利用Tet-on系統建構植物可誘導轉位子

Using the Tet-on System to Construct an Inducible Transposon in Plant

翁瑞鍵(Ruei-Chien Weng) ； 常玉強(Yuh-Chyang Charng)

《作物、環境與生物資訊》 14卷1期 (2017/03) Pp. 17-30

https://doi.org/10.30061/CEB.201703_14(1).0003

摘要

本試驗利用微生物Tet-on 系統結合Ac轉位子，期望建立一有效且精準控制之誘導系統，驅動轉位子並有效創造突變體。首先以Tetpro 啟動子結合轉位酶基因建構於Ac轉位子之端點內，而成TetAc 可誘導轉位子系統。該系統以農桿菌轉殖法在水稻中創造轉殖系，利用各轉殖系之癒傷組織進行誘導並以PCR 技術檢測。發現水稻癒傷組織未受誘導劑doxycycline 誘導而可自行轉位，以南方氏墨點法分析自行轉位大多發生於T-DNA 多拷貝數之轉殖水稻。因此，判斷Tetpro 啟動子在植物中不宜作為單獨誘導之工具；為了延伸Tet-on 系統在植物之實用性，本試驗更進一步建立TetPR 雙重調控系統，即結合PR-1a 及Tet-on 啟動子系統及luciferase (LUC)報導基因，需以兩種誘導劑水楊酸 (Salicylic acid) 與Doxycycline 同時誘導，才能驅動報導基因。該系統轉入水稻，發現水稻癒傷組織之標的基因已無自行表現的情況，且在經由兩種誘導劑同時誘導下最高可增加16.81 倍的LUC 表現。

關鍵字

水稻 ； 可誘導啟動子 ； 報導基因

並列摘要

In this study, the Tet-on system from microorganism was intergrated with Ac transposon in an attempt to construct an efficient and inducible transposon system in plant. We combined the Ac transposase with the inducible Tetpro promoter based on Tet-on system to create the inducible transposon, TetAc. The system was introduced into rice plants by agro-transformation. The transgenic calli of TetAc system were induced with several concentrations of doxycycline and induction periods and subsequently analyzed by PCR and DNA blot techniques. We found spontaneous transposition of TetAc in the transgenic rice lines mostly containing multiple copies of TetAc construct. Transgenic rice lines containing sinlge and non-spontaneous transposed TetAc were demonstrated to occure transposition events by doxycycline induction. In order to establish a double control system ＂TetPR＂, we used luciferase (LUC) and rtTA gene to fuse with the Tetpro and PR-1a inducible promoter, respectively. The TetPR system was introduced into rice by agro-transformation. No spontaneous expression was detected. After induced with doxycycline and salicylic acid in the rice calli of TetPR system, the levels of LUC activity of the rice calli could achieve 16.82-fold compared with non-induction.

並列關鍵字

Rice ； Inducible promoter ； Reporter gene