Regulation of amino acid metabolism: gene expression during seed development and the possible roles of GCN2.

• Other Authors: Ma, Junhao.
• Format: Others
• Language: English; Chinese
• Published: 2004
• Subjects: Rice; Amino acids > Metabolism; Gene expression
• Online Access: http://library.cuhk.edu.hk/record=b5892142; http://repository.lib.cuhk.edu.hk/en/item/cuhk-324935

Ma Junhao. === Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. === Includes bibliographical references (leaves 111-123). === Abstracts in English and Chinese. === Thesis Committee --- p.i === Statement --- p.ii === Abstract --- p.iii === Acknowledgements --- p.vii === General Abbreviations --- p.ix === Abbreviations of Chemicals --- p.xi === Table of Contents --- p.xii === List of Figures --- p.xvi === List of Tables --- p.xix === Chapter 1. --- Literature review --- p.1 === Chapter 1.1 --- Importance of amino acid metabolism --- p.1 === Chapter 1.1.1 --- Rice as the important source of essential amino acids --- p.1 === Chapter 1.1.2 --- Rice seeds are nutritionally incomplete --- p.2 === Chapter 1.1.3 --- The nitrogen source for aspartate family amino acid synthesis --- p.3 === Chapter 1.1.4 --- Synthesis of aspartate family amino acids in plants --- p.4 === Chapter 1.1.5 --- Regulation of the aspartate pathway at free amino acid level --- p.12 === Chapter 1.2 --- Regulation of amino acid metabolism during seed development --- p.14 === Chapter 1.3 --- Signalling system for nitrogen metabolism --- p.17 === Chapter 1.3.1 --- Nitrogen signalling in plants --- p.18 === Chapter 1.3.2 --- GCN signalling: another nitrogen signalling pathway --- p.21 === Chapter 1.3.2.1 --- Mechanism of GCN signalling pathway in yeast --- p.21 === Chapter 1.3.2.2 --- GCN in mammalian --- p.24 === Chapter 1.3.2.3 --- GCN in higher plant --- p.24 === Chapter 1.3.3 --- Relationship between carbon and nitrogen metabolic signaling in plants --- p.26 === Chapter 1.3.4 --- Paradigm for elucidating new signal transduction pathways --- p.29 === Chapter 1.4 --- Hypothesis of this thesis work --- p.30 === Chapter 2. --- Materials and Methods --- p.32 === Chapter 2.1. --- Materials --- p.32 === Chapter 2.1.1 --- Plants --- p.32 === Chapter 2.1.2 --- Bacterial strains and vectors --- p.32 === Chapter 2.1.3 --- Chemicals and reagents --- p.33 === Chapter 2.1.4 --- Buffer，solution and gel --- p.33 === Chapter 2.1.5 --- Commercial kits --- p.33 === Chapter 2.1.6 --- Equipments and facilities used --- p.33 === Chapter 2.1.6 --- Growth medium --- p.34 === Chapter 2.2 --- Methods --- p.34 === Chapter 2.2.1 --- Profiling genes expression pattern in developing rice seeds --- p.34 === Chapter 2.2.1.1 --- Growth conditions of rice --- p.34 === Chapter 2.2.1.2 --- Collection of developing rice seeds --- p.35 === Chapter 2.2.1.3 --- Total RNA extraction from rice seeds --- p.37 === Chapter 2.2.1.4 --- Total RNA extraction from plant leaf --- p.37 === Chapter 2.2.1.5 --- Gel electrophoresis --- p.38 === Chapter 2.2.1.6 --- First strand cDNA synthesis from rice total RNA --- p.39 === Chapter 2.2.1.7 --- Search for the coding sequence of rice genes related to amino acid metabolism --- p.39 === Chapter 2.2.1.8 --- Alignment of homologous coding sequence between family member genes --- p.42 === Chapter 2.2.1.9 --- Primer design --- p.42 === Chapter 2.2.1.10 --- Quantitation of total RNA and determination of internal control --- p.45 === Chapter 2.2.1.11 --- PCR to amplify the DNA fragments --- p.45 === Chapter 2.2.1.12 --- DNA Sequencing --- p.46 === Chapter 2.2.1.13 --- Generation and testing of single-stranded DIG-labelled DNA probes --- p.46 === Chapter 2.2.1.14 --- Northern blot --- p.47 === Chapter 2.2.1.15 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.48 === Chapter 2.2.2 --- Expression assay of selected genes in herbicide treated plants --- p.49 === Chapter 2.2.2.1 --- Growing conditions and herbicide treatments --- p.49 === Chapter 2.2.2.2 --- GCN2 homologue in Arabidopsis and rice --- p.51 === Chapter 2.2.2.3 --- RT-PCR to analyze the change in expression level of selected genes in herbicide treated plants --- p.53 === Chapter 2.2.3 --- Generation of transgenic Arabidopsis --- p.56 === Chapter 2.2.3.1 --- Preparation of T-vector for T-ligation --- p.56 === Chapter 2.2.3.2 --- Cloning of Arabidopsis GCN2 gene --- p.56 === Chapter 2.2.3.3 --- Transformation of the plasmid into DH5a competent cell --- p.57 === Chapter 2.2.3.4 --- Screening of right recombinants --- p.58 === Chapter 2.2.3.5 --- Construction of chimeric AtGCN2 genes --- p.59 === Chapter 2.2.3.6 --- Transformation of electro-competent Agrobacterium cell --- p.60 === Chapter 2.2.3.7 --- Transformation of Arabidopsis by vacuum infiltration --- p.61 === Chapter 2.2.3.8 --- Selection of hemizygous and homozygous transgenic plants --- p.61 === Chapter 2.2.3.9 --- Screening of the T3 transformants --- p.63 === Chapter 2.2.3.10 --- Expression analysis of homozygous AtGCN2 transgenic Arabidopsis --- p.63 === Chapter 3. --- Results --- p.63 === Chapter 3.1 --- Profiling genes expression pattern in developing rice seeds --- p.63 === Chapter 3.1.1 --- Quantification of total RNA from seeds at different developing stages --- p.63 === Chapter 3.1.2 --- DNA sequence analysis --- p.66 === Chapter 3.1.3 --- Profiling the gene expression in developing rice seeds --- p.67 === Chapter 3.1.3.1 --- Expression profiles of nitrogen assimilation related genes --- p.67 === Chapter 3.1.3.2 --- Expression profiles of aspartate pathway genes --- p.72 === Chapter 3.1.3.3 --- Expression profiles of branched-chain amino acid synthesis pathway genes --- p.78 === Chapter 3.2 --- Relationship between GCN2 and amino acid metabolism in plants --- p.82 === Chapter 3.2.1 --- GCN2 homologue in A. thaliana and rice --- p.82 === Chapter 3.2.2 --- GCN2 and amino acid starvation --- p.85 === Chapter 3.2.3 --- Effects of amino acid starvation on GCN2 expression --- p.90 === Chapter 3.2.3 --- Changes in the expression level AK and BCAT genes in herbicide treated rice and A. thaliana --- p.93 === Chapter 3.3 --- Characterization of GCN2 transgenic A. thaliana --- p.96 === Chapter 3.3.1 --- Construct of pBI121-AtGCN2 --- p.96 === Chapter 3.3.2 --- Construction of GCN2 transgenic A. thaliana --- p.96 === Chapter 3.3.3 --- Expression of GCN2 in transgenic A. thaliana --- p.97 === Chapter 3.3.4 --- Expression level changes of AK and BCAT in transgenic A. thaliana --- p.99 === Chapter 4. --- Discussions --- p.101 === Chapter 4.1 --- Expression pattern of selected metabolic genes in developing plant seeds --- p.101 === Chapter 4.1.1 --- Most genes studied displayed a similar pattern --- p.101 === Chapter 4.1.2 --- Regulation of gene expression in developing rice seeds --- p.105 === Chapter 4.2 --- GCN2 and its role in higher plants --- p.106 === Chapter 4.2.1 --- The existence of the GCN2 gene in rice --- p.106 === Chapter 4.2.2 --- GCN2 responses solely to amino acid starvation --- p.106 === Chapter 5. --- Conclusion and Prospective --- p.109 === Reference --- p.111 === Appendix I: Chemicals and reagents --- p.124 === "Appendix II: Buffer, solution and gel" --- p.126 === Appendix III: Commercial kits --- p.128 === Appendix IV: Equipments and facilities used --- p.128