published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

Wong, Hon-Kit. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 119-139). / Abstract in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vii / General abbreviations --- p.ix / Abbreviations of chemicals --- p.x / List of figures --- p.xii / Table of contents --- p.xvi / Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Nitrogen assimilation and regulation in plants --- p.1 / Chapter 1.2 --- Asparagine metabolism and its gene regulation in plants --- p.2 / Chapter 1.2.1 --- A brief introduction of asparagine --- p.2 / Chapter 1.2.2 --- Asparagine synthetase gene family in A. thaliana --- p.3 / Chapter 1.2.3 --- Reciprocal regulation of ASN1 and ASN2 gene --- p.3 / Chapter 1.2.4 --- Primary structure difference of ASN1 and ASN2 protein --- p.4 / Chapter 1.2.5 --- "ASN1 overexpressor support the notion that it is a major gene regulating the free asparagine levels in plant tissues, while ASN may play different physiological function(s)" --- p.2 / Chapter 1.2.6 --- Evidence support ammonium-dependent AS in plant --- p.6 / Chapter 1.3 --- Ammonium toxicity and mechanism of ammonium toxicity to plant --- p.7 / Chapter 1.3.1 --- Ammonium toxicity --- p.7 / Chapter 1.3.2 --- Mechanism of ammonium toxicity --- p.9 / Chapter 1.4 --- "Relationship among asparagine, ammonium, and stress physiology" --- p.12 / Chapter 1.4.1 --- Ammonium accumulates under stress conditions --- p.12 / Chapter 1.4.2 --- Asparagine accumulates under stress conditions --- p.14 / Chapter 1.5 --- Relationship of asparagine metabolism and photorespiration --- p.17 / Chapter 1.5.1 --- A brief introduction of photorespiratory pathway --- p.17 / Chapter 1.5.2 --- Involvement of Asn in the photorespiration nitrogen cycle --- p.18 / Chapter 1.5.3 --- Reassimilation of ammonium released from photorespiration --- p.19 / Chapter 1.5.4 --- Photorespiration and stress physiology --- p.21 / Chapter 1.6 --- Role of amino acids in abiotic stress resistance --- p.23 / Chapter 1.6.1 --- Overview --- p.23 / Chapter 1.6.2 --- Proline accumulation and plant adaptation to water deficits and salinity stress --- p.24 / Chapter 1.6.3 --- Role of amino acids as precursors of quaternary ammonium compounds serving as compatible osmolytes --- p.28 / Chapter 1.7 --- A brief history of protoplast transient expression systems --- p.35 / Chapter 1.8 --- Advantages of mesophyll protoplast transient expression systems --- p.37 / Chapter 1.9 --- Hypothesis and main idea of this study --- p.38 / Chapter 2 --- Methods and Materials --- p.39 / Chapter 2.1 --- Materials --- p.39 / Chapter 2.1.1 --- Plants --- p.39 / Chapter 2.1.2 --- Bacterial strains and plasmid vector --- p.39 / Chapter 2.1.3 --- Primer used --- p.39 / Chapter 2.1.4 --- Chemicals and reagents used --- p.40 / Chapter 2.1.5 --- Solution used --- p.40 / Chapter 2.1.6 --- Commercial kits used --- p.40 / Chapter 2.1.7 --- Equipment and facilities used --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Growth medium and condition --- p.41 / Chapter 2.2.1.1 --- Normal growth condition --- p.41 / Chapter 2.2.1.2 --- Growth medium and stresses treatments --- p.41 / Chapter 2.2.1.3 --- Plant growth in Azaserine medium --- p.43 / Chapter 2.2.2 --- Biochemical Assay --- p.44 / Chapter 2.2.2.1 --- Ammonium assay --- p.44 / Chapter 2.2.2.2 --- Ammonium extraction for ammonium assay --- p.46 / Chapter 2.2.2.3 --- Soluble protein determination --- p.46 / Chapter 2.2.2.4 --- Detection of chlorophyll content --- p.47 / Chapter 2.2.3 --- Molecular techniques --- p.47 / Chapter 2.2.3.1 --- Bacterial cultures for recombinant DNA --- p.47 / Chapter 2.2.3.2 --- Recombinant DNA techniques --- p.48 / Chapter 2.2.3.3 --- Transformation of DH5a Competent cell --- p.48 / Chapter 