大豆的豐富營養和經濟價值使它成為重要的農產品。但是，土壤鹽漬化影響著大豆的產量。這個問題在沿岸地方特別嚴重。若要改善大豆的耐鹽能力，必先增加對大豆耐鹽機理的了解。 / 本實驗室從大豆中發現了一個受鹽脅迫誘導表達的基因GmSAL1。以往透過體外酶反應分析法， GmSAL1蛋白被介定為一個能作用於1,4,5-三磷酸肌醇 (IP₃) 的肌醇磷酸-5-磷酸酶。這有別於在擬南芥中的SAL1同源蛋白AtSAL1, AtSAL1是一個肌醇磷酸-1-磷酸酶。由於IP₃是信號傳導途徑中的重要分子，本課題對與IP₃信號及耐鹽性相關的GmSAL1蛋白功能進行研究。 / 本課題旨在：（一） 研究GmSAL1對於細胞質內IP₃的累積的體內作用； （二） 研究 GmSAL1 在脫落酸 (ABA) 的信號傳導中的可能角色； （三） 研究 GmSAL1 在鹽脅迫下的保護作用。 / 本研究利用體內報告系統證明了 GmSAL1 對於減少細胞質內IP₃的累積的作用。這種影響IP₃水平的功能減弱了由 ABA 信號所引起的氣孔關閉和種子萌發抑制。利用 GmSAL1 轉基因煙草細胞 (BY-2) 和擬南芥，證明 GmSAL1 在鹽脅迫下起著短暫的保護作用。GmSAL1在鹽脅迫下的保護功能可能是由於蒸騰作用的局部恢復和細胞鈉離子區室化的作用。 / 本研究展示了肌醇信號，ABA信號和鹽脅迫反應三者之間的關係。這是在以前的研究中未被清楚闡釋的。 / Soybean is nutritionally and economically important. However, high soil salinity, particularly in coastal regions, impedes the production of soybean. Understanding the salt tolerance mechanism is the first step towards the enhancement of salt tolerance of soybean. / Our laboratory identified a salt-responsive gene from soybean namely GmSAL1. Previous in vitro enzyme assay suggested that the GmSAL1 protein is an inositol 5’-phopsphatase acting on inositol 1,4,5-trisphosphate (IP₃), which is different from the enzymatic activity reported for the SAL1 homologue (AtSAL1) in Arabidopsis thaliana (A. thaliana) which is an inositol 1-phopsphatase. Since IP₃ is an important molecule involved in signal transduction pathways, this project is to explore the in vivo functions of GmSAL1 in relation to IP3 signaling and salinity tolerance. / The specific objectives of this research are (1) to study the in vivo role of GmSAL1 on cytosolic IP₃ accumulation; (2) to study the possible involvement of GmSAL1 in ABA signaling; (3) to study the protective roles of GmSAL1 under salt stress. / In this project, the function of GmSAL1 to reduce cytosolic IP₃ was demonstrated using an in vivo reporter system. This activity on IP₃ levels reduced the sensitivity of stomatal closure and seed germination inhibition mediated by ABA signals. A transient protection effect against the ionic effect under salt stress by GmSAL1 was shown by gain-of-function tests in transgenic BY-2 cells and transgenic A. thaliana. The protective effect conferred by GmSAL1 may be due to a partial resuming of transpiration through reduction of ABA signals and compartmentalization of Na⁺ into vacuoles. / The study of GmSAL1 in this research demonstrated the link among inositol signaling, ABA signaling, and salinity response which was not well addressed in previous reports. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ku, Yee Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 97-104). / Abstracts also in Chinese. / Statement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.v / General Abbreviations --- p.vii / Abbreviations of Chemicals --- p.ix / Table of Contents --- p.xi / List of Figures --- p.xviii / List of Tables --- p.xxi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction to salinity and agriculture --- p.1 / Chapter 1.1.1 --- Adverse effects of salinity on plants --- p.2 / Chapter 1.1.1.1 --- Osmotic stress --- p.2 / Chapter 1.1.1.2 --- Ionic stress --- p.3 / Chapter 1.1.1.3 --- Separation of ionic effect from osmotic effect --- p.3 / Chapter 1.1.1.4 --- Oxidative stress --- p.4 / Chapter 1.1.2 --- Major physiological responses of plants to achieve salt tolerance --- p.5 / Chapter 1.1.2.1 --- Maintenance of cellular ion homeostasis --- p.5 / Chapter 1.1.2.2 --- Balance between Na⁺ and K⁺ influx --- p.5 / Chapter 1.1.2.3 --- Efflux of Na⁺ from