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標題: | 水稻肌動蛋白去聚合因子OsADF2及OsADF11基因異位表達於阿拉伯芥中之功能性分析 Functional Analysis of Rice Actin Depolymerizing Factor OsADF2 and OsADF11 by Heterologous Expression in Arabidopsis |
作者: | Row-Yu Ma 馬若瑀 |
指導教授: | 張孟基(Men-Chi Chang) |
關鍵字: | 水稻肌動蛋白去聚合因子,細胞骨架,異位表達,植物生長發育,非生物逆境, Actin depolymerizing factor,Cytoskeleton,Heterologous expression,development and growth,abiotic stresses, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 肌動蛋白去聚合因子 (Actin Depolymerizing Factor, ADF) 為一與肌動蛋白結合之蛋白質,其功能為藉由與actin結合將F-actin解聚為G-actin,在細胞中參與了細胞骨架之動態性變化。目前已知ADFs和植物細胞之生長發育、形態、細胞分裂、訊息傳導及生物性逆境相關,然而ADFs與植物非生物逆境耐受性之關連及其功能仍有待探討。因此本論文之實驗以異位表達之方式,利用35S啟動子過量表現水稻OsADF2及OsADF11基因於阿拉伯芥中,並觀察轉殖株與野生型外表型及其在非生物逆境下的反應。結果顯示T3同型結合子之OsADF2和OsADF11轉殖株在一般生長情況下轉殖株葉片大小較野生型阿拉伯芥為大,其抽苔時間與開花時間也較野生型為早。一般環境下發芽時間與野生型無差異。然而在150 mM NaCl的環境下,35S::OsADF2及35S::OsADF11轉殖株的發芽時間較野生型阿拉伯芥為早。另外將5天大及10天大之阿拉伯芥移植至含有150 mM NaCl之培養基後發現OsADF2及OsADF11轉殖株之根部生長及存活率皆大於野生型阿拉伯芥。另外以乾旱、高鹽、低溫逆境處理21天大之OsADF2及OsADF11轉殖株成株,結果顯示OsADF2及OsADF11皆較野生型耐旱,然而OsADF11在低溫處理後植物組織有受傷的情形發生,但此現象只在子葉部分觀察到,而新生葉則與野生型無明顯差異。利用紅外線熱影像檢測植株葉溫之變化,在一般條件下轉殖株與野生型葉溫無差異。另一方面以200 mM NaCl 處理三周大之OsADF2及OsADF11轉殖株及野生型阿拉伯芥,則發現OsADF2轉殖株之葉片溫度與野生型無顯著差異,而OsADF11轉殖株之葉溫則是較野生型為高。利用玉米ubiquitin promoter表現水稻OsADFs基因家族之GFP融合蛋白,接著以暫時性表達分析水稻OsADFs之次細胞定位,結果顯示水稻OsADF2、OsADF3及OsADF11與GFP之融合蛋白於細胞核及細胞質中皆有表現,其中OsADF2與GFP之融合蛋白在葉綠體中也有表現,而OsADF1、OsADF4、OsADF5、OsADF6與GFP之融合蛋白則是皆表現於葉綠體中。由以上之結果推論水稻OsADF2及OsADF11可能與植物面臨高鹽逆境以及乾旱逆境時的耐受機制有關。 Actin Depolymerizing Factors (ADFs) are small actin-binding proteins in cytoskeleton remodeling in cells. Recently, ADFs were reported to play a role in plant growth, development, cell division, signal transduction and pathogen resistance. However the functions of rice ADFs (OsADFs) under abiotic stress still remained to explore. In this thesis, we took heterologous gene expression approach to overexpress rice OsADF2 and OsADF11 in Arabidopsis, and observed the phenotype difference between transgenic and wild type plants under normal growth and abiotic stress. The phenotype of OsADF2 and OsADF11 transgenic plants showed bigger leaves size than Col-0, moreover, the bolting time and flowering time was earlier. The germination rate of transgenic plants had no difference compared to wild-type under normal condition while the germination rate of transgenic plants was faster after treatment with 150 mM NaCl. We also transferred the 5-day old and 10-day old seedlings to the 150 mM NaCl medium and found that the root growth and survival ratio of transgenic plants were both higher than wild-type. Next we grown 21-day old OsADF2, OsADF11 transgenic and wild-type plants under drought, salt and cold stresses, and OsADF2 and OsADF11 transgenic plants tended to be drought tolerance while injury tissue occurred in OsADF11 transgenic plant under cold treatment. Using non-destructive infrared thermography camera recorded the leaf temperature change under salt stress condition, the result showed that no obvious differences were found between transgenic and wild-type plants under normal condition. However OsADF11 transgenic plants showed higher leaf temperature compared to wild-type but not OsADF2 after treatment with 200 mM NaCl. In addition, to understand the subcellular localization of rice OsADFs, we used maize ubiquitin promoter to express rice OsADFs-GFP fusion protein through rice protoplast mediated transformation. The result showed that OsADF3-GFP and OsADF11-GFP were located in the nucleus and cytosol, and OsADF2-GFP was located in thel nucleus, the cytosol and also in chloroplasts. OsADF1, OsADF4, OsADF5 and OsADF6 were located in the chloroplasts. Taken together from above results, our study implied that rice OsADF2 and OsADF11 maybe involve in the plant response to drought and salt stress. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5087 |
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顯示於系所單位: | 農藝學系 |
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