2013-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/692336摘要：本研究欲探索二個 AP2/ERF 超級家族內之轉錄因子基因ERF1 與AtERF#018。這二個 轉錄因子是篩選自過度表達阿拉伯芥之ERF 轉錄因子之轉殖種子組中獲得，並且它的 非生物逆境反應不同於野生型。AtERF#018 (At1g74930) 是 CBF/DREB 次家族之成員， 而ERF1 (At3g23240)屬於ERF 次家族。當AtERF#018 過渡表達時轉殖株矮小並延遲開 花，節間短，同時亦較野生型對乾旱敏感。初步實驗中我們發現ABI1 誘導AtERF#018 基因的表現，而ABI2 則是AtERF#018 的下游基因。AtERF#018 可以吸附到ABI2 基因 啟動子上面一個新的cis-element 序列CGACCAAT。這些結果顯示AtERF#018 是ABI1 和 ABI2 之間的連結者，並且在乾旱逆境下調控ABA 的反應。微陣分析顯示AtERF#018 直接下游基因含有LOX, AOS, AOC, OPR3 和JAR1 等均是JA 和JA-isoleucine 的合成有 關。本計劃中我們要回答下面的問題。1.在乾旱逆境下，細胞內有多少ABI2 基因表現 是因為AtERF#018 蛋白質所調控的? 為了此一目的 AtERF#018, ABI1 和 ABI2 等突變 株都會用於乾旱逆境下測試AtERF#018 蛋白這個路徑在ABI2 基因表現上的重要性。 2. AtERF#018 蛋白是否真的結合到LOX, AOS, AOC, OPR3 和JAR1 等基因啟動子上的 假設性cis-element 序列? 染色質免疫沉澱方法將會被使用到。3. AtERF#018 基因上游 的轉譯子是什麼? 酵母菌一次雜合測試會用來探討不知道的轉譯子如此將可以提供受 傷之早期訊息傳遞和下游JA 合成之間的連結。知道ERF1 參與病毒的防禦已經有一段 時間，並且充當乙烯與JA 訊息傳導的下游分子，扮演一個調控防禦反應的重要因素。 然而ERF1 在非生物逆境方面的功能則完全未知。我們初步的實驗結果顯示35S:ERF1 轉殖株比起野生型更為耐旱耐熱耐鹽。同時ERF1 蛋白在感受不同環境刺激扮演多重 角色，因為它可以結合到啟動子上不同的cis-element 序列並且誘導不同基因表現。本 研究計劃內我們將以修正過的酵母菌一次雜合技術尋找在ABA 及鹽逆境處理下能與 ERF1 結合的特殊調控因子並且共同結合到P5CS1 基因啟動子上面的DRE 序列。這個 研究將可回答為什麼一個特殊的cis-element 兩側的序列在基因表現上是如此重要。同 時針對 microRNAs 如何在逆境之下調控ERF1 與 AtERF#018 二個轉錄子亦將加以研 究。 表<br> Abstract: In this study we intend to investigate two AP2/ERF superfamily transcription factor genes, ERF1 and AtERF#018 which were obtained from screening a collection of transgenic lines that over-express HA-tagged ORFs of the Arabidopsis ethylene response factor (ERF) transcription factors, and exhibited modified abiotic-stress responses when compared with wild-type plants. ERF1 (At3g23240) belongs to the ERF subfamily; and AtERF#018 (At1g74930) belongs to the CBF/DREB subfamily. AtERF#018 overexpression transgenic plant exhibited smaller plant size, late flowering time as well as sensitive to drought stress than wild type plants. In the preliminary study, we found that AtHB6 together with ABI1 cooperatively activate the expression of AtERF#018, and ABI2, another negative regulator of ABA, was downstream of AtERF#018. We also discovered that AtERF#018 binds to a new cis-element, CGACCAAT in the promoter of the ABI2 gene. These results suggest that AtERF#018 is a linker between ABI1 and ABI2, and play a role under drought stress and modulate ABA response. Microarray study revealed the directly up-regulated genes of AtERF#018 includes LOX, AOS, AOC, OPR3 and JAR1 indicating the involvement of AtERF#018 in JA and JA-isoleucine biosynthesis. In this project, we want to answer the following questions. 1. What is the proportion of the ABI2 activation in the cell under drought stress dependent on AtERF#018 protein? AtERF#018, ABI1 and ABI2 mutant lines will be used under dehydration stress to see how important the AtERF#018 signaling pathway in the expression of ABI2. 2. Does AtERF#018 protein bind to the proposed cis-elements in the promoters of the LOX, AOS, AOC, OPR3 and JAR1 genes? The chromatin immuno-precipitation (CHIP) assay will be adopted. 3. What is the upstream transcription factor of the AtERF#018 gene? Yeast one hybrid assay will be used to uncover the unknown transcription factor which provides the link between early signaling of wounding or herbivore attack and the downstream JA biosynthesis. ERF1 is known involved in the defense of pathogens for a long time, and acts downstream of the intersection between the ethylene and JA pathways, a key element in regulating defense response. However, the function of ERF1 in response to abiotic stresses has never been reported. Our preliminary study indicated that dehydration-, heat-shockand salt-stress tolerance in the 35S:ERF1 plants. Also, the ERF1 protein plays multiple roles in sensing different environmental stimuli and binds to different cis-elements to activate a suite of specific gene groups, resulting in crosstalk between different signals. In this project, the specific ERF1- interacting partners binding to the DRE element in the promoter of P5CS1 (Δ1 -pyrroline-5-carboxylate synthetase1, the key enzyme involved in Pro synthesis) under ABA and salt treatments will be investigated using modified yeast one hybrid assay. This study can answer why the flanking region of a specific cis-element is important for the gene expression. Also microRNAs that regulate ERF1 and AtERF#018 under stress conditions will be explored.AP2/ERFs與其它轉錄調控因子參與植物逆境的分子機制-AP2/ERF基因家族之二個轉錄調控因子參與非生物逆境反應之特性分析(1/3)