cell --- p.9 / Chapter 1.1.2.4 --- Enhanced compartmentalization of Na⁺ and Cl⁻ in vacuole --- p.11 / Chapter 1.1.2.5 --- Enhanced vacuolar ion compartmentalization --- p.13 / Chapter 1.1.2.6 --- Biosynthesis of osmolytes --- p.13 / Chapter 1.2 --- Signal transduction under salt stress --- p.14 / Chapter 1.2.1 --- General introduction to signal transduction under salt stress --- p.14 / Chapter 1.2.2 --- ABA signaling under salinity --- p.15 / Chapter 1.2.2.1 --- General introduction to ABA signaling --- p.15 / Chapter 1.2.2.2 --- IP₃ and ABA signaling --- p.16 / Chapter 1.2.2.3 --- Introduction to inositol phosphate --- p.16 / Chapter 1.2.2.4 --- Phosphatidylinositol-3-monophosphate --- p.18 / Chapter 1.2.2.5 --- Phosphatidylinositol-4-monophosphate --- p.18 / Chapter 1.2.2.6 --- Phosphatidylinositol-5-monophosphate --- p.18 / Chapter 1.2.2.7 --- Phosphatidylinositol (3,5) bisphosphate --- p.19 / Chapter 1.2.2.8 --- Phosphatidylinositol (4,5) bisphosphate --- p.19 / Chapter 1.2.2.9 --- Inositol (1,4,5) trisphosphate (IP₃) --- p.19 / Chapter 1.2.2.10 --- Inositol metabolism under salt stress --- p.19 / Chapter 1.2.2.11 --- The involvement of IP₃ in ABA signaling --- p.20 / Chapter 1.2.3 --- General introduction to Ca²⁺ signaling --- p.22 / Chapter 1.2.4 --- Ca²⁺ channels --- p.23 / Chapter 1.2.4.1 --- Ligand-gated Ca²⁺ channels --- p.23 / Chapter 1.2.4.1.1 --- IP₃ gated Ca²⁺ channels --- p.23 / Chapter 1.2.4.1.2 --- Cyclic nucleotide gated channels (CNGCs) --- p.24 / Chapter 1.2.4.1.3 --- Glutamate receptor homologs (GLRs) --- p.24 / Chapter 1.2.4.2 --- Voltage-gated Ca2⁺ channels --- p.25 / Chapter 1.2.4.2.1 --- Two-pore channels (TPCs) --- p.25 / Chapter 1.2.4.2.2 --- Mechanosensitive Ca2²⁺permeable channels (MSCCs) --- p.25 / Chapter 1.2.4.2.3 --- Ca²⁺ and ABA signaling --- p.26 / Chapter 1.2.5 --- ABA, IP₃ and Ca²⁺ signaling --- p.26 / Chapter 1.2.5.1 --- Ca²⁺ signaling under salt stress --- p.30 / Chapter 1.2.5.2 --- Ca²⁺ signal mediated cellular responses --- p.30 / Chapter 1.3 --- Introduction to inositol phosphatases --- p.30 / Chapter 1.3.1 --- Previous studies on inositol phosphatases in plant --- p.33 / Chapter 1.3.1.1 --- Inositol polyphosphate 1-phosphatase --- p.33 / Chapter 1.3.1.2 --- Inositol polyphosphate 5-phosphatase --- p.34 / Chapter 1.4 --- Previous research on GmSAL1 in Prof. Hon-Ming Lam’s lab --- p.37 / Chapter 1.5 --- Objective and Significance of this project --- p.38 / Chapter Chapter 2 --- Materials and methods --- p.39 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Plants, bacterial strains and vectors --- p.39 / Chapter 2.1.2 --- Chemicals and enzymes --- p.40 / Chapter 2.1.3 --- Buffer, medium and solution --- p.41 / Chapter 2.1.4 --- Primers --- p.41 / Chapter 2.1.5 --- Equipments and facilities --- p.44 / Chapter 2.1.6 --- Software --- p.44 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Measurement of osmolarity --- p.44 / Chapter 2.2.2 --- Plant growth and treatment conditions --- p.45 / Chapter 2.2.2.1 --- NaCl, PEG and ABA treatment on soybean plant --- p.45 / Chapter 2.2.3 --- Artificial crossing of A. thaliana --- p.46 / Chapter 2.2.3.1 --- Screening of double homozygous transgenic A. thaliana lines --- p.46 / Chapter 2.2.4 --- Transformation of tobacco BY-2 cells --- p.47 / Chapter 2.3 --- Molecular techniques --- p.48 / Chapter 2.3.1 --- DNA extraction --- p.48 / Chapter 2.3.2 --- PCR --- p.48 / Chapter 2.3.2.1 --- Screening of transgenes --- p.48 / Chapter 