Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22552
標題: 以蛋白質交互作用分析耐鹽植物冰花腎形細胞累積耐鹽相關mcSKD1蛋白及其參與之高等植物耐鹽機制
Characterization of bladder cell-specific mcSKD1 and its interacting proteins in halophyte Mesembryanthemum crystallinum L.
作者: 周映孜
Jou, YingTzy
關鍵字: Mesembryanthemum crystallinum
冰花
yeast two-hybrid
蛋白質交互作用
出版社: 生命科學系所
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摘要: 冰花(Mesembryanthemum crystallinum L.)是一種具有C3-CAM光合作用轉換形式植物可生存於高鹽環境的耐鹽模式植物,表皮特化的腎型細胞在高鹽環境下液胞會迅速膨大,具有儲存水分及區隔過量鈉離子的功能。利用酸性下會呈紅色的染劑neutral red浸染不同時期的冰花表皮細胞,觀察到冰花腎型細胞液胞pH值除了會隨著發育逐漸由酸性轉變為中性外,幼年期的腎型細胞在照光時液胞pH值亦會快速升高,顯示腎型細胞的代謝旺盛並非僅供儲存之用,還具有調節離子平衡之功能。冰花的mcSKD1 (suppressor of K+ transport growth defect)是一個會在冰花的腎型細胞表現的鹽誘導基因,mcSDK1蛋白與酵母菌的液胞運送相關蛋白VPS4 (vacuolar protein sorting 4)和阿拉伯芥AAA-type ATPase (ATPase associated with a variety of cellular activities)具有高度的相似性,並觀察到mcSKD1主要分布在冰花細胞質之ER/Golgi network。為了更進一步瞭解mcSKD1蛋白之功能,以酵母菌雙雜交系統分別由阿拉伯芥基因庫中篩選出與SKD1具有蛋白質交互作用的過氧化逆境相關的catalase 3、細胞壁結構的AGP21 (arabinogalactan protein 21)等蛋白。以及由冰花根部加鹽處理三天所製成的基因庫篩選出與SKD1具有蛋白質交互作用蛋白mcSNF1 (sucrose non-fermenting 1)及mcCPN1 (copine 1)等蛋白。SNF1已知為一參與醣類代謝及逆境相關訊息傳遞作用之蛋白激酶,mcSNF1還具有UBA (ubiquitin associated) domain,mcCPN1蛋白具有與細胞膜蛋白質運送相關之VWA domain及具有E3 ubiquitin ligase活性之RING domain,mcSNF1與mcCPN1也會受加鹽誘導提高蛋白累積量。免疫共沈澱證明了mcSKD1與mcSNF1與mcCPN1具有蛋白質交互作用,以pull-down的蛋白產物比例計算發現,加鹽處理會使mcSKD1與mcSNF1結合的比例提高三倍。利用蔗糖梯度分離microsome蛋白以及細胞免疫染色法,發現加鹽處理會快速改變mcSKD1、mcSNF1和mcCPN1在細胞中分佈的位置,且colocolization的比例提高。當以破壞細胞骨架的藥劑處理細胞時,mcSKD1及mcSNF1在細胞中的分布改變,累積在細胞邊緣,推測細胞骨架參與mcSKD1在細胞中的運輸。鹽處理會造成阿拉伯芥snf1或copine1突變株中atSKD1不正常的聚集,並對鹽更加敏感。實驗結果推測冰花mcSKD1蛋白透過與逆境訊息傳遞mcSNF1、細胞膜相關蛋白mcCPN1的交互作用,參與鹽逆境時ubiquitin相關的蛋白質運送或是逆境訊息傳遞,幫助耐鹽植物冰花在高鹽環境下維持正常生理代謝。
The halophyte Mesembryanthemum crystallinum L. (ice plant) is an inducible CAM and a model plant for studying salt-tolerant mechanisms in higher plants. It contains specialized epidermal bladder cells (EBCs) which rapidly expend under salt stress. The major functions of EBCs are maintaining ion homeostasis and water storage. During plant development, increase in vacuolar pH was observed in EBCs. In addition, light-induced rapid change in vacuolar pH was found in juvenile stage of ice plant indicating EBCs are metabolic active cells. A salt-induced gene mcSKD1 (suppressor of K+ transport growth defect) is highly expressed in EBCs. It has high homology to yeast VPS4 (vacuolar protein sorting 4) and Arabidopsis AAA-type ATPase (ATPase associated with a variety of cellular activities). Immunofluorescence labeling showed mcSKD1 protein was located in cytoplasm around ER/Golgi network. Yeast two-hybrid screen was performed to identify mcSKD1-interacting proteins. Using a library constructed from Arabidopsis, catalase 3 and AGP21 (arabinogalactan protein 21) were identified. Using a library constructed from salt-treated ice plant roots, mcSNF1 (sucrose non-fermenting 1) and mcCPN1 (copine 1) were further characterized. Yeast SNF1 is a ser/thr kinase that plays an important role in carbon metabolism and stress signaling. The SNF1 protein identified in ice plant contained an extra UBA (ubiquitin associated) domain. Sequence analysis of mcCPN1 showed it contains a VWA domain for membrane trafficking and a RING domain for protein ubiquitination. The accumulation of mcSNF1 and mcCPN1 was both induced by salt stress. Co-immunoprecipitation experiment showed mcSKD1 interacted with both mcSNF1 and mcCPN1 in vitro. In vivo pull-down assay showed a 3-fold increase in the association between mcSKD1 and mcSNF1 under salt stress. Microsomal fractionation and immunolabeling experiments showed salt induced rapid changes in cellular localization of mcSKD1, mcSNF1, and mcCPN1. When the cytoskeleton was disrupted, the distribution of mcSKD1 and mcSNF1 was altered, as seen by abnormal aggregation around plasma membrane. The result suggested that mcSKD1 is trafficking along the cytoskeleton. Arabidopsis snf1 and copine 1 mutants showed more salt sensitive than the wild type and aggregation of atSKD1 inside mutant cells. The results suggested that mcSNF1 and mcCPN1 function together with mcSKD1 in ubiquitin-related protein trafficking and stress signal transduction in order to maintain normal growth under high salinity.
URI: http://hdl.handle.net/11455/22552
其他識別: U0005-2008200715562100
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2008200715562100
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