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

Suen Pui Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 154-168). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / List of Figures --- p.vii / List of Tables --- p.ix / List of Abbreviations --- p.x / Chapter Chapter 1: --- Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.3 / Chapter 2.1 --- Phosphorus and Plants --- p.3 / Chapter 2.1.1 --- Importance of phosphorus --- p.3 / Chapter 2.1.2 --- Phosphorus is a limiting nutrient --- p.3 / Chapter 2.2 --- Responses of Plants to Phosphate Deficiency --- p.4 / Chapter 2.2.1 --- Morphological changes of plants during phosphate deficiency --- p.5 / Chapter 2.2.1.1 --- Modification of the root system --- p.5 / Chapter 2.2.1.2 --- Symbiotic association of roots with mycorrhiza --- p.6 / Chapter 2.2.2 --- Maintenance of phosphate levels in plants during phosphate deficiency --- p.7 / Chapter 2.2.2.1 --- Phosphate homeostasis in plants --- p.7 / Chapter 2.2.2.2 --- "Enhancement of Pi scavenging, recycling and uptake" --- p.9 / Chapter 2.2.2.3 --- Pi-limited metabolism --- p.11 / Chapter 2.2.3 --- Hormones and phosphate starvation responses --- p.12 / Chapter 2.2.4 --- Regulation of gene expression during phosphate starvation --- p.14 / Chapter 2.2.4.1 --- The pho regulon in bacteria and yeast --- p.14 / Chapter 2.2.4.2 --- The coordination of phosphate starvation induced genes in plants --- p.19 / Chapter 2.2.4.3 --- Signaling phosphate starvation --- p.19 / Chapter 2.2.4.4 --- Phosphite and phosphate starvation --- p.21 / Chapter 2.2.4.5 --- Transcriptional regulation during phosphate starvation --- p.22 / Chapter 2.3 --- Acid Phosphatases in Higher Plants --- p.26 / Chapter 2.3.1 --- Enzymatic properties of acid phosphatases --- p.26 / Chapter 2.3.2 --- Localization and function of acid phosphatases --- p.27 / Chapter 2.3.3 --- Expression of acid phosphatases --- p.28 / Chapter 2.4 --- Purple Acid Phosphatases --- p.29 / Chapter 2.4.1 --- Properties of purple acid phosphatases --- p.29 / Chapter 2.4.2 --- Regulation and expression of plant purple acid phosphatase --- p.32 / Chapter 2.5 --- Tomato Purple Acid Phosphatases --- p.33 / Chapter 2.6 --- Promoter Analysis --- p.35 / Chapter 2.6.1 --- Structure of an eukaryotic promoter --- p.35 / Chapter 2.6.2 --- Promoter analysis by deletion mapping --- p.37 / Chapter 2.6.3 --- The computational approaches in promoter analysis --- p.38 / Chapter 2.6.4 --- Transient expression assay and transgenic expression assay --- p.39 / Chapter 2.7 --- Transcriptional Regulation of Tomato Purple Acid Phosphatase Expression --- p.40 / Chapter 2.8 --- Hypothesis --- p.41 / Chapter Chapter 3: --- Materials and Methods --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Materials --- p.44 / Chapter 3.2.1 --- Chemicals --- p.44 / Chapter 3.2.2 --- Plant materials --- p.44 / Chapter 3.2.3 --- Plasmid vectors and bacterial strains --- p.44 / Chapter 3.2.4 --- Primers design --- p.45 / Chapter 3.2.5 --- Confirmation of sequence fidelity --- p.46 / Chapter 3.3 --- Cloning of the TPAP1 Promoter Fragments --- p.46 / Chapter 3.3.1 --- Genomic DNA extraction --- p.46 / Chapter 3.3.1.1 --- Materials --- p.46 / Chapter 3.3.1.2 --- Procedures --- p.47 / Chapter 3.3.2 --- Cloning strategy of TPAP1 promoter --- p.47 / Chapter 3.3.3 --- TPAP1 promoter cloning --- p.48 / Chapter 3.3.3.1 --- Long-distance PCR --- p.48 / Chapter 3.3.4 --- Chimeric gene constructs --- p.48 / Chapter 3.3.4.1 --- Chimeric gene construction for particle bombardment --- p.51 / Chapter 3.3.4.2 --- Chimeric gene construction for tobacco transformation --- p.51 / Chapter 3.4 --- Transient Expression Assay of the TPAP1 Promoter Fragments --- p.54 / Chapter 3.4.1 --- TPAP1 promoter activity assay --- p.54 / Chapter 3.4.2 --- Preparation of MS culture medium --- p.54 / Chapter 3.4.3 --- Growing tomato seedlings in MS liquid medium --- p.56 / Chapter 3.4.4 --- Biolistic bombardment --- p.56 / Chapter 3.4.5 --- GUS histochemcial staining --- p.57 / Chapter 3.4.5.1 --- Materials --- p.57 / Chapter 3.4.5.2 --- Procedures --- p.57 / Chapter 3.5 --- Transgenic Assay of the TPAP1 Promoter Fragments --- p.58 / Chapter 3.5.1 --- Materials for tobacco transformation --- p.58 / Chapter 3.5.2 --- Agrobacterium tumefaciens preparation --- p.58 / Chapter 3.5.3 --- Tobacco transformation and regeneration --- p.59 / Chapter 3.5.4 --- Promoter activity analysis --- p.60 / Chapter 3.5.4.1 --- Materials --- p.60 / Chapter 3.5.4.2 --- Procedures --- p.60 / Chapter 3.5.5 --- Southern blot analysis --- p.61 / Chapter 3.5.6 --- RNA isolation --- p.61 / Chapter 3.5.6.1 --- Materials --- p.61 / Chapter 3.5.6.2 --- Procedures --- p.61 / Chapter 3.5.7 --- Northern blot analysis --- p.62 / Chapter 3.6 --- Biochemical Analysis of Acid Phosphatase Activities --- p.63 / Chapter 3.6.1 --- Excretion of acid phosphatase into the environment --- p.63 / Chapter 3.6.2 --- Growing tomato seedlings in MS medium --- p.63 / Chapter 3.6.3 --- Acid phosphatase activity assay by p-nitrophenyl phosphate --- p.64 / Chapter 3.6.4 --- Activity-gel detection --- p.65 / Chapter 3.6.4.1 --- Materials --- p.65 / Chapter 3.6.4.2 --- Procedures --- p.65 / Chapter 3.7 --- "Sequence Analysis of the TPAP1 gene, cDNA and promoter" --- p.66 / Chapter 3.7.1 --- Isolation of TPAPl cDNA --- p.66 / Chapter 3.7.1.1 --- Rapid amplification of cDNA ends (RACE) --- p.66 / Chapter 3.7.1.2 --- RT-PCR --- p.67 / Chapter 3.7.2 --- Isolation of TPAP1 gene --- p.67 / Chapter 3.7.2.1 --- PCR amplification of the TPAP1 gene --- p.67 / Chapter 3.7.2.2 --- TPAP1 gene sequence determination --- p.68 / Chapter 3.7.3 --- Sequence analysis --- p.69 / Chapter 3.8 --- Statistical analysis --- p.70 / Chapter Chapter 4: --- Results --- p.72 / Chapter 4.1 --- "Cloning of the TPAP1 Promoter Fragments, Gene and cDNA" --- p.72 / Chapter 4.1.1 --- TPAP1 promoter fragment constructs --- p.72 / Chapter 4.1.2 --- TPAP1 cDNA cloning --- p.72 / Chapter 4.1.3 --- TPAP1 gene cloning --- p.72 / Chapter 4.2 --- "Sequence analysis of the TPAP1 promoter, gene, cDNA and predicted amino acid sequence" --- p.76 / Chapter 4.2.1 --- "The DNA sequence of the TPAP1 promoter, gene and cDNA" --- p.76 / Chapter 4.2.2 --- Properties of TPAP1 cDNA and protein --- p.83 / Chapter 4.2.3 --- Identification of potential metal ligating residues on TPAP1 --- p.85 / Chapter 4.2.4 --- Phylogenetic relationship of TPAPl to other plant PAPs --- p.86 / Chapter 4.2.5 --- Sequence comparison of 5'UTR ofTPAPl and NtPAP12 --- p.89 / Chapter 4.3 --- APase Activity Assay --- p.90 / Chapter 4.3.1 --- p-NPP APase activity assay --- p.90 / Chapter 4.3.2 --- Activity-gel detection --- p.90 / Chapter 4.4 --- "Comparison of TPAP 1, IAP，SAP 1 and SAP2" --- p.96 / Chapter 4.5 --- Potential Cis-acting Regulatory Elements (CAREs) on the TPAP1 Promoter --- p.100 / Chapter 4.5.1 --- Search for potential CAREs --- p.100 / Chapter 4.5.2 --- Functions of CAREs --- p.100 / Chapter 4.6 --- Transient Expression Analysis --- p.102 / Chapter 4.6.1 --- Biolistic bombardment of TPAP1 promoter