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研究生:鄭執甫
研究生(外文):Chin-Fu Cheng
論文名稱:於水稻中持續表現植物硫鐵蛋白可增強水稻光合作用碳固定效率
論文名稱(外文):Constitutive Expression of a Plant Ferredoxin-like Protein (pflp) in Transgenic Rice Enhances Capacity of Photosynthetic Carbon Assimilation
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:2010
畢業學年度:98
語文別:英文
論文頁數:39
中文關鍵詞:植物硫鐵蛋白光合作用產量
外文關鍵詞:crop yieldphotosynthesisplant ferredoxin – like protein (PFLP)
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光合作用主要是植物吸收光能後將二氧化碳固定成各種醣類,例如: 作為能量利用的葡萄糖與果糖,作為運輸用的蔗糖與儲存用的澱粉。第一型硫鐵蛋白廣泛分布在植物綠色組織中,其主要在電子傳遞鏈中扮演傳遞電子的角色。從甜椒萃取之植物硫鐵蛋白(Plant Ferredoxin-Like Protein,PFLP)經蛋白質序列比對後發現,其序列與阿拉伯芥、蕃茄、水稻及菠菜之第一型硫鐵蛋白有高度同源性。先前研究發現pflp轉殖植物在病原菌感染後可大幅產生過氧化物(Reactive oxygen species, ROS),進而誘導過敏反應產生。由於PFLP屬於光合作用第一型硫鐵蛋白,此暗示過表現植物硫鐵蛋白於轉殖植物中可能經由加強光合作用與糖類代謝來增加抵抗病原菌的能力。為了探討上述機制的可能性,花椰菜鑲嵌病毒35S啟動子(CaMV 35S promoter)被用來大量表現PFLP蛋白於水稻中,並且分析轉殖水稻的光合作用效率。經由反轉錄聚合酶鏈式反應及西方墨點法確認pflp轉基因以成功表現並累積PFLP蛋白於轉殖水稻中。實驗結果顯示轉殖水稻具有較高的電子傳遞能力與氣體交換速率。在醣類含量方面,轉殖水稻均具有高含量的果糖,葡萄糖,蔗糖與澱粉。而轉殖水稻中醣類合成相關基因轉錄活性大多高於野生型水稻2至4倍。上述結果證實轉殖水稻含有較高之醣類累積是導因於其具有較高電子傳遞效率、較高氣體交換速率與較強醣類合成相關基因表現。在產量分析方面,轉殖水稻無論是在單株實粒重、單株分穗數或種子內澱粉含量均高於野生種水稻。此外,以病原菌接種植株後,偵測氣體交換速率變化,發現轉殖株在此期間可維持氣體交換速率;而野生型之氣體交換速率則明顯的下降。綜合上述結果,本篇論文指出於水稻中持續表現pflp可增強光合作用碳固定效率,並進而提升水稻產量。
Photosynthesis is the major metabolic pathway that converts carbon dioxide (CO2) into organic compounds, such as fructose, glucose, sucrose, and starch in plant. Ferredoxin-I (Fd-I) protein, known as an electron carrier in photosynthesis, generally exists in green tissue. The protein sequence of the plant ferredoxin – like protein (PFLP), isolated from sweet peppers, shows high homology to the Fd-I sequence of Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Spinacia oleracea. Previous studies reported that constitutive expression of pflp in transgenic plants exhibit host resistance to virulent bacterial pathogens. The disease resistance of transgenic plants result from the induction of reactive oxygen species (ROS) generation. It is possible that PFLP could enhance photosynthetic efficiency. In order to assess this possibility, pflp transgenic rice was generated and studied their capacity of photosynthetic carbon assimilation. In this report, two transgenic lines of rice (pflp-1 and pflp-2) showed that the pflp transgenic rice exhibit highly photosynthesis efficiency. As comparing the photosynthetic gas exchange rate, the pflp-1 and the pflp-2, respectively, exhibited 1.3- and 1.2-fold higher than the wild type (WT). The capacity of photosynthetic electron transport of two transgenic lines are significantly higher than WT. Additionally, the fructose, glucose, sucrose and starch contents in the pflp-1 and pflp-2 transgenic lines were higher than those of the WT. The transcript activity of carbohydrate metabolism related genes in transgenic lines were higher than those that of WT. In crop yield, on grain weight per plant the pflp-1 and the pflp-2 showed 1.7- and 1.3-fold higher than WT. As for the number of panicles per plant, the pflp-1 and the pflp-2 are increased 1.4- and 1.5-fold higher than the WT. In addition, we demonstrated that the transgenic rice maintain normal photosynthetic gas exchange rate on the period of pathogen infected. Taken altogether; we demonstrate that constitutive expression of pflp in transgenic rice could enhance capacity of photosynthetic carbon assimilation and increase crop yield.
Table of contents
List of figures
Chinese abstract
English abstract
Chapter I Introduction
Chapter II Materials and methods
2.1 Plant materials and growth condition
2.2 Genomic DNA extraction and Genomic DNA PCR2.3 RNA extraction, Reverse transcription PCR and Quantitative reverse transcription
PCR
2.4 Determination the content of chlorophyll, protein and western blot analysis
2.5 Gas exchange rate and chlorophyll fluorescence measurement
2.6 Soluble sugars and starch contents assay
2.7 Crop yield assay
2.8 Bacterial blight inoculation
2.9 Statistical
Chapter III Results
3.1 Constitutive expression of pflp in transgenic rice
3.2 PFLP can increase the capacity of photosynthetic electron transport
3.3 PFLP can enhance rate of photosynthetic gas exchange rate in pflp transgenic lines

3.4 Highly contents of soluble sugar and starch accumulate in transgenic rice results from relative high expression pattern of carbohydrate metabolism related gene3.5 Analysis of crop yield in pflp transgenic lines
3.6 pflp transgenic lines maintain photosynthetic gas exchange rate after a bacterial pathogen infection
Chapter IV Discussion
Chapter V Reference
Chapter VI Supplemental table
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