Piu Wong. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 126-146). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.viii / General abbreviations --- p.xi / Abbreviations of chemicals --- p.xiii / List of figures --- p.xv / List of Tables --- p.xvii / Table of contents --- p.xviii / Chapter 1 --- Literature review / Chapter 1.1 --- Overviews --- p.1 / Chapter 1.1.1 --- Nutritional and economical significance of aspartate family amino acidsin human and animal nutrition --- p.1 / Chapter 1.1.2 --- Synthesis of aspartate family amino acids in plants --- p.2 / Chapter 1.2 --- Regulation of aspartate family amino acids between sink and source organs --- p.6 / Chapter 1.2.1 --- Co-ordination of genes/enzymes involved in amide amino acid metabolism to channel aspartate for aspartate family amino acid synthesis --- p.6 / Chapter 1.2.2 --- Sink-source regulation as a general mechanism in plants --- p.9 / Chapter 1.3 --- Source regulation at free amino acid level --- p.11 / Chapter 1.3.1 --- Regulation of free methionine synthesis --- p.11 / Chapter 1.3.1.1 --- Competition for OPHS between TS and CGS --- p.11 / Chapter 1.3.1.2 --- Turnover of CGS mRNA --- p.12 / Chapter 1.3.1.3 --- Post-translational regulation of CGS enzyme --- p.13 / Chapter 1.3.2 --- Regulation of lysine synthesis and catabolism --- p.15 / Chapter 1.3.2.1 --- Feedback regulation loop --- p.15 / Chapter 1.3.2.2 --- Possible intracellular compartmentalization of enzymes and metabolitesin regulating lysine level --- p.21 / Chapter 1.3.2.3 --- Co-ordination of gene/enzyme in aspartate kinase pathway in regulating flux to Lys --- p.21 / Chapter 1.3.3 --- Significance of lysine catabolism in mammals and plants --- p.24 / Chapter 1.3.3.1 --- Complex developmental regulation and stress response of LKR/SDH gene expression --- p.28 / Chapter 1.3.3.2 --- Regulation through a novel composite locus LKR-SDH --- p.28 / Chapter 1.3.3.3 --- Post-translational control of LKR-SDH activity --- p.31 / Chapter 1.3.3.4 --- Implication of two metabolic flux in Lys catabolism --- p.34 / Chapter 1.4 --- Source (free lysine) enhancement in transgenic plants --- p.36 / Chapter 1.4.1 --- Expression of feedback insensitive enzyme in transgenic plants to enhance free lysine supply in transgenic plant --- p.36 / Chapter 1.4.2 --- Reducing or eliminating lysine catabolism to enhance free lysine poolin transgenic plants --- p.40 / Chapter 1.5 --- Sink regulation --- p.41 / Chapter 1.5.1 --- Engineering transgenic plants through expression of seed storage protein (sink) --- p.41 / Chapter 1.5.2 --- "Dynamic relationship between sink protein, nitrogen metabolism and sulphur metabolism" --- p.45 / Chapter 1.6 --- Transgenic plants with improved source or enhanced sinks related to aspartate family amino acids available for our research --- p.47 / Chapter 1.6.1 --- Enhanced source: ASN1 over-expressers --- p.47 / Chapter 1.6.2 --- Enhanced source: metL transgenic plants --- p.47 / Chapter 1.6.3 --- Altered source: RNAi line --- p.47 / Chapter 1.6.4 --- Effective sink: LRP transgenic plants --- p.48 / Chapter 1.7 --- Overall concept of this study --- p.48 / Chapter 2 --- Materials and methods --- p.50 / Chapter 2.1 --- Materials and growth conditions --- p.50 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.50 / Chapter 2.1.2 --- Chemicals and reagents used --- p.53 / Chapter 2.1.3 --- Solutions used --- p.53 / Chapter 2.1.4 --- Commercial kits used --- p.53 / Chapter 2.1.5 --- Equipment and facilities used --- p.53 / Chapter 2.1.6 --- Growth condition --- p.53 / Chapter 2.1.7 --- Tagging of A. thaliana siliques of different developmental stage --- p.54 / Chapter 2.2 --- Methods --- p.55 / Chapter 2.2.1 --- Expression pattern analysis --- p.55 / Chapter 2.2.1.1 --- RNA extraction --- p.55 / Chapter 2.2.1.2 --- Generation of single-stranded DIG-labelled ASN1 DNA probes --- p.55 / Chapter 2.2.1.3 --- Testing the concentration of DIG-labelled probes --- p.56 / Chapter 2.2.1.4 --- Northern blot --- p.57 / Chapter 2.2.1.5 --- Hybridization --- p.58 / Chapter 2.2.1.6 --- Stringency washes --- p.58 / Chapter 2.2.1.7 --- Chemiluminescent detection --- p.58 / Chapter 2.2.2 --- Amino acid analysis and nitrogen determination --- p.60 / Chapter 2.2.2.1 --- Free amino acids in A. thaliana --- p.60 / Chapter 2.2.2.2 --- Phloem exudates collection from A. thaliana --- p.60 / Chapter 2.2.2.3 --- Soluble Protein quantitation --- p.61 / Chapter 2.2.2.4 --- Extraction of salt and water soluble