2.2.3.4 --- Gel electrophoresis --- p.49 / Chapter 2.2.3.5 --- DNA and RNA extraction from plant tissues --- p.50 / Chapter 2.2.3.6 --- Generation of cRNA probes for Northern blot analyses --- p.52 / Chapter 2.2.3.7 --- Northern blot analysis --- p.53 / Chapter 2.2.3.8 --- PCR techniques --- p.54 / Chapter 2.2.3.9 --- Sequencing --- p.55 / Chapter 2.2.4 --- Genetic techniques --- p.56 / Chapter 2.2.4.1 --- Backcross of Azaserine resistant mutant --- p.56 / Chapter 2.2.4.2 --- Phenotype screening of backcross progenies --- p.56 / Chapter 2.2.5 --- Transient gene expression --- p.57 / Chapter 2.2.5.1 --- Protoplast isolation from Arabidopsis leave --- p.57 / Chapter 2.2.5.2 --- Protoplast transformation --- p.58 / Chapter 2.2.5.3 --- Gus protein extraction from protoplasts --- p.59 / Chapter 2.2.5.4 --- Gus assay --- p.60 / Chapter 2.2.5.5 --- MU calibration standard --- p.60 / Chapter 2.2.5.6 --- Sample assay --- p.60 / Chapter 3 --- Result --- p.61 / Chapter 3.1 --- Expression of ASN2 and ammonium assay in Arabidopsis thaliana under various stress conditions and senescence --- p.61 / Chapter 3.1.1 --- Ammonium assay of wild type seedlings under stress conditions --- p.61 / Chapter 3.1.2 --- Kinetic studies of ASN2 expression under different stresses treatments --- p.65 / Chapter 3.1.3 --- Ammonium assay of wild type seedlings under stress conditions --- p.70 / Chapter 3.2 --- NH4+ regulation on expression of ASN2 promoter --- p.73 / Chapter 3.2.1 --- The cloning ASN2 promoter --- p.73 / Chapter 3.2.1.1 --- Defining of ASN2 promoter region --- p.73 / Chapter 3.2.1.2 --- PCR amplification of ASN2 promoter from genomic sequence --- p.77 / Chapter 3.2.1.3 --- Cloning ASN2 promoter into transient gene expression vector (pBI221 vector) --- p.80 / Chapter 3.2.2 --- Transient gene expression --- p.84 / Chapter 3.2.2.1 --- Arabidopsis leave mesophyll protoplasts isolation --- p.84 / Chapter 3.2.2.2 --- Transformation and GUS expression assay --- p.87 / Chapter 3.3 --- Characterization ASN2 transgenic plants under stress conditions --- p.91 / Chapter 3.3.1 --- Construction of ASN2 transgenic plants --- p.91 / Chapter 3.3.2 --- Characterization of ASN2 transgenic plants --- p.93 / Chapter 3.3.2.1 --- Ammonium assay of ASN2 transgenic plant under different concentration of ammonium --- p.93 / Chapter 3.3.2.2 --- Ammonium assay of ASN2 transgenic plant under high light irradiance --- p.93 / Chapter 3.4 --- Characterization of mutant plants (AzaR) that showed altered ASN2 expression --- p.97 / Chapter 3.4.1 --- Phenotype of azaserine resistant mutant --- p.97 / Chapter 3.4.2 --- ASN2 expression level up-regulated in azaserine resistant mutant --- p.99 / Chapter 3.4.3 --- Checking for linkage between azaserine resistance and ASN2 overexpression --- p.101 / Chapter 3.4.4 --- Crossing the mutant with Landsberg for mapping the azaserine resistant mutant --- p.106 / Chapter 4 --- Discussion --- p.108 / Chapter 4.1 --- ASN2 may relate to ammonium metabolism --- p.108 / Chapter 4.2 --- ASN2 transgenic plants and their response under stresses conditions --- p.111 / Chapter 4.3 --- ASN2 promoter studies by transient gene expression method --- p.112 / Chapter 4.3.1 --- Identification of promoter region --- p.113 / Chapter 4.3.2 --- Isolation of protoplasts from Arabidopsis leaf --- p.114 / Chapter 4.3.3 --- Studies of ASN2 promoter transient gene expression in A thaliana protoplasts --- p.114 / Chapter 4.4 --- Azaserine Resistant Mutant --- p.115 / Chapter 4.4.1 --- Overexpression of ASN2 gene in Azaserine resistant mutant and checking for linkage --- p.115 / Chapter 4.4.2 --- Cross of Azaserine Resistant mutants with Lersberg ecotype for mapping --- p.116 / Chapter 5 --- Conclusion and prospective --- p.118 / References --- p.119 / Appendix --- p.140

