Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90056
DC FieldValueLanguage
dc.contributorWei-Ming Leuen_US
dc.contributor呂維茗zh_TW
dc.contributor.author蔡曉倩zh_TW
dc.contributor.authorShiao-Chien Tsaien_US
dc.contributor.other生物科技學研究所zh_TW
dc.date2014zh_TW
dc.date.accessioned2015-12-09T02:07:04Z-
dc.identifier.citation1.林忠威 (2007). 水稻結鈣激活酵素基因群之表現分布與功能性分析. 國立中興大學, 台中市. 2.詹益維 (2013). 評估 OsCPK26 抑制花粉萌發的機制與尋找水稻花粉中 CDPKs 的下游受質蛋白. 國立中興大學, 台中市. 3.陳婉潔 (2003). 水稻花粉結鈣激酶 OsCPK1 之基因表現、蛋白胞內分布位置與基因轉殖植物分析. 國立中興大學, 台中市. 4.歐天永 (2010). 水稻 OsCPK 及其交互作用蛋白 OIP30 於調控花粉發育、花粉萌發與花藥開裂之角色探討. 國立中興大學, 台中市. 5.Arakaki, N., Nagao, T., Niki, R., Toyofuku, A., Tanaka, H., Kuramoto, Y., Emoto,Y., Shibata, H., Magota, K., and Higuti, T. (2003). Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells. Molecular cancer research : MCR 1, 931-939. 6.Asai, S., Ichikawa, T., Nomura, H., Kobayashi, M., Kamiyoshihara, Y., Mori, H.,Kadota, Y., Zipfel, C., Jones, J.D., and Yoshioka, H. (2013). The variable domain of a plant calcium-dependent protein kinase (CDPK) confers subcellular localization and substrate recognition for NADPH oxidase. 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dc.identifier.urihttp://hdl.handle.net/11455/90056-
dc.description.abstract水稻中有二十九個 CDPKs (calcium dependent protein kinase) 基因成員,其中七個專一表現於花粉。先前實驗室發現將 OsCPK26 大量表現於水稻花粉,會產生雄不稔性狀,另外,若大量表現於百合花花粉,則會強烈抑制花粉萌發,萌發率僅 11.5%,但 OsCPK26(G2A) 卻均無上述現象。為了找出 OsCPK26 抑制花粉萌發的關鍵性胺基酸或蛋白質結構,利用暫時性大量表現 OsCPK26-ECFP 融合蛋白於百合花花粉,發現附膜是 OsCPK26 抑制花粉萌發的必要非充分條件。於 domain swapping 和 C-terminal serial-deletion 實驗中,發現OsCPK26 僅需蛋白 N 端的 7 個胺基酸便可附膜,且至少需其 1-59 a.a.片段,才具有抑制能力,最後透過 point mutation 和 gain-of-function 策略,證實 46-58 a.a.間的 α-helix 為抑制花粉萌發的重要結構。 儘管鈣離子訊號在花粉萌發及延長上扮演重要的角色,但目前只找到少數花粉專一的 CDPKs 之下游受質蛋白。為了尋找下游受質蛋白,本實驗利用 pull-down親和力純化受質蛋白,並以 LC-MS/MS 鑑定其身分,獲得 OsCPK29 之候選受質蛋白,於眾多可能的蛋白中,選擇粒線體的 ATP synthase β-subunit 做進一步研究,將之命名為 ATPSB1。雖然 ATPSB1 普遍存在於水稻各組織中,但 ATPSB1 的相對轉錄量與蛋白表現量,皆於花粉中最高。本實驗發現 ATPSB1 基因靜默水稻會產 生花粉發育不全和花藥無法開裂之性狀,最後導致稔實極低 (約 1%),另外,暫時性表現 ATPSB1-EYFP 融合蛋白於百合花花粉,可知其分布於粒線體中,令人驚訝的是,會附膜的 OsCPK29 和 ATPSB1 共表現時,出現 ATPSB1 從細胞質轉移到細胞膜的現象,暗示這兩個蛋白質間可能有交互作用。進一步將 ATPSB1 與 α-subunit(ATPSA1) 共表現,則發現 ATPSA1 與 ATPSB1 行為相似,均與 OsCPK29 結合於細胞膜,但其他同樣會附膜的 OsCPK21 與 OsCPK26 則均無上述現象。這些結果 說明 ATPSB1 可能於花粉發育或萌發上扮演重要角色,且可能OsCPK29 之下游受質蛋白,未來可更進一步調查 ATPSB1 於細胞膜上之生化特性與生理角色。zh_TW
dc.description.abstractIn rice, CDPK (calcium dependent protein kinase) genes are constituted by twenty nine members with seven of them expressed predominantly in pollen. Previous studies revealed that overexpression of the pollen-predominant OsCPK26, but not its non-membrane bound G2A form, caused male-sterility in rice and strongly inhibited the germination rate of lily pollen to be less than 11.5%. We characterized the protein motifs and amino acid residues in OsCPK26 critical for the inhibition effect. By tracing the transiently overexpressed OsCPK26-ECFP fusion protein in lily pollen, we found that membrane-binding is a prerequisite but is not sufficient for the inhibition effect. The domain-swapping and C-terminal serial deletion constructs revealed that even the N-terminal 7-a.a. fragment is enough for the membrane-binding of OsCPK26. However,the minimal length for the inhibition effect of OsCPK26 require at least its 1-59 a.a.fragment. Through point mutation and gain-of function strategies, an α-helix reside between 46-58 a.a. region was demonstrated to be critical. Although calcium signaling is known to play critical roles in pollen germination and tip growth, only a few proteins had been identified as substrates downstream to pollen-predominant CDPKs so far. Using affinity pull-down, candidate substrate proteins bound by OsCPK29 were identified by LC-MS/MS. A mitochondrial ATP synthase β-subunit, named as ATPSB1, was chosen for further study. Although ATPSB1 is expressed ubiquitously, relatively high transcript and protein level were detected in the mature pollen grains than all other tissues. Silence expression of ATPSB1 disrupted pollen development and anther dehiscence, finally resulted in an extremely low fertility rate (~1%). Transient expression of EYFP-fused ATPSB1 in lily pollen revealed its location within mitochondria. Surprisingly, co-expression of the membrane-bound OsCPK29 recruited ATPSB1 onto the plasma membrane, suggest a strong interaction between these two proteins. Moreover, the α-subunit of ATP synthase (ATPSA1) was also co-distributed with the beta subunit and OsCPK29. None of the other membrane-bound OsCPKs, including OsCPK21 and OsCPK26, possess the affinity with ATP synthase. These results suggest that the ATPSB1 play important roles in pollen development/germination and ATPSB1 may be a native downstream substrate for OsCPK29 in pollen. The functional relevance of ATP synthase on the plasma membrane require further investigations.en_US