protein from A. thaliana seeds --- p.61 / Chapter 2.2.2.5 --- Purification and amino acid analysis of protein extracts from A. thaliana seeds --- p.62 / Chapter 2.2.2.6 --- Total amino acid determination in mature dry seeds --- p.63 / Chapter 2.2.3 --- Generation of crossing progenies --- p.64 / Chapter 2.2.3.1 --- Artificial crossing of A. thaliana --- p.64 / Chapter 2.2.3.2 --- CTAB extraction of genomic DNA --- p.64 / Chapter 2.2.3.3 --- PCR screening for successful crossing --- p.65 / Chapter 2.2.4 --- Generation of transgenic plants --- p.67 / Chapter 2.2.4.1 --- Cloning of E.coli dapA gene --- p.67 / Chapter 2.2.4.2 --- Preparation of recombinant plasmid --- p.68 / Chapter 2.2.4.3 --- Gene sequencing --- p.68 / Chapter 2.2.4.4 --- Homology search of differentially expressed genes --- p.69 / Chapter 2.2.4.5 --- Construction of chimeric dapA genes (TP-Phas-dapA) --- p.69 / Chapter 2.2.4.6 --- Transformation of electro-competent Agrobacterium cell --- p.73 / Chapter 2.2.4.7 --- Transformation of A. thaliana through vacuum infiltration --- p.73 / Chapter 2.2.4.8 --- Selection of hemizygous and homozygous transgenic plants --- p.74 / Chapter 2.2.4.9 --- Expression analysis of homozygous LRP/dapA transgenic plants --- p.75 / Chapter 3 --- Results --- p.77 / Chapter 3.1 --- Characterization of ASN1 over-expressers --- p.77 / Chapter 3.1.1 --- Overexpression of the ASN1 gene enhances the sink-source relationship of asparagine transport under regular daylight cycle --- p.88 / Chapter 3.1.2 --- Spatial distribution of total free amino acids under normal daylight cycle --- p.88 / Chapter 3.1.3 --- Over-expression of the ASN1 gene affects free amino acid level quantitatively under normal daylight cycle --- p.89 / Chapter 3.1.4 --- Over-expression of the ASN1 gene affects composition of total amino acid under normal daylight cycle --- p.89 / Chapter 3.2 --- Construction of dapA transgenic Arabidopsis --- p.91 / Chapter 3.2.1 --- Construction of chimeric gene for expression of the dapA gene --- p.91 / Chapter 3.2.2 --- Transformation of p1300/Phas-dapA into Arabidopsis and selection of homozygous progenies --- p.91 / Chapter 3.3 --- Generation of transgenic plants with altered sink-source relationship through crossing and in-planta transformation --- p.96 / Chapter 3.3.1 --- Rationale in methods for generating transgenic plants with different combination of sources and sinks --- p.96 / Chapter 3.3.2 --- Screening for double homozygous progenies through crossing --- p.98 / Chapter 3.3.3 --- Screening for F1 progenies of successful crossing --- p.100 / Chapter 3.3.4 --- Selection of homozygous crossing progenies --- p.102 / Chapter 3.3.5 --- Screening for homozygous dapA/LRP transgenic plants --- p.104 / Chapter 3.4 --- Amino acid composition analysis --- p.109 / Chapter 3.4.1 --- The change of aspartate family amino acids in mature seeds of transgenic plants with altered sources --- p.113 / Chapter 3.4.2 --- The change of aspartate family amino acids in mature seeds of transgenic plants with improved sink --- p.114 / Chapter 3.4.3 --- The change of aspartate family amino acids in mature seeds of transgenic plants with improved sink --- p.115 / Chapter 4. --- Discussion / Chapter 4.1 --- Characterization of ASN1 over-expressers --- p.116 / Chapter 4.1.1 --- Possible regulation of ASN1 mRNA stability through level of asparagine --- p.117 / Chapter 4.1.2 --- Over-expression of ASN1 gene may improve nitrogen remobilisation from source to sink tissues --- p.118 / Chapter 4.1.3 --- Over-expression of ASN1 gene has modified the composition of amino acidsin sink organs --- p.119 / Chapter 4.2 --- ASN1 RNAi transgenic plants increases the relative contents of lysine in the seeds --- p.122 / Chapter 4.2.1 --- Role of ASN1 in supplying or competing aspartate in developing seeds --- p.122 / Chapter 4.2.2 --- Possible role of glutamate receptor --- p.123 / Chapter 4.3 --- Lysine catabolism may strictly control the level of lysine --- p.123 / Chapter 4.3.1 --- Possible role of lysine-tRNA in protein synthesis --- p.124 / Chapter 5. --- Conclusion and prospective --- p.125 / References --- p.126 / Appendix --- p.147
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324393 |
| Date | January 2003 |
| Contributors | Wong, Piu., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xxv, 155 leaves : ill. (some col.) ; 30 cm. |
| 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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