994)。為了降低此現象,本實驗利用nuclear localization signal (NLS)可將蛋白質帶入細胞核中的特性,將玉米Ac轉位子中轉位酶和一典型之NLS融合的構築在可誘導的啟動子PR1-a驅動下,送進含非自動轉位子Ds的轉殖菸草中,使其表現轉位。當含NLS之轉位酶基因受到水楊酸(salicylic acid)誘導時,會使得Ds跳出Luciferase或β-glucuronidase報導基因(reporter gene),利用Lucifersae分析和GUS分析可以測到轉位效率及發生的位置。另外,運用了特定的引子(primer)做不同的聚合酶連鎖反應(Polymerase chain reaction)來確認轉殖株中的轉位情形,發現72個獨立轉殖株中有56株已自動轉位。此結果顯示,可能是由於植物內生的水楊酸誘導轉位酶產生,且在一被轉譯出來時就馬上被送進細胞核中表現,降低了過多轉位酶在核膜上聚集的情形,使得轉位效率提高。在Ac轉位機制的研究上,希望此方法能提供一些資訊,並且使轉位子系統更適於用來釣取高等植物的基因(gene tagging)。

Transposable elements Activator (Ac) was first discovered in maize by Barbara McClintock (1947). She found that Ac transposed autonomously between genes and trans-activated another non-autonomous element Dissociation (Ds). Ac-Ds was cloned and sequenced by Starlinger (1984). Further studies showed that: (1) Ac is 4565 bp long and codes for a single product, the transposase. (2) generally, Ds elements are internal deletion derivatives of Ac (3) A fully active minimal Ds element may contain only 300 bp of each end of Ac element. Gene tagging technique using the transposable element as an insertional mutagen for the isolation of important plant genes has been proven to be a useful tool.
A curious aspect of Ac transposon is that the accumulation of high levels of the Ac transposase may inhibit subsequent transposon excision, which termed as “inverse dosage effect”. This effect was saw in maize, tobacco and tomato but not Arabidopsis. It was hypothesized that high level of the transposase might aggregate on the nuclear membrane. As a result of this, the transposase cannot transport into the nuclear to perform the transposition events. In this study, a classical nuclear localization signal (NLS) was fused with the transposase gene under the control of promoter of the inducible gene for pathogenesis-related protein 1a (PR-1a). The purpose of the NLS fusion is to help the entrance of the transposase into the nucleus for the subsequent transposition events. Excision of non-autonomous transposable element(Ds) from luciferase(LUC) and β-glucu- ronidase(GUS) reporter gene constructs was employed to analyze the induction of the Ac transposase containing NLS. Spontaneous excision has been primarily identified by polymerase chain reaction in 56 out of 72 independent transgenic tobacco plants. This result suggested that transposase could be transported into the nucleus immediately by induction of the internal salicylic acid stimuli. An alternative inducible transposon strategy for functional genomic is suggested.

第一章 緒論 01
1-1 轉位子的發現 01
1-2 轉位子的分類 02
1-3 Ac/Ds轉位子的簡 02
1-4 Ac/Ds轉位子的研 04
1-5 Ac轉位機制 06
1-6 轉位作用的調控 08
1-7 Ac轉位子送入其他植物中的情形 10
1-8 轉位子在基因釣取上(Gene Tagging)的應用 12
1-9 控制轉位子:誘導轉位子的產生 14
1-10 增強轉位酶穿入細胞核的能力 15
第二章 材料 17
第三章 方法 27
第四章 結果 39
4-1 構築結果 39
4-2 電導及Triparental mating結果 40
4-3 菸草轉殖結果 40
4-4 PCR結果 41
4-5 Southern Blot結果 41
第五章 討論 43
圖表 47
參考文獻 64

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