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研究生:何美萱
研究生(外文):Mei-Hsuan Ho
論文名稱:在水稻中過表達植物硫鐵蛋白基因會增加其對高鹽的耐受性
論文名稱(外文):Overexpression of a Plant Ferredoxin-like Protein in Transgenic Rice Improves Tolerance against High Salinity
指導教授:葛孟杰
指導教授(外文):Mang-Jye Ger
學位類別:碩士
校院名稱:國立高雄大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:65
中文關鍵詞:植物硫鐵蛋白鹽害逆境過氧化物
外文關鍵詞:Plant Ferredoxin-like Proteinsalinity stressReactive oxygen species
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植物處在高鹽的環境當中會造成其體內的滲透壓失衡及離子毒性,嚴重地影響到植物的生長發育及農作物產量。為了抵抗逆境環境的侵害,植物會有一套自我保護的機制,例如過氧化物( ROS )的產生、透過訊息傳遞路徑誘導下游逆境相關基因的表現、植物賀爾蒙的誘導、滲透保護物質(osmoprotectants)的累積及antiporters的調控等。其中ROS不只是代謝機制中的副產物,ROS也參與了許多訊息傳遞路徑。PFLP (Plant Ferredoxin-like Protein)是由甜椒中分離出來的蛋白,與光合作用當中的Ferredoxin-1有高度相似性。先前研究指出過表達PFLP可以透過調控ROS的產生使植物能夠對抗病原菌侵害與促進植物根毛之發育。但PFLP在非生物性逆境中扮演的角色尚未明瞭。因此本篇論文在於研究過表達PFLP在水稻中是否會影響其對高鹽逆境的抗性,並且進一步探討其調控機制。實驗結果顯示PFLP轉殖水稻相較於野生型水稻具有較高之抗鹽害能力。鹽逆境中可以較早誘導轉殖水稻中ROS的產生,並透過訊息傳遞的交互作用提高ABA生成及其相關合成基因的表現。轉殖水稻也擁有較高的抗氧化酵素活性( POD、SOD )來降低過多的ROS帶來的侵害。此外,轉殖水稻在鹽逆境下也呈現較高的SOS相關基因( OsSOS1, OsCIPK24, OsNHX2 )的表現,使得葉部的鈉離子含量相對少於野生型植株。綜合上述結果,本篇研究指出PFLP轉殖水稻可以透過較早產生ROS、提高ABA含量及逆境相關基因的表現來增進其在鹽害逆境中的抗性。
High salinity stress is the main factor of reducing crops productivity. Plants possess a number of strategies against this damage including ROS generation, induction of stress-related genes expression, accumulation of ABA and proline and up-regulation of antiporters. Reactive oxygen species (ROS) are not only the by-product of metabolism but also involving signal transduction pathway. Previous studies have been reported that continuously expression of PFLP in transgenic plants encourages the generation of ROS and participates in pathogen-resistant mechanism and root-hair development. However, the function of PFLP in plants response to abiotic stress is unclear. In order to investigate the role of PFLP under high salinity stress, pflp-transgenic rice have been generated and studied. In this report, we demonstrated that pflp-transgenic plants exhibit higher salt tolerance compared with TNG67 rice. Earlier ROS production, higher activities of antioxidant enzyme, more ABA accumulation and up-regulated expression in stress-related genes including OsRBOHa, Cu/Zn SOD, OsAPX, OsNCED, OsSOS, OsCIPK24 and OsNHX2 were found in pflp-transgenic plants under high salinity condition. Additionally, the Na+ content of leaf tissues in pflp-transgenic rice were lower than that of TNG67. Here, we demonstrate overexpression of pflp in transgenic rice improves tolerance against high salinity.
Table of contents……………………………………………………………………………Ⅰ
List of figures………………………………………………………………………………Ⅲ
摘要………………………………………………………………………………………………1
Abstract…………………………………………………………………………………………2
Chapter I Introduction………………………………………………………………………4
Chapter II Material and Methods…………………………………………………………10
2.1 Plant material and stress conditions ……………………………………………10
2.2 RNA extraction and Reverse transcription PCR …………………………………10
2.3 Quantitative reverse transcription PCR …………………………………………11
2.4 H2O2 content ……………………………………………………………………………12
2.5 Peroxidase activity assay……………………………………………………………12
2.6 Superoxide dismutase activity assay………………………………………………13
2.7 DAB stain…………………………………………………………………………………13
2.8 Proline content…………………………………………………………………………14
2.9 ABA content………………………………………………………………………………14
2.10 Na+ analysis……………………………………………………………………………15
Chapter III Results…………………………………………………………………………16
3.1 Isolation and characterization of pflp transgenic lines……………………16
3.2 Overexpression of pflp enhances tolerance to high salinity stress
in rice…………………………………………………………………………………………16
3.3 H2O2 content of TNG67 and transgenic plants after high salinity
treatment………………………………………………………………………………………17
3.4 Antioxidant enzymes activities of TNG67 and transgenic plants after high salinity treatment …………………………………………………………………………18
3.5 The expression patterns of redox-regulated genes ……………………………19
3.6 ABA concentration and related synthesis gene expression after high salinity stress………………………………………………………………………………20
3.7 Proline contents and proline synthesis-related genes expression of TNG67 and transgenic plants………………………………………………………………………21
3.8 Na+/H+ antiporter related-gene expression under high salinity stress …22
3.9 Na+ contents in leaf and root tissues of TNG67 and transgenic lines after salt stress treatment………………………………………………………………………22
Discussion ……………………………………………………………………………………24
Reference………………………………………………………………………………………47
Supplemental table …………………………………………………………………………S1
List of Abbreviation ………………………………………………………………………S1
PCR primers……………………………………………………………………………………S2
The kimura B solution………………………………………………………………………S4
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