2.3.2.2 --- Synthesis of DIG-labeled DNA probe for northern blot analysis --- p.49 / Chapter 2.3.3 --- DNA gel electrophoresis --- p.49 / Chapter 2.3.4 --- RNA extraction --- p.50 / Chapter 2.3.5 --- Northern blot analysis --- p.51 / Chapter 2.3.5.1 --- RNA treatment --- p.51 / Chapter 2.3.5.2 --- Electrophoresis --- p.51 / Chapter 2.3.5.3 --- RNA blotting --- p.51 / Chapter 2.3.5.4 --- GmSAL1 mRNA detection --- p.52 / Chapter 2.4 --- Cell viability assay --- p.52 / Chapter 2.5 --- Na⁺ compartmentalization assay --- p.53 / Chapter 2.6 --- ABA sensitivity assays --- p.53 / Chapter 2.6.1 --- Seed germination assay --- p.53 / Chapter 2.6.2 --- Stomatal opening assay --- p.54 / Chapter Chapter 3 --- Results --- p.55 / Chapter 3.1 --- Differential response of GmSAL1 expression level to NaCl and PEG treatment --- p.55 / Chapter 3.2 --- The expression of GmSAL1 in host plant is responsive to ABA --- p.59 / Chapter 3.3 --- Effect of GmSAL1 on cytosolic IP₃ level in vivo --- p.62 / Chapter 3.4 --- Overexpression of GmSAL1 down-regulates in planta IP₃ level in guard cell --- p.62 / Chapter 3.5 --- Ectopic expression of GmSAL1 in A. thaliana alters stomatal aperture in the presence of ABA in a Ca²⁺ dependent manner --- p.67 / Chapter 3.6 --- Ectopic expression of GmSAL1 in A. thaliana reduces the ABA inhibitory effect on seed germination --- p.71 / Chapter 3.7 --- Overexpression of GmSAL1 transiently protects A. thaliana against ionic effect under salinity --- p.76 / Chapter 3.8 --- Overexpression of GmSAL1 enhances the survival of tobacco BY-2 cells under salt treatment but not near iso-osmotic PEG treatment --- p.80 / Chapter 3.9 --- Overexpression of GmSAL1 confers enhanced vacuolar compartmentalization of Na⁺ in NaCl treated BY-2 cells --- p.85 / Chapter Chapter 4 --- Discussion --- p.90 / Chapter 4.1 --- GmSAL1 as a novel inositol 5-phosphatase --- p.90 / Chapter 4.2 --- The effect of GmSAL1 expression on ABA signaling --- p.91 / Chapter 4.3 --- Involvement of GmSAL1 in tolerance toward ionic effect under salt stress --- p.92 / Chapter 4.4 --- The protective function of GmSAL1 under salinity --- p.93 / Chapter Chapter 5 --- Conclusion --- p.96 / References --- p.97 / Chapter Appendix I --- Chemicals --- p.105 / Chapter Appendix II --- Formulations of buffer, medium and solution --- p.107 / Chapter Appendix III --- Equipments and facilities --- p.110 / Chapter Appendix IV --- Osmolarity of solutions --- p.111 / Chapter Appendix V --- Result of biological repeat of northern blot analysis of GmSAL1 in soybean leaf under NaCl --- p.113 / Chapter Appendix VI --- Result of biological repeat of northern blot analysis of GmSAL1 in soybean root under NaCl --- p.114 / Chapter Appendix VII --- Result of biological repeat of northern blot analysis of GmSAL1 in soybean leaves under 100μM ABA treatment for 0hr, 0.5hr, 1hr, 2hr and 4hr --- p.115 / Chapter Appendix VIII --- Result of biological repeat experiment on the survival rate of tobacco BY-2 cell in 150mM NaCl supplemented MS medium --- p.116 / Chapter Appendix IX --- Result of biological repeat experiment on the survival rate of tobacco BY-2 cell in 13.3% PEG-6000 supplemented MS medium --- p.117
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328579 |
| Date | January 2012 |
| Contributors | Ku, Yee Shan., 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 (xxi, 117 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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