為滿足人類對微量元素硒的需求，在本研究中，我們進一步完善優化利用少硒化肥強化水稻的生物農業性狀，並且全面地檢測了富硒稻米中硒的生物有效性和生物利用度。首先，我們發現低濃度的亞硒酸鈉（2毫克/升）在提高水稻幼苗生長方面有顯著的成效。通過抽穗後葉面噴施亞硒酸鈉生產富硒稻米及調控低量的亞硒酸鈉（10.5克硒/公頃）能顯著增加水稻籽粒中硒含量高達51倍；同時，水稻產量也上調了1.24倍。此外，通過硒形態分析、體外胃腸消化和抗氧化實驗來評估，在富硒稻米中，硒的主要富集形態是硒代蛋氨酸；同時，富硒稻米具有明顯較高的抗氧化生物活性。這種富硒大米在人類補硒方面具有巨大潛力。 / 硒對植物生長的作用有兩方面，既有有利作用又有毒副作用。水稻種植應用低濃度的亞硒酸鈉能促進生長，而較高濃度的亞硒酸鈉則抑制生長。為詳細解釋這種兩面性影響機制，我們應用二維凝膠電泳（2-DE）結合基質輔助鐳射解吸離子化-飛行時間質譜（MALDI-TOF/TOF MS）進行蛋白質組學研究。將硒處理組與對照組水稻幼苗之間的凝膠圖像進行比較，確定了莖葉和根中分別有66和97個差異表達的蛋白質。基因聚類分析顯示，水稻的中心代謝，光合作用和氧化還原平衡高度受硒處理影響。低硒處理（2和6毫克/升亞硒酸鈉）啟動抗氧化系統，增強光合作用和初級代謝。而較高的硒處理（10毫克/升的亞硒酸鈉）則抑制光合作用和初級代謝。此項研究在未來生產富硒水稻方面具有指導性意義。 / 為了更好地瞭解水稻穀粒中硒富集的生物機制，我們應用2-DE結合MALDI-TOF/TOF MS及1-DE結合傅裏葉變換離子迴旋共振質譜（FTMS）進行了水稻穀粒的蛋白質組學研究。這項研究提供了最全面的稻米穀粒蛋白質表達圖譜。通過硒處理和對照之間的比較，62和250個差異表達的蛋白質分別被雙電離飛行時間質譜和傅裏葉變換質譜所鑒定。通過基因功能分類，在成熟的稻穀中，硫代謝，碳代謝，細胞的氧化還原調控，和種子的營養儲存過程中涉及的蛋白質受到硒富集的高度影響。此外，有6個蛋白被檢測具有含硒氨基酸片斷，這是高等植物中含硒蛋白的首次鑒定。 / 富硒水稻的基因工程能提高人類的補硒預期。因此，為獲得可以應用於基因工程改造的合適的水稻基因，我們通過在經典模型植物擬南芥中過表達水稻O-乙醯絲氨酸硫解酶（OASTL）的基因，包括在胞漿中表達的OASTLA基因、在質體中表的OASTLB基因和線粒體中表達的OASTLC基因，用以研究這些基因在轉基因植株中對硒富集的影響。在不同硒濃度處理下，與野生型植物相比，此三個基因均表達顯著提高轉基因植物中的硒含量。即時定量反轉錄PCR分析結果顯示，由於過表達的水稻OASTL基因，硒同化的整個代謝途徑被啟動，尤其是與半胱氨酸和蛋氨酸合成有關的基因被啟動，這可能就是引起更多的硒富集在轉基因植物裏的原因。此外，過表達水稻OASTL基因也啟動穀胱甘肽還原酶，這可能增強富硒轉基因植物的抗氧化系統從而提高抗逆性並增加產量。因此，OASTL基因在基因工程生產富硒稻米方面具有重要潛在價值。 / To fulfill the natural human needs of selenium (Se), I further improved the agronomic biofortification of rice (Oryza sativa) with less Se fertilizers and comprehensively evaluated Se bioaccessibility and bioavailability in the Se-enriched rice. Se-enriched rice grains were prepared by foliar application of selenite after rice heading. As compared with control, low amount of sodium selenite (10.5 g Se/ha) significantly increased Se content in rice grains by up to 51 times; at the same time, rice yield was also up-regulated by up to 1.24 times. Furthermore, by Se speciation analysis, in vitro gastrointestinal digestion and antioxidant assays, the Se-enriched rice grains contain readily absorbable selenomethionine as the major Se species and have significantly higher antioxidant bioactivities. This Se-enriched rice has enormous potential for Se supplementation in humans. / Se shows both beneficial and toxic effects on plant growth. Treatments with lower concentrations of sodium selenite enhanced the growth of rice seedlings, whereas higher concentrations of sodium selenite repressed seedling growth. To reveal the regulatory mechanisms underlying these effects, a comparative proteomics study combining 2-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization (MALDI)-tandem time of flight (TOF/TOF) mass spectrometry (MS) were performed. By comparison of gel images between Se treatments and control, 66 and 97 differentially expressed proteins were identified in shoot and root, respectively. Gene Ontology and Clustering analysis reveal primary metabolism, photosynthesis and redox homeostasis are the most highly affected biological processes by Se treatments. Lower Se treatments (2 and 6 mg/L sodium selenite) activated antioxidative system, enhanced photosynthesis and primary metabolism. However, higher Se treatment (10 mg/L sodium selenite) damaged photosynthesis apparatus, inhibited photosynthesis and primary metabolism. This study provided novel insights into Se response in rice at the proteome level, which are expected to be highly useful for dissecting the Se response pathways in higher plants and for producing of Se enriched rice cultivars in the future. / To better understand the regulatory mechanism under Se accumulation in rice grains, a comparative proteomics study using 2-DE coupled MALDI-TOF/TOF MS and 1-dimensional gel electrophoresis (1-DE) coupled liquid chromatography (LC) - Fourier transform-ion cyclotron resonance (FT-ICR) MS were carried out. By comparison of Se treatments and control, 62 and 250 differentially expressed proteins were identified by 2-DE/MALDI-TOF/TOF MS and 1-DE/LC-FT-ICR MS, respectively. By gene functional classification, proteins involved in the processes of sulfur metabolism, carbon metabolism, cell redox regulation, and seed