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標題: | 利用阿拉伯芥Di19-2蛋白研究阿拉伯芥鈣離子依存性激酶之基質專一性 Study of Substrate Specificity of Arabidopsis Calcium-Dependent Protein Kinases Using Arabidopsis Drought-Induced Protein 19 as a Substrate |
作者: | Chia-Lun Chang 張家綸 |
指導教授: | 張英? |
關鍵字: | 鈣離子依存性激酶,親緣分析,AtDi19蛋白,基質專一性,定點突變,ACC合成酶, calcium-dependent protein kinases,phylogenetic analysis,AtDi19 protein,substrate specificity,site-direct mutagenesis,ACC synthase, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 鈣離子依存性激酶 (Calcium-dependent protein kinase; CDPK) 是植物細胞內十分重要的鈣離子感應器,它在接收到鈣離子訊號後會對下游的基質進行磷酸化,將鈣離子訊號傳遞下去。根據阿拉伯芥 (Arabidopsis thaliana) CDPK與CRK (CDPK-related kinase) 的胺基酸序列所做的親緣分析 (phylogenetic analysis),34個CDPK可分為四子群 (subgroup),且第四子群與CRK的親緣關係較為接近;而這樣的現象在匯入其他物種的CDPK與CRK同樣保持,且發現若進行區域 (domain) 的獨立分析,也觀察到相同現象。阿拉伯芥AtDi19 (drought-induced protein 19) 蛋白是近幾年被發現新的CDPK基質,AtDi19蛋白在阿拉伯芥中有七個家族成員,分別為AtDi19-1~7,具有兩個可能會與DNA結合的類鋅手指結構區域 (zinc-finger like motif),目前發現AtDi19-2可能與高鹽逆境有關。前人發現AtDi19-2上找到一個磷酸化位點在Ser109,並命名為AtDi19-2-2。且AtDi19-2-2具有基質專一性 (substrate specificity),只會被AtCPK16所磷酸化而不被其他CDPK磷酸化,在本研究中利用含有磷酸化位點的融合胜肽 (fusion peptide) 之定點突變 (site-directed mutagenesis) 株發現Di19-2-2上Ser109前後-1~+2對於AtCPK16的辨認十分重要。另外在本研究中,找到兩個尚未發表的阿拉伯芥CDPK的基質,分別為阿拉伯芥ACC合成酶5和7 (AtACS5, AtACS7)。 Calcium-dependent protein kinases (CDPKs) play important roles in the Ca2+ signal transduction in plants. CDPKs activated by Ca2+ signal phosphorylate proteins involved in the Ca2+ signal transduction. According to the phylogenetic analysis of 34 CDPKs and 8 CRKs (CDPK-related kinase) in Arabidopsis thaliana, CDPKs were divided into 4 subgroups, and there is the similarity among the CDPKs of subgroup IV and CRKs. This study includes the CDPKs and CRKs of other species, and showed the similar result. Moreover, the phylogenetic analysis of the domains also divided into 4 subgroups. The result of grouping is consistent in planta. In a previous study, the Arabidopsis thaliana AtDi19 (drought-induced protein 19) protein was found to be the substrate of CDPK. The AtDi19 gene family encodes 7 proteins, AtDi19-1~7, which contain two zinc-finger like motifs. AtDi19-2 could be involved in the salt stress response. Based on another research, Ser109 is the phosphorylation site of the AtDi19-2, namely AtDi19-2-2. In particular, AtDi19-2-2 was only phosphorylated by AtCPK16 but not by other CDPKs. The result showed the substrate specificity of CDPKs. In order to identify which amino acids are important for the recognition of AtCPK16, site-directed mutagenesis of fusion peptide containing the phosphorylation site revealed that -1~+2 amino acids next to Ser109 are important for the recognition. In addition, we discovered two additional substrates of Arabidopsis CDPKs, Arabidopsis ACC synthase 5 and 7 (AtACS5, AtACS7). |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47870 |
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顯示於系所單位: | 植物科學研究所 |
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