構築含無啟動子報導基因之可誘導轉位子

Construction of an Inducible Transposon with Promoterless Reporter Gene

常玉強(Yuh-Chyang Charng) ； 謝嘉珊(Chia-Shan Hsieh)

《作物、環境與生物資訊》 1卷2期 (2004/06) Pp. 89-101

https://doi.org/10.30061/CEB.200406.0003

摘要

轉位子(Activator, Ac)為研究功能基因體學之重要工具，然而大多數轉位子或T-DNA必須創造同源突變體後才能研究功能基因。為了可在異源突變體中研究功能基因，本試驗將先前構築之可誘導轉位子INAc改造成含有報導基因的轉位子，稱為INAcEGFP。此報導基因為一無啟動子(promoterless)之報導基因，當轉位子以正方插入一個啟動子下游附近時，報導基因就會表現。試驗中並將INAcEFP轉殖於水稻中，共得到51個獨立的轉殖系，其中有5個轉殖系之轉位子自動轉位，自動轉位比率為9.8%。並以水楊(salicylic acid, SA)和5-Azacytidine(5-Azac)為誘導劑處理11個轉殖系之水稻癒合組織細胞，成功地誘導INAcEGFP轉位。更進一步，INAcEGFP轉位子或T-DNA所插入之水稻基因也成功的定序出來。因此，利用此系統不只可成功的應用在基因釣取(gene tagging)上，亦可應用於啟動子捕捉上(promoter trap)。

關鍵字

水稻 ； 可誘導轉位子 ； 基因釣取 ； 基因捕捉 ； 報導基因

並列摘要

Transposon tagging has become one of the powerful tools to create mutants for isolating new genes. A fusion of the SA inducible promoter (PR-1a) with the Ac transposase gene was constructed and designated INAc previously. Its excision can be induced by inducer (SA). In this study, a promoterless reporter gene EGFP was inserted into INAc to generate a new inducible transposon, termed INAcEGFP. This new construct allows the possibility to study gene function in heterologous mutant. INAcEGFP was transformed into rice plant and 51 independent transgenic lines were generated. Spontaneous transposition events were detected in 5 out of these transgenic lines. The other transgenic rice lines were induced by SA and yielded high transposition frequencies. Furthermore, the flanking sequence of T-DNA and transposed INAcEGFP could be obtained. Thus, the INAcEGFP system could be a powerful tool for promoter/gene trap.

並列關鍵字

Rice ； Transposon ； Inducible promoter ； Reporter gene