dc.description.tableofcontents壹、前言 ............................................... 1 貳、前人研究 ........................................... 2 一、 結鈣激酶 (CDPKs) 之功能 .............................2 二、 花粉管生長的重要因子..................................2 三、 結鈣激酶與其下游受質..................................4 四、 ATP synthase β-subunit 與 eATP ....................5 參、材料方法 ........................................... 7 一、 定點突變............................................7 (一) 聚合酶鏈鎖反應 ..................................... 7 (二) Dpn I 清除背景質體 ................................ 7 二、 基因槍轉殖百合花花粉與萌發率計數.......................7 (一) 金粉製備 ...........................................7 (二) 基因槍轟擊用子彈製備 ............................... 7 (三) 基因槍轟擊用花粉前處理 .............................. 8 (四) 基因槍轟擊操作 ..................................... 8 (五) 花粉 in vitro 萌發 ................................ 8 (六) 花粉萌發率計數 ..................................... 8 三、 Pull-down 釣取受質蛋白流程 ..........................9 四、 Real-time PCR .....................................9 五、 植物蛋白質萃取.......................................9 六、 西方點墨法..........................................9 七、 水稻轉殖...........................................10 八、 轉殖水稻之性狀觀察..................................10 (一) 花粉形態 ..........................................10 (二) 花粉存活率 ....................................... 10 (三) 花粉 in vivo 萌發 ................................ 10 (四) 花藥開裂 ......................................... 11 (五) 稔實率 ............................................11 肆、結果 .............................................. 12 第一部份:OsCPK26 蛋白 N 端之序列功能分析 ................12 一、 OsCPK26 結合細胞膜與抑制花粉萌發之關聯 .............. 12 二、 OsCPK 家族成員之細胞定位與萌發率分析 ................ 12 三、 OsCPK26 之蛋白區塊與抑制抑制花粉萌發之關聯 ...........13 四、 分析 OsCPK26 V domain 結合細胞膜的重要胺基酸 ....... 13 五、 定位 OsCPK26 V domain 內抑制花粉萌發的關鍵胺基酸區間 ....... 14 六、 評估 V domain 上的 α-helix 在抑制花粉萌發所扮演的角色 ........... 14 七、 證實含有 α-helix 的胺基酸區域會抑制花粉萌發 ............................. 15 第二部份:ATP synthase 為水稻花粉 OsCPK29 之潛力受質蛋白 ..................15 一、 以 pull –down 策略獲得 OsCPKs 下游受質蛋白 .............................. 16 二、 以 E. coli 表現 ATPSB1 蛋白與其抗原製備 ........................16 三、 ATPSB1 於水稻各個組織中之基因與蛋白質表現量 ....................... 16 四、 分析水稻中 ATP synthase α-與 β-subunit 之基因成員 ...................... 17 五、 ATPSB1 與 ATPSB2 基因同時靜默之水稻轉殖株性狀分析 ........... 17 六、 ATPSB1 大量表現於百合花花粉中之性狀及細胞內定位分析 ....... 18 七、 OsCPK29 改變 ATPSB1 之細胞內分布位置 ..................................... 18 八、 OsCPK29 使 ATPSB1 與 ATPSB2 均聚集於細胞膜上 ..................... 19 伍、討論 ............................................. 20 第一部份 : OsCPK26 蛋白 N 端之序列功能分析 ..............................................20 OsCPKs 之脂肪酸修飾訊號與蛋白附膜之相關性 ..................................... 20 OsCPKs 附膜與抑制萌發之相關性 ......................... 20 抑制萌發可能因 OsCPK26 與細胞膜上特殊受質或成分結合所致 .......... 20 OsCPK26 V domain 抑制萌發可能因蛋白質本身結構或特殊修飾所致 .. 21 第二部份:ATP synthase 為水稻花粉 OsCPK29 之潛力受質蛋白 ..................22 ATPSB1 異常表現導致水稻花粉的種種性狀 ............................................. 22 位於細胞膜之 ATP synthase 可能與 extracellular ATP 有關....................... 23 OsCPK29 與 ATPSB1 間上下游之關聯 ...................... 23 陸、參考文獻 ..................................................... 25zh_TW
dc.language.isozh_TWzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2018-05-11起公開。zh_TW
dc.subjectzh_TW
dc.subjectnoen_US
dc.titleCharacterization and functional identification of rice pollen-specific OsCPK and its potential downstream substrateen_US
dc.title水稻花粉特有 OsCPK 與其潛力受質之特性分析與功能鑑定zh_TW
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2018-05-11zh_TW
dc.date.openaccess2018-05-11-
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