https://scholars.lib.ntu.edu.tw/handle/123456789/185079
標題: | 構築數種可誘導轉位子以建立一植物基因釣取系統 | 作者: | 常玉強 | 關鍵字: | 可誘導轉位子;可誘導啟動子;基因釣取;transposable element;inducible promoter;PR-1a;gene tagging | 公開日期: | 31-七月-2002 | 出版社: | 臺北市:國立臺灣大學農藝學系暨研究所 | 摘要: | 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 (transposase) 基因 (2)Ds 與Ac 之差別僅在於Ds 缺少(或突變)了 Ac 之轉位 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 表現Ac 轉位頻量率降低(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轉過的多位聚 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 和一NL典S 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 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 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 產生, 且在一被轉譯上膜細胞核中表現,聚降低了過多轉位 集在核的 情形,使得轉位效率提高。在Ac 轉位機制的研上, 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-glucuronidase (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. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/19831 | 其他識別: | 902313B002275 | Rights: | 國立臺灣大學農藝學系暨研究所 |
顯示於: | 農藝學系 |
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