Bibliography: leaves 89-104. In this study an EMS-mutated Arabidopsis mutant pho2, which accumulates Pi in leaves, was used to study Pi uptake and transport by comparing it to wild-type seedlings. The study aimed to define the physiological lesions in pho2 mutant and to obtain evidence regarding the function of the PHO2 gene in P nutrition in higher plants. Accumulation of Pi in leaves of pho2 was found to reside in the symplast and was not related to Zn-deficiency. The physiology of the pho2 mutant is consistent with either a block in Pi transport in phloem from shoots to roots or an inability of shoot cells to regulate internal Pi concentration. Southern block analysis revealed that the two transporter genes, APT1 and APT2 were not responsible for the pho2 mutant. Data from the mapping of the PHO2 gene along with information from the Arabidopsis genome sequencing will form the basis for cloning the PHO2 gene in the future.

published_or_final_version / abstract / Botany / Master / Master of Philosophy

This project was designed to investigate the impact of natural levels of ultraviolet-B radiation on the genomic mutation rate in Arabidopsis thaliana. UV-B radiation is a known mutagen, but plants may have evolved mechanisms to cope with any genomic damage induced by routine exposure to this radiation. In an attempt to determine whether the genomic mutation rate in a plant species is elevated in the presence of UV-B, two eleven generation mutation accumulation studies were preformed. One study incorporated levels of UV-B similar to that encountered on a clear mid-summer's day, while the other was performed in the absence of this mutagen. Mutation rate estimates, obtained primarily from maximum likelihood analysis of phenotypic data, were not significantly greater than zero, both in the presence and absence of UV-B. No evidence was found to support the notion that the genomic mutation rate is increased by exposure to natural levels of UV-B.

Bibliography: leaves 89-104. / vi, 104 leaves, [15] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / In this study an EMS-mutated Arabidopsis mutant pho2, which accumulates Pi in leaves, was used to study Pi uptake and transport by comparing it to wild-type seedlings. The study aimed to define the physiological lesions in pho2 mutant and to obtain evidence regarding the function of the PHO2 gene in P nutrition in higher plants. Accumulation of Pi in leaves of pho2 was found to reside in the symplast and was not related to Zn-deficiency. The physiology of the pho2 mutant is consistent with either a block in Pi transport in phloem from shoots to roots or an inability of shoot cells to regulate internal Pi concentration. Southern block analysis revealed that the two transporter genes, APT1 and APT2 were not responsible for the pho2 mutant. Data from the mapping of the PHO2 gene along with information from the Arabidopsis genome sequencing will form the basis for cloning the PHO2 gene in the future. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1999

No description available.

