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標題: | 水稻CBLs和CIPK15在糖訊息調控的交互作用 Interaction of CBLs and CIPK15 in the sugar signaling pathway in rice |
作者: | Ming-Yang Ho 何銘洋 |
指導教授: | 賀端華(Tuan-Hua David Ho) |
關鍵字: | Calcineurin B-like (CBL) 蛋白,CBL-interacting protein kinases (CIPKs),NAF/FISL motif,alpha-澱粉水解酶,N-myristoylation motif,暫時性表現試驗,糖反應核酸區域, Calcineurin B-like (CBL) protein,CBL-interacting protein kinases (CIPKs),NAF/FISL motif,alpha-amylase,N-myristoylation motif,transient expression assay,sugar response complex (SRC), |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 植物特有的鈣離子感受器Calcineurin B-like (CBL) 蛋白,會藉由和CBL-interacting protein kinase (CIPK) 交互作用而活化後者激酶的活性。近年的研究發現,在水稻中CIPK15會在缺氧的環境下,經由糖訊息調控來提升alpha-澱粉水解酶 (alpha-amylase)的表現。然而,目前在糖訊息調控的路徑當中,和CIPK15交互作用的CBL還未被發現。因此,在本篇研究中,我們利用酵母菌雙雜合 (yeast two-hybrid)技術鑑定出CBL1、CBL4和CBL7會藉由CIPK15的NAF/FISL區域交互作用。這三個在N端具有N-myristoylation motif的CBLs在水稻原生質體轉染 (protoplast transfection)中被發現位於細胞膜上;CIPK15則是位於細胞質及核內。當CBL1、CBL4或CBL7和CIPK15共同表現時,CIPK15在細胞內的位置會轉移到細胞膜上。但是去除N-myristoylation 修飾的CBL1/4/7 (G2A)會轉而分佈在細胞質及核內,同時也失去了把CIPK15定位於細胞膜的能力。另外,藉由逆轉錄聚合酶鏈式反應 (RT-PCR),我們發現CBL4、CBL7和CIPK15在細胞缺糖時會被誘導表現。而在暫時性表現試驗 (transient expression assay)中,CBL1或CBL7會和CIPK15共同活化alphaAmy3糖反應核酸區域 (SRC)啟動子。另一方面,不在細胞膜上的CBL1/4/7 (G2A)無法和CIPK15共同活化SRC啟動子;而暫時性表現兩個持續活化狀態的CIPK15 (T171D)和CIPK15 (T171DdeltaNAF)顯示前者比後者更有能力活化SRC啟動子。這些結果顯示CIPK15可能是藉由和CBL1/4/7交互作用使激酶活化,並同時定位在細胞膜上傳遞糖訊息調控。 Calcineurin B-like (CBL) proteins, plant-specific calcium sensors, activate CBL-interacting protein kinases (CIPKs) by releasing its autoinhibition. In rice (Oryza sativa), CIPK15 is necessary for up-regulation of alpha-amylase expression through the sugar starvation signaling pathway under oxygen deficiency conditions. However, CBLs which interact with CIPK15 in the sugar signaling pathway have not been identified. By yeast two-hybrid assays, we identified CBL1, CBL4, and CBL7 that interact with the NAF/FISL motif in CIPK15. These three CBLs, bearing N-myristoylation motifs in their N-termini, were localized on the plasma membrane, whereas CIPK15 was localized in both cytosol and nucleus. Levels of CBL4/7 and CIPK15 mRNAs increased under sugar starvation. When co-expressed with CBL1, CBL4, or CBL7, CIPK15 was detected mainly on the plasma membrane. However, the N-myristoylation defected CBL1/4/7 (G2A) neither was localized on plasma membrane nor brought CIPK15 to the plasma membrane. In transient expression assay, co-overexpression of CBL1 or CBL7 with CIPK15 activated alphaAmy3 SRC promoter, whereas CBL1/4/7 (G2A) lost its ability to co-activate alphaAmy3 SRC promoter with CIPK15. These results suggested that CBL1, together with enhanced expression of CBL4 and CBL7, may both activate and recruit CIPK15 to the plasma membrane to initiate the sugar starvation signaling and induce the expression of alpha-amylase. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38617 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物科學研究所 |
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