fragments into tomato roots --- p.102 / Chapter 4.7 --- Transgenic Expression Analysis --- p.104 / Chapter 4.7.1 --- Transformation of tobacco --- p.104 / Chapter 4.7.2 --- Northern and RT-PCR analysis of GUS expression --- p.110 / Chapter 4.7.3 --- GUS activity analysis --- p.114 / Chapter 4.7.4 --- Histochemical staining of GUS --- p.123 / Chapter Chapter 5: --- Discussions --- p.135 / Chapter 5.1 --- Properties ofTPAPl --- p.135 / Chapter 5.1.1 --- "Structure of the TPAP1 promoter, gene and cDNA" --- p.135 / Chapter 5.1.2 --- Potential flmction(s) ofTPAPl --- p.135 / Chapter 5.1.3 --- The potential relationship between TPAP1 and NtPAP12 --- p.137 / Chapter 5.2 --- Induction of Secretory APases during Pi Starvation --- p.137 / Chapter 5.3 --- Putative Protein Encode by theTPAP 1 cDNA --- p.138 / Chapter 5.4 --- Promoter Analysis of TPAP1 --- p.140 / Chapter 5.4.1 --- Construct preparation --- p.140 / Chapter 5.4.2 --- Potential CAREs located on the TPAP1 promoter --- p.141 / Chapter 5.4.3 --- Transient expression analysis --- p.142 / Chapter 5.4.4 --- Transgenic expression analysis --- p.143 / Chapter 5.4.4.1 --- Northern analysis and RT-PCR analysis of GUS expression --- p.143 / Chapter 5.4.4.2 --- GUS activity analysis --- p.143 / Chapter 5.4.4.3 --- Histochemical staining of GUS --- p.145 / Chapter 5.5 --- Hypothetical Model for TPAP1 Promoter Activities --- p.146 / Chapter 5.5.1 --- Model for expression level --- p.146 / Chapter 5.5.2 --- Models for spatial expressions --- p.148 / Chapter 5.6 --- Future Perspectives --- p.150 / Chapter Chapter 6: --- Conclusions --- p.152 / References --- p.154

O milho geneticamente modificado MON810, que expressa a proteína Cry1Ab de Bacillus thuringiensis Berliner, está em fase de avaliação e aprovação para liberação comercial no Brasil. Sendo assim, o objetivo da presente pesquisa foi o de estudar os efeitos de MON810 sobre a entomofauna em Barretos/SP e Ponta Grossa/PR no período de 1999 a 2001. O levantamento de insetos foi realizado por meio de diferentes armadilhas (alçapão, bandeja d'água, cartão adesivo e rede de varredura) e contagem de insetos nas plantas de milho, visando avaliar o efeito do milho MON810 sobre a comunidade de insetos, guildas tróficas e dinâmica populacional das espécies predominantes, incluindo organismos benéficos e pragas não-alvo. A interação tritrófica envolvendo milho MON810, Spodoptera frugiperda (J.E. Smith) e Doru luteipes (Scudder) também foi avaliada no presente trabalho. Adicionalmente, um estudo comparativo da comunidade geral de insetos nas diferentes safras de milho foi realizado com o uso de armadilha luminosa. Os tratamentos avaliados foram o milho geneticamente modificado MON810 (MON), milho convencional com aplicação de inseticidas (CCI) e milho convencional sem aplicação de inseticida (CSI). Foi coletado um total de 957.081 espécimes e 409 diferentes espécies. Baseado na análise faunística e índices de riqueza, diversidade, eqüitabilidade e similaridade, não foram observadas diferenças entre os tratamentos na comunidade de insetos. Estes resultados foram também confirmados com as análises de componentes principais e de Kruskal-Wallis. Não foram observadas diferenças significativas entre os tratamentos quanto à proporção relativa de diferentes guildas tróficas analisadas (predadores, parasitóides, polinizadores, decompositores, sugadores e mastigadores). Também não foi observado efeito do milho MON810 na dinâmica populacional das espécies predominantes de aranhas e insetos de diferentes