利用Tet-on系統建構植物可誘導轉位子

Abstract

本實驗利用微生物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，驅動轉位子轉位，快速且大量創造突變體。 在實驗中，利用PR-1a與Tet-on系統的Tetpro可誘導啟動子結合Ac轉位子與luc報導基因創造出三種系統，分別為TetLUC單一調控系統 (由Tetpro啟動子結合luc基因)、TetAc可誘導轉位子系統 (由Tetpro啟動子結合轉位酶基因) 與TetPR雙重調控系統 (由PR-1a啟動子結合rtTA基因，Tetpro啟動子結合luc基因)。 以農桿菌轉殖法在水稻 (單子葉植物) 與菸草 (雙子葉植物) 中創造出各系統之轉殖系。利用各轉殖系之癒傷組織進行誘導與檢測，發現TetLUC單一調控系統在水稻與菸草癒傷組織中，未誘導之Tetpro啟動子有自行啟動的情況，其比例在水稻轉殖系中約佔88.8 %。不過以誘導劑Doxycycline誘導，仍可增加水稻與菸草中LUC表現比例約2 ~ 92.7倍與2 ~ 24.63倍 (相對於未誘導之轉殖癒傷組織)。另外，TetAc可誘導轉位子系統中的水稻癒傷組織同樣發現自行表現的情況，使轉位子自行轉位，其比例約佔90.47 %。 為了解決自行轉位狀況，本實驗更進一步建立TetPR雙重調控系統。誘導發現，此系統的水稻癒傷組織啟動子已無自行表現的情況，且在經由兩種誘導劑水楊酸 (Salicylic acid) 與Doxycycline同時誘導下有最高增加16.81倍的LUC表現，不過其誘導後LUC表現量較弱。因此，未來研究重點為：提高誘導之表現量，以增強此系統在功能基因的利用性。

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. As a control, the inducible Tetpro promoter was also fused with luc gene, termed as TetLUC system. Each system was introduced into rice and tobacco by agro-transformation. The transgenic calli of TetAc system were induced with several concentrations of doxycycline and induction periods. These inductions were applied to the control, TetLUC system. We found about 88.8 percent spontaneous transposition of TetAc in the transgenic rice lines. The Tetpro promoter of the TetLUC was also observed for about 90.47 percent spontaneous expression in transgenic rice lines. However, the Tetpro promoter of the TetLUC system could be induced by doxycycline and increased for about 2 ~ 92.7 -fold and 2 ~ 24.43 -fold of luc gene expression ratio (compare with uninduced transgenic line) in the rice and tobacco calli, respectively. In order to establish a double control system “TetPR”, we used luc and rtTA gene to fuse with the Tetpro and PR-1a inducible promoter, respectively. The TetPR system was indroduced into rice and tobacco by agro-transformation. No spontaneous expression was detected. After induced with doxycycline and salicylic acid in the rice calli of TetPR system, the LUC expression of the rice calli could achieve 16.82 -fold compared with non-induction. However, the luc gene expression is relatively low compared with the luc gene which was driven by 35S promoter. Further experiments should find a way which can increase the expression of luc gene and the availability of the inducible transposon system in the future.