阿拉伯芥啟動子在基因轉殖的表現行為分析;Exploration of Arabidopsis promoters for stable gene expression

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Title:	阿拉伯芥啟動子在基因轉殖的表現行為分析;Exploration of Arabidopsis promoters for stable gene expression
Authors:	陳怡仁;Chen, I-Jen
Contributors:	生命科學系
Keywords:	阿拉伯芥;啟動子;基因轉殖植物;基因靜默;Arabidopsis;promoter;transgenic plants;gene silence
Date:	2021-07-12
Issue Date:	2021-12-07 11:21:14 (UTC+8)
Publisher:	國立中央大學
Abstract:	科學家利用基因工程將植物基因進行改造或在植物中表達外源基因，這些改造過的植物稱為基因轉殖植物，基因轉殖植物有許多應用潛力，例如研究有用基因功能、增強植物對極端氣候的抵抗力、增加營養價值或用於生產有用的化合物等。這些基因轉殖植物通常透過所選的啟動子控制內源或外源基因的表達，花椰菜嵌紋病毒35S就是常用的啟動子之一。然而，外源性的35S啟動子所驅動的基因在基因轉殖植物生長、繁衍數個世代後，往往會發生基因靜默的現象，導致基因無法表現，這大大地阻礙了基因轉殖植物在應用上的穩定性，因此急需尋找到可以替代35S的穩定啟動子。為此，我們選擇5個內源性啟動子進行基因轉殖阿拉伯芥的建構，並使用阿拉伯芥的第2代至第5代基因轉殖植物來進行實驗測試所選定啟動子的強度和穩定性。我們的結果顯示： AT4G05320 (UBQ10)、AT5G60390 (EF1α)、AT4G31700 (RPS6a) 和 AT1G13320 (PP2A)四個啟動子在各個組織均可表達，受這些啟動子所驅動的eGFP表現基因在第二代至第五代的轉植株中也可以穩定表達。其中，EF1α和UBQ10啟動子的活性強度與35S相似，是替代35S的最佳候選內源啟動子。另一方面，RPS6a和PP2A啟動子則是具有中度與低度活性，這些具體成果可以提供未來建構基因轉殖植物時所需啟動子的多重選擇。出乎意料的是，我們所選的啟動子之一AT1G13440 (GAPC2)驅動的表達基因在基因轉殖植物繁殖幾代時也出現基因靜默現象。這表示利用內源性基因本身的啟動子和外源性啟動子（如35S）都可能在基因轉殖植物中引發基因靜默。總結而言，我們的研究提供未來應用於基因轉殖植物時，具有表達穩定性和廣泛活性的植物啟動子替代選擇。;Plants can be genetically modified (termed transgenic plants) to meet special needs, such as increased resistance to extreme weather, increased nutritious values, or the production of the useful compounds. These often involve the expression of endogenous or exogenous genes under the control of chosen promoters. Cauliflower mosaic virus (CaMV) 35S is one of the promoters commonly used. However, genes driven by the foreign 35S promoter often suffer from gene silencing after the transgenic plants grow for a few generations, greatly hampers the stable application of transgenic plants produced. Therefore, there are urgent needs of identifying stable promoters that can replace 35S. For this purpose, we selected and experimentally tested the strength and stability of 5 endogenous promoters in Arabidopsis in 2nd (T2) to 5th (T5) generations of transgenic plants. Our results revealed that promoters AT4G05320 (UBQ10), AT5G60390 (EF1α), AT4G31700 (RPS6a), and AT1G13320 (PP2A) have ubiquitous activities and can drive stable expression of the reporter gene eGFP in T2 to T5 generations of the transgenic lines. Among them, promoter strength of EF1α and UBQ10 is similar to that of 35S thus are optimal candidates to replace 35S. RPS6a, and PP2A promoters, on the other hand, have intermediate to low activities, allowing for versatile choices in expressing transgenes of interests. Unexpectedly, the reporter gene driven by one of the promoters selected, AT1G13440 (GAPC2), also exhibited gene silencing behaviors when transgenic plants were propagated for several generations. This indicated that both origin endogenous (which construct from endogenous promoters) and heterologous (such as 35S) promoters can elicit gene silencing in transgenic plants. In conclusion, our study revealed alternative choices of plant promoters with expression stability and wide-range of activities for generating transgenic plants.
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