Cheung Ming-yan. / Thesis submitted in 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 169-185). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.x / List of figures --- p.xii / List of tables --- p.xv / Table of Contents --- p.xvii / Chapter Chapter 1 --- General Introduction - Using transgenic plants as bioreactor --- p.1 / Chapter 1.1 --- Plant as Bioreactor --- p.1 / Chapter 1.1.1 --- Plant transformation historical milestones --- p.1 / Chapter 1.1.2 --- Applications of transgenic plants --- p.5 / Chapter 1.1.2.1 --- Examples of in situ Application --- p.5 / Chapter 1.1.2.2 --- Examples of ex situ application of transgenic plant --- p.9 / Chapter 1.2 --- Plant Hosts for Transformation: Arabidopsis thaliana and Glycine max --- p.18 / Chapter 1.2.1 --- Essential components for plant transformation --- p.18 / Chapter 1.2.1.1 --- Marker genes --- p.18 / Chapter 1.2.1.2 --- Promoters --- p.18 / Chapter 1.2.2 --- Arabidopsis thaliana --- p.20 / Chapter 1.2.2.1 --- Agrobacterium-mediated transformation --- p.20 / Chapter 1.2.2.2 --- Transformation methods for A. thaliana --- p.21 / Chapter 1.2.3 --- Glycine max (soybean) --- p.22 / Chapter 1.2.3.1 --- Soybean cultivars for transformation --- p.23 / Chapter 1.2.3.2 --- Soybean regeneration systems --- p.24 / Chapter 1.2.3.3 --- Soybean transformation systems --- p.26 / Chapter 1.3 --- Target Pharmaceutical and Agricultural Proteins: Lymphocytic choriomeningitis virus and Goldfish Growth hormones I and II --- p.29 / Chapter 1.3.1 --- Production of pharmaceutical proteins --- p.29 / Chapter 1.3.1.1 --- Lymphocytic choriomeningitis virus --- p.30 / Chapter 1.3.1.2 --- Nucleoprotein of LCMV --- p.33 / Chapter 1.3.2 --- Agricultural protein category --- p.34 / Chapter 1.3.2.1 --- Carassius auratus --- p.34 / Chapter 1.3.2.2 --- Growth hormones I and II --- p.35 / Chapter 1.4 --- Hypothesis and Objectives --- p.37 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.38 / Chapter 2.1.2 --- Chemicals and Regents --- p.43 / Chapter 2.1.3 --- Commercial kits --- p.44 / Chapter 2.1.4 --- Primers and Adaptors --- p.45 / Chapter 2.1.5 --- Equipments and Facilities used --- p.47 / Chapter 2.1.6 --- "Buffer, solution and medium" --- p.47 / Chapter 2.2 --- Methods --- p.48 / Chapter 2.2.1 --- Molecular Techniques --- p.48 / Chapter 2.2.1.1 --- Bacterial cultures for recombinant DNA and plant transformation --- p.48 / Chapter 2.2.1.2 --- Recombinant DNA techniques --- p.48 / Chapter 2.2.1.3 --- "Preparation and transformation of DH5a, DE3 and Agrobacterium competent cells" --- p.49 / Chapter 2.2.1.4 --- Gel electrophoresis --- p.52 / Chapter 2.2.1.5 --- "DNA, RNA and protein extractions" --- p.53 / Chapter 2.2.1.6 --- Generation of cRNA probes for Southern and Northern blot analyses --- p.56 / Chapter 2.2.1.7 --- Southern blot analysis --- p.56 / Chapter 2.2.1.8 --- Northern blot analysis --- p.57 / Chapter 2.2.1.9 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) in bacterial system --- p.58 / Chapter 2.2.1.10 --- Western blot analysis for LCMV NP --- p.59 / Chapter 2.2.1.11 --- Protein dot blot for detecting the presence of recombinant LCMV-NP generated from transgenic plants --- p.62 / Chapter 2.2.1.12 --- PCR techniques --- p.62 / Chapter 2.2.1.13 --- Sequencing --- p.63 / Chapter 2.2.2 --- Plant tissue culture and transformation --- p.64 / Chapter 2.2.2.1 --- Arabidopsis thaliana --- p.64 / Chapter 2.2.2.2 --- Soybean --- p.65 / Chapter 2.2.3 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.68 / Chapter 2.2.3.1 --- In vitro transcription of target genes with with Ribomix