建構雙重誘導之植物基因表現系統

Construction of a Dual Inducible Plant Gene Expression System

龎靖(Ching Pang) ； 常玉強(Yuh-Chyang Charng)

《作物、環境與生物資訊》 10卷3期 (2013/09) Pp. 200-208

https://doi.org/10.30061/CEB.201309_10(3).0004

摘要

基因轉殖植物常需在特定組織或時間表現標的基因，學者曾建構可誘導系統，希望藉由可誘導型啟動子控制標的基因的表現；但有時單一可誘導啟動子不易精確操控，因此本研究利用可受Dexamethasone（DEX）誘導之pINDEX系統結合PR-1a啟動子（受水楊酸誘導），並以luciferase（luc）及barnase（brs）基因為標的（前者為冷光基因，後者為致死基因），期望建立一有效之雙重調控系統，命名為pI4PRLUC、pI4PRbrs。上述構築以農桿菌轉殖法轉入菸草創造出各轉殖系，並利用菸草各轉殖系之葉片及癒傷組織進行誘導與檢測。pI4PRLUC菸草葉片經誘導並以in vivo及in vitro冷光酶試驗後，當SA濃度為1與5 mM及DEX濃度為1與10 μM時發現冷光酶有表現。pI4PRbrs菸草癒傷組織經誘導後，以barnase為報導基因觀察發現，當SA濃度為1 mM及DEX濃度為1、10與100 μM時，癒傷組織皆有出現褐化且死亡性狀。本試驗結果證明DEX之誘導及SA與DEX之雙重誘導可使轉基因植物表現標的基因。

關鍵字

菸草 ； 可誘導啟動子 ； 報導基因

並列摘要

In this study, we established a dual inducible system to control the gene expression in plants based on the plant inducible pINDEX vector, which is composed of two components: one constitutive expressed gene yielding GVG protein to activate another gene by the existence of the inducer, dexamethasone (DEX). The dual inducible system was achieved by replacing the constitutive promoter of pINDEX with the PR-1a promoter to trigger either a luciferase (luc) or a barnase (brs) gene, yielding the plasmids pI4PRLUC and pI4PRbrs. Accordingly, the expression of the target genes requires the existence of both DEX and salicylic acid (SA). The plasmids described above were introduced into plants by agro-transformation. No spontaneous expression was detected in transgenic tobacco with the dual inducible systems. After induction, pI4PRLUC system could be induced by DEX and SA and increased for about 25 to 41 folds of luc gene expression ratio in transgenic tobacco. Furthermore, the pI4PRbrs transgenic tobacco calli showed lethal phenotype when DEX and SA were applied.

並列關鍵字

Tobacco ； Inducible promoter ； Reporter gene