2013-01-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/683502摘要：本研究我們欲探索二個AP2/ERF家族內之轉錄因子，它們在環境逆境反應中扮演負調控者的角色。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。AtDS1 (At1g74930) 是ERF家族中之DREB A-5亞族之成員。當AtDS1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。初步實驗中比較AtDS1的基因表現在abi1KO與abi1-1顯性變異系時發現ABI1作用在AtDS1之上游。我們亦得知AtHB6 與ABI1會一起活化AtDS1基因的表現。ABI2是另一個 ABA的負調節者則會受到AtDS1的表現所影響。同時我們也觀察到在AtDS1OE與AtDS1-RNAi之變異株中ABI2 的表現會分別上昇與下降。我們認為ABI2 是AtDS1的直接下游基因。但是在ABI2 的啟動子序列上找不到AP2/ERF轉錄因子可以與之結合的cis-element。已知道只有41 % 的AP2/ERF轉錄因子家族成員可結合到GCC box, DRE, RAV1與ANT-AP2R1R2等cis-element上。而在ABI2的啟動子上找不到任何已知的cis-element。此顯示有可能AtDS1結合到一個新的cis-element上。同時上述之初步結果亦顯示AtDS1可能扮演著ABI1 與 ABI2之間的連接者並且在乾旱逆境與ABA處理下有其功能。我們將使用各項反向遺傳方法來研究AtDS1.的功能。又組織專一性表現，細胞內的位置，基因破損與基因過度表達變異株之生理現象均將加以研究。此外pull-down與co-immunoprecipitation等技術研究可能與AtDS1有交互作用的蛋白質也將加以探究。微陣技術則可顯示AtDS1過度表達變異株較之野生型受到誘導與受到抑制的基因群。為了探討AtDS1 蛋白質可以結合的cis-element 會使用 chromatin immuno-precipitation (CHIP) 的技術並使用electrophoretic mobility shift 技術來確定驗証所獲得的cis-element。ABI1與ABI2二者均為ABA 的共同接受者或主要調控基因。這一個研究將使ABA訊息傳導多增加了一層新的關係。 Aintegumenta-Like 7 (AIL7, At5g55610) 則是受到鹽與滲透逆境所誘導但對ABA不敏感。在初步實驗中得知AIL7OE較野生型對乾旱與鹽逆境更為敏感。我們在短期測試中發現全長的AIL7基因與基因之C端不具有轉錄活性，而AIL7基因之N端片段則具明顯的轉錄活性。我們希望執行此計晝將使我們了解這二個基因之分子功能，看它們如何在面對逆境反應中扮演負調控者的角色。 <br> Abstract: In this study we intend to investigate two unknown function AP2/ERF family transcription factor genes which serve as negative regulator in response to environmental stresses. These two genes were obtained from screening a collection of transgenic lines that over-express HA-tagged ORFs of the Arabidopsis ethylene response factor (ERF) transcription factors. AtDS1 (At1g74930) is a member of the DREB A-5 subfamily of ERF/AP2 transcription factor family. AtDS1 overexpression transgenic plant exhibited smaller plant size, late flowering time, short internode as well as sensitive to drought stress than wild type plants. In the preliminary study, we found that ABI1 might act as an upstream gene of AtDS1 by comparing the AtDS1 expression level in abi1KO and abi1-1 dominant mutant lines. We also learned that AtHB6 together with ABI1 cooperatively activate the expression of AtDS1. Interestingly, gene expression of ABI2, another negative regulator of ABA, was influenced by the expression level of AtDS1. Thus in AtDS1OE and AtDS1-RNAi mutant lines the expressions of ABI2 were increased and decreased, respectively. We suggest that ABI2 is one of the direct targets of AtDS1. No AP2/ERF protein binding cis-element was found in the ABI2 promoter. Only 41 % AP2/ERF family proteins bind to known AP2/ERF binding sites, like GCC box, DRE, RAV1, and ANT-AP2R1R2, and there is no known cis-element existed in the promoter of the ABI2. This indicated that AtDS1 might bind to a new cis-element on ABI2 promoter. These results suggest that AtDS1 is a linker between ABI1 and ABI2, and play a role under drought stress and ABA treatment. We will use various reverse-genetic approaches to study the function of AtDS1. Tissue specific expression, cell localization, the physiology of knock-out and over-expressed plant will be studied. Also pull-down and co-immunoprecipitation will be used to study any protein would interact with AtDS1 in vitro and in vivo, respectively. Microarray will be used to reveal the up-regulated and down-regulated gene using AtDS1 over-expressing plants. To reveal the cis-element of AtDS1 protein could bind, the chromatin immuno-precipitation (CHIP) will be adopted and electrophoretic mobility shift assay will be used to confirm the identity of the cis-element. ABI1 and ABI2 both are the master gene or co-receptor of ABA and central element is ABA signaling. This knowledge will provide a new layer of ABA signaling which has never been mentioned in the literature. Aintegumenta-Like 7 (AIL7, At5g55610) was induced by salt and osmotic stresses but not by ABA. In the preliminary test, AIL7OE was more sensitive to drought and salt when compared to WT plants. It is interesting to find the full length of AIL7 and C-terminal had no transcriptional activity by transient assay, while N-terminal fragment showed distinct transcriptional activity. Eventually I hope to explicitly understand the molecular function of these two genes under stress and how they play a negative regulatory component in the stress response.阿拉伯芥轉錄子Aintegumenta-Like 7AtDS1乾旱誘導負向調控者Arabidopsis thalianaAintegumenta-Like 7AtDS1ERF/AP2 transcription factornegative regulator