large scale RNA production systems-T7 and SP6 (Promega) --- p.68 / Chapter 2.2.3.2 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract --- p.69 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- Expression of Lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) and goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1 --- Expression of LCMV-NP in transgenic Arabidopsis thaliana --- p.71 / Chapter 3.1.1.1 --- Cloning of the gene encoding LCMV NP into the binary vector system W104 --- p.71 / Chapter 3.1.1.2 --- Transformation of W104-LCMV-NP into the Agrobacterium GV3101/pMP90 --- p.78 / Chapter 3.1.1.3 --- Transformation of LCMV-NP cDNA into Arabidopsis thaliana --- p.80 / Chapter 3.1.1.4 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.83 / Chapter 3.1.1.5 --- Production of recombinant LCMV-NP protein in DE3 cells --- p.90 / Chapter 3.1.1.6 --- Detection of recombinant LCMV-NP protein in transgenic A.thaliana --- p.98 / Chapter 3.1.2 --- Expression of goldfish growth hormones I and II (GHI and GHII) in transgenic Arabidopsis thaliana --- p.105 / Chapter 3.1.2.1 --- "Screening of homozygous lines of goldfish, Carassius auratus, growth hormones transgenic Arabidopsis thaliana" --- p.105 / Chapter 3.1.2.2 --- Southern blot and Northern blot analyses of transgenic plant containing the LCMV-NP cDNA --- p.109 / Chapter 3.1.2.3 --- Detection of recombinant GHI and GHII from transgenic plant --- p.112 / Chapter 3.2 --- In vitro transcription and translation of target genes in rabbit reticulocyte and wheat germ systems --- p.117 / Chapter 3.2.1 --- Subcloning of target genes in pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.1 --- Subcloning of LCMV-NP fragment into pGEM-3Zf(+) vector --- p.117 / Chapter 3.2.1.2 --- Subcloning of goldfish GHI and GHII fragments into pGEM-3Zf(+) vector --- p.120 / Chapter 3.2.2 --- In vitro transcription of target genes with Ribomix large scale RNA production systems-T7 and SP6 --- p.125 / Chapter 3.2.3 --- In vitro translation with rabbit reticulocyte lysate and wheat germ extract systems --- p.128 / Chapter 3.3 --- Establishment of Glycine max regeneration and transformation systems --- p.130 / Chapter 3.3.1 --- The Establishment of soybean regeneration system --- p.130 / Chapter 3.3.2 --- Establishment of soybean transformation system --- p.133 / Chapter 3.3.2.1 --- Definition of transformation efficiency --- p.133 / Chapter 3.3.2.2 --- Effects of plant hosts --- p.136 / Chapter 3.3.2.3 --- Effects of Agrobacterium strains --- p.138 / Chapter 3.3.2.4 --- The application of vacuum infiltration --- p.139 / Chapter 3.3.2.5 --- Effect of kanamycin --- p.140 / Chapter 3.3.2.6 --- Effect of cocultivation duration and light/ dark treatment during germination --- p.141 / Chapter 3.3.2.7 --- Application of the detergent Silwet-77 --- p.142 / Chapter 3.3.3 --- Verification of transformation results by PCR screening --- p.143 / Chapter Chapter 4 --- Discussion --- p.147 / Chapter 4.1 --- "Expression of LCMV-NP, GHI and GHII in A. thaliana" --- p.148 / Chapter 4.2 --- Establishing a soybean transformation system --- p.157 / Chapter 4.2.1 --- Plant hosts and explants --- p.158 / Chapter 4.2.2 --- Regeneration of explants --- p.159 / Chapter 4.2.3 --- Agrobacterium strains --- p.161 / Chapter 4.2.4 --- Bacteria-plant interaction --- p.161 / Chapter 4.2.5 --- Transient versus stable transformation --- p.165 / Chapter 4.3 --- Conclusion and perspective --- p.167 / References --- p.169 / Appendix --- p.186

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

Abstract not available