阿拉伯芥ABF3在鹽及離層酸作用之下調控ABI5基因表現之研究

Abstract

在植物中，bZIP 是一群大的轉錄因子（TF）基因家族。先前的研究發現，bZIPs參與許多非生物逆境反應。bZIP會藉由調節許多下游基因，進而去對抗環境逆境。然而，對於植物抵抗環境壓力的轉錄機制仍然不是很清楚。 14-3-3蛋白是一種鷹架蛋白，其與標的蛋白結合可導致酶的激活/去激活、改變座落位置，細胞核，線粒體和葉綠體等胞器之進出、防止或刺激蛋白質的降解。ABF3及ABI5皆受鹽分誘導表現。為了研究ABF3這一個bZIP類的轉錄因子，在阿拉伯芥中參與的轉錄機制，以及和14-3-3蛋白之間的交互關係，本研究使用了螢光素酶測試法檢測轉錄活性。本實驗結果顯示共同表現ABF3及14-3-3能夠提升以ABI5啟動子驅動的螢光素酶表現。染色質免疫沉澱 (ChIP) 實驗結果分析發現ABF3會與ABI5啟動子結合。利用試管內激酶活性分析，鈣離子依存性激酶可磷酸化ABF3；並找到三個磷酸化位點分別為Thr-128，Ser-134及Thr-451。總體而言，本研究發現在阿拉伯芥中14-3-3與轉錄因子ABF3共表現下，可調節下游基因ABI5之表現，在阿拉伯芥當中是否可透過此調控方式抵禦鹽分逆境，則需要更多後續研究。

Basic region/leucine zippers (bZIPs) are transcription factors (TFs) encoded by a big gene family in plants. Previous studies showed that bZIPs participate in many abiotic stress responses. bZIPs can regulate many down-stream genes, which could response against the environment stresses. However, the transcriptional network and the transcription mechanism in plants remained unclear. 14-3-3 proteins are scaffold proteins, which can result in activation/deactivation of enzymes, alteration of the translocation into/out of organelles like the nucleus, mitochondria and chloroplasts, prevention or stimulation of proteolytic breakdown of protein. The gene expression of ABF3 and ABI5 are induced by salt. In order to investigate the relationship between ABF3, a bZIP TF, and 14-3-3 protein in the transcriptional network in Arabidopsis, This research carried by protoplast transactivation assay using luciferase as a reporter. The results showed that the expression of both 14-3-3 and ABF3 can up-regulate ABI5 gene expression. Chromatin immunoprecipitation (ChIP) analysis results showed that ABF3 binds to the promoter of ABI5. In vitro kinase assay showed that calcium dependent protein kinase can phosphorylate ABF3. Thr-128, Ser-134 and Thr-451 phosphorylation sites were identified. In conclusion, 14-3-3 and ABF3 together regulate the transcription level of down-stream ABI5 gene. Whether this regulation pathway is involved in salt stress response requires further studies.