- 學位論文
以阿拉伯芥內源啟動子元件建立基因過量表現之誘導系統
Establishing an inducible system by endogenous promoter elements for gene overexpression in Arabidopsis
摘要
花椰菜鑲嵌病毒(Cauliflower Mosaic Virus )之35S promoter能於植物中驅動轉殖基因的大量表現，常被作為基改作物中轉殖基因的啟動子。G10-90 promoter 為另一可在植物中驅使基因大量表現之合成啟動子(synthetic promoter)，其利用4重複「G-box10」之順式元件(cis-element) 徵召RNA pol II，使其能結合後端35S minimal promoter (-90至 -1，相對於轉錄起始+1)中之TATA box，有效驅動轉錄起始。Zuo等人所建構pER8載體中的XVE誘導系統，便以G10-90 promoter驅動合成轉錄因子(synthetic transcription factor )XVE大量表現來驅動受LexA-operator控制的目標基因表現。然而XVE誘導系統中，目標基因常於轉殖植物後代有表現下降的情形，推測可能因G10-90 promoter帶有35S minimal promoter而有受植物甲基化修飾靜默(Silencing)的現象。為建立世代交替後仍能持續穩定表現的轉殖基因系統，我將pER8載體中可能造成表觀遺傳學靜默之DNA片段置換成阿拉伯芥內生性元件，利用阿拉伯芥持家基因(housekeeping gene) 包括-RIBOSOMAL PROTEIN S6A (RPS6A)、RUBISCO SMALL SUBUNIT 2B (RBCS2B) 及UBIQUITIN 10 (UBQ10)的啟動子分別驅動XVE表現，此外亦利用RPS6A上游(-127至 -1)取代XVE系統中驅動目標基因的LexA-operator後端35S minimal promoter。利用阿拉伯芥幼苗暫時性表現實驗測試所得構築，我發現所挑選之RPS6A 編碼區上游片段的確可做為RNA pol II起始轉錄用的minimal promoter，三種構築皆可測得目標基因的誘導表現，其中又以UBQ10啟動子(pUBQ10)驅動目標基因的表現最高。本論文成功以阿拉伯芥內生性啟動子建立誘導表現用之構築，未來將可進一步應用其構築作為轉殖基因的載體，有助於建立不受植物表觀靜默抑制之轉殖品系。
並列摘要
The cauliflower mosaic virus (CaMV) 35S promoter is broadly used in driving the overexpression of transgenes in plants. A 35S-derived G10-90 comprises four tandem repeats of ‘G-box10’ for recruiting RNA pol II and a TATA-box containing 35S minimal promoter (-90 to -1, as the transcription starts at +1) for initiating transcription. The G10-90 promoter is frequently utilized due to its strong activity in driving transcription. Zuo et al. adapted G10-90 in the binary vector pER8 to drive the synthetic transcription factor XVE, which drives the transcription of a transgene under the control of LexA-operator. Nevertheless, the induction level of the transgene is usually decreased in the progeny generations of the transgenic lines. The 35S minimal promoter used in the pER8 vector might be a target epigenetically silenced by the chromatin methylation. To establish a robust transgenic system, I used promoters of housekeeping genes including RIBOSOMAL PROTEIN S6A (RPS6A), RUBISCO SMALL SUBUNIT 2B (RBCS2B) and UBIQUITIN 10 (UBQ10) to control the expression of XVE individually in the vector. Furthermore, I replaced LexA-operator downstream 35S minimal promoter with the RPS6A upstream -127 to -1 fragment in pER8. The constructs were then examined by a transient expression system in Arabidopsis seedlings. All three constructs showed the capability of transgene induction, indicating that the minimal upstream fragment of RPS6A is a functional minimal promoter for RNA pol II to initiate the transcription. The promoter of UBQ10 is the strongest promoter among the three constructs. This study has successfully established inducible constructs for transgene expression by using endogenous promoters of Arabidopsis. Such constructs can be applied to produce robust transgenic lines across generations.
參考文獻
延伸閱讀
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