Fusion of the Transposase with a Classical Nuclear Localization Signal to Increase the Transposition Efficiency of Ac Transposon

Ac/Ds轉位子為研究植物功能基因體學之重要工具。在機制上，轉位酶合成後必須靠核定位信號(NLS)進入細胞核才能執行轉位。轉位酶雖含有內生NLS，但並非典型有效。本研究建構兩種含SV40病毒之NLS之轉位酶之構築，配合先前建立之「可誘導轉位子」系統，期望增強轉位酶穿入細胞核之能力，也因而提高轉位子之轉位效率。兩構築分別為：全長之轉位酶與SV40病毒之NLS融合（稱SV40TPase構築）及原轉位酶缺失一段（含內生NLS）再與SV40之NLS融合（稱SV39TPase構築）。檢測轉位效率方法為：將一被動轉位子Ds建構於冷光報導基因與啟動子之間，Ds受轉位酶驅動後轉位而使報導基因表現而發冷光。結果發現含NLS之轉位酶之兩構築均可驅動Ds轉位，且轉位效率較原轉位酶之轉位效率高。此外，SV40TPase構築之驅動能力高於SV39TPase構築。本文最後討論轉位酶與典型NLS融合後增加轉位效率之策略，配合已建立之「可誘導轉位子」系統，對於研究植物功能基因體學之影響。

關鍵字

Ac轉位酶；可誘導轉位子；冷光報導基因；核定位信號

並列摘要

A new strategy was applied to improve the transposition efficiency of the maize transposon Activator (Ac) in heterologous plants. The Ac transposase was fused with a classical nuclear localization signal (NLS) of SV40 to promote the transport of transposase into a nucleus. Base on this, two NLS-TPase constructs were yielded, one containing the full length transposase gene (termed as SV40TPase), the other containing the truncated transposase gene (lacking its first NLS-like signal, termed as SV39TPase). These two NIS-TPase genes were expressed in transgenic tobacco plants under the control of PR-1a promoter. Excision of non-autonomous transposable element (Ds) from luciferase (LUC) reporter gene constructs was employed to analyze the induction of Ac transposase containing NLS. Applying the LUC assay and PCR analysis, these new NIS-TPase sources triggered higher Ds excision efficiencies then the native transposase. Furthermore, the SV40TPase showed more ability then the SV39TPase to trigger the Ds element. The usage of this new inducible transposon for plant functional genomics is discussed.

並列關鍵字

Ac transposase ； Inducible transposon ； Luciferase reporter gene ； Nuclear localization signal

國際替代計量

Fusion of the Transposase with a Classical Nuclear Localization Signal to Increase the Transposition Efficiency of Ac Transposon

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