nutritional storage were the most highly affected by Se accumulation in mature rice grains. In addition, there were 6 proteins identified to contain fragments of selenoamino acid modification, which was the first identification of selenoproteins in higher plants. / Genetic engineering of Se-enriched rice will have important implications for human health in Se deficient regions. Therefore, to acquire appropriate rice genes as candidates for bioengineering of Se-enriched rice cultivars, I overexpressed three of the rice O-Acetylserine(thiol)lyase (OASTL) genes encoding cytosolic OASTLA, plastic OASTLB and mitochondrial OASTLC, individually in the model plant Arabidopsis (Arabidopsis thaliana) to characterize the effects of Se accumulation in transgenic plants. The results showed that compared to the wild type plants, overexpression of all these genes significantly increased Se content in transgenic plants under treatments of different selenite concentrations. By real-time RT-PCR analysis, I found that the whole metabolic pathway of selenite assimilation was activated by overexpressing rice OASTL genes, especially the genes involved in cysteine and methionine biosynthesis, which may give rise to more Se accumulation in the transgenics. In addition, overexpression of rice OASTL genes also activated the antioxidative system by activating the glutathione reductase, which may be responsible for the increased biomass of Se-enriched transgenic plants. Therefore, OASTL genes could be good candidate for the future genetic engineering of Se-enriched rice. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Yudong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 149-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / 摘要 (Abstract in Chinese) --- p.v / List of Abbreviations --- p.vii / List of Figures --- p.x / List of Tables --- p.xi / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- Implications of Se for human health and its metabolism in plants --- p.1 / Chapter 1.2. --- Systemic study of Se metabolism and regulation in rice --- p.3 / Chapter 1.3. --- Candidate genes for genetic engineering of Se-enriched rice --- p.4 / Chapter 1.4. --- Objectives of this project --- p.7 / Chapter Chapter 2: --- Generation of selenium-enriched rice with enhanced grain yield, selenium content and bioavailability through fertilization with selenite --- p.9 / Chapter 2.1. --- Introduction --- p.9 / Chapter 2.2. --- Materials and methods --- p.12 / Chapter 2.2.1. --- Reagents --- p.12 / Chapter 2.2.2. --- Plant materials and growth conditions --- p.12 / Chapter 2.2.3. --- Se speciation analysis --- p.14 / Chapter 2.2.4. --- Antioxidant assays --- p.16 / Chapter 2.2.5. --- Data analysis --- p.18 / Chapter 2.3. --- Results and discussion --- p.18 / Chapter 2.3.1. --- Effects of fertilization of selenite on the growth and Se content of rice seedlings --- p.18 / Chapter 2.3.2. --- Effects of fertilization of selenite on the antioxidant activity of rice seedlings --- p.19 / Chapter 2.3.3. --- Effect of fertilization of selenite on Se content in rice products --- p.20 / Chapter 2.3.4. --- Effect of fertilization of selenite on rice yield --- p.21 / Chapter 2.3.5. --- Analysis of Se bioaccessibility in Se-enriched rice grains --- p.22 / Chapter 2.3.6. --- Analysis of Se bioavailability in Se-enriched rice grains --- p.23 / Chapter 2.4. --- Conclusion --- p.24 / Chapter Chapter 3: --- Proteomics analysis reveals multiple regulatory mechanisms in response to selenium in rice --- p.37 / Chapter 3.1. --- Introduction --- p.37 / Chapter 3.2. --- Materials and methods --- p.39 / Chapter 3.2.1. --- Plant materials and growth conditions --- p.39 / Chapter 3.2.2. --- Physiological measurements --- p.40 / Chapter 3.2.3. --- Total protein extraction --- p.40 / Chapter 3.2.4. --- 2-DE separation, gel staining and image analysis --- p.40 / Chapter 3.2.5. --- Trypsin digestion, mass spectrometry and protein identification --- p.41 / Chapter 3.2.6. --- Protein functional classification and hierarchical cluster analysis --- p.43 / Chapter 3.2.7. --- Statistical analysis --- p.43 / Chapter 3.3. --- Results and discussion --- p.43 / Chapter 3.3.1. --- Effects of Se on rice seedlings --- p.43 / Chapter 3.3.2. --- Effects of Se on shoot and root proteomes of rice seedlings --- p.44 / Chapter 3.3.3. --- Gene ontology analysis of Se-responsive proteins --- p.46 / Chapter 3.3.4. --- Clustering analysis revealed the dynamics of functional protein groups under Se treatment --- p.47 / Chapter 3.3.5. --- Se treatment induced redox and stress related proteins --- p.48 / Chapter 3.3.6. --- Se-responsive proteins preferentially associated with primary metabolism and photosynthesis --- p.50 / Chapter 3.3.7. --- Post translational modifications involved in plant Se-response --- p.52 / Chapter 3.4. --- Conclusion --- p.53 / Chapter Chapter 4: --- Comparative proteomics analysis of selenium responses in selenium-enriched rice grains --- p.79 / Chapter 4.1. --- Introduction --- p.79 / Chapter 4.2. --- Materials and methods --- p.82 / Chapter 4.2.1. --- Plant materials and growth conditions --- p.82 / Chapter 4.2.2. --- Total protein extraction --- p.83 / Chapter 4.2.3. --- 2-DE separation, gel staining and image analysis --- p.83 / Chapter 4.2.4. --- Trypsin digestion, mass spectrometry and protein identification --- p.84 / Chapter 4.2.5. --- Preparative SDS-PAGE separation and trypsin digestion --- p.85 / Chapter 4.2.6. --- NanoLC-FT-ICR MS and protein identification --- p.86 / Chapter 4.2.7. --- Label-free quantitation of identified proteins --- p.87 / Chapter 4.2.8. --- Functional classification of Se-responsive proteins --- p.87 / Chapter 4.3. --- Results and discussion --- p.88 / Chapter 4.3.1. --- Effects of foliar application of selenite in rice grain production --- p.88 / Chapter 4.3.2. --- 2-DE/MALDI-TOF/TOF MS analysis of Se-enriched rice grains --- p.89 / Chapter 4.3.3. --- Label-free 1-DE/LC-FT-ICR-MS analysis of Se-enriched rice grains --- p.89 / Chapter 4.3.4. --- Gene ontology analysis of rice grain proteome and Se-responsive proteins --- p.90 / Chapter 4.3.5. --- Sulfur metabolism were highly repressed in Se-enriched rice --- p.91 / Chapter 4.3.6. --- Proteins involved in redox regulation were induced in Se-enriched rice --- p.92 / Chapter 4.3.7. --- Se-responsive proteins are preferentially associated with carbon metabolism --- p.93 / Chapter 4.3.8. --- Proteins involved in seed nutritional storage --- p.95 / Chapter 4.4. --- Conclusion --- p.97 / Chapter Chapter 5: --- Overexpressing rice O-Acetylserine(thiol)lyase Genes Enhances Selenium Accumulation in Arabidopsis --- p.123 / Chapter 5.1. --- Introduction --- p.123 / Chapter 5.2. --- Materials and methods --- p.126 / Chapter 5.2.1. --- DNA constructs --- p.126 / Chapter 5.2.2. --- Transient gene expression and subcellular localization --- p.127 / Chapter 5.2.3. --- Arabidopsis plant transformation and growth --- p.127 / Chapter 5.2.4. --- Selenium treatment and physiological measurements --- p.127 / Chapter 5.2.5. --- Total Se content assay --- p.127 / Chapter 5.2.6. --- RT-PCR analysis --- p.128 / Chapter 5.3. --- Results and discussion --- p.128 / Chapter 5.3.1. --- Phenotypes of OASTL-transgenic Arabidopsis --- p.128 / Chapter 5.3.2. --- Se accumulated in OASTL-transgenic Arabidopsis under Se treatment --- p.129 / Chapter 5.3.3. --- Overexpression of rice OASTL genes activated Se assimilation pathways --- p.130 / Chapter 5.3.4. --- ATSAT genes were highly expressed in OASTL-transgenics --- p.132 / Chapter 5.3.5. --- Overexpression of rice OASTL genes activated the antioxidative system --- p.133 / Chapter 5.3.6. --- Methionine synthesis was enhanced in OASTL-transgenics --- p.134 / Chapter 5.4. --- Conclusion --- p.134 / Chapter Chapter 6: --- Conclusion --- p.146 / References --- p.149 / Chapter Appendix I: --- Publications --- p.160
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328142 |
| Date | January 2012 |
| Contributors | Wang, Yudong, Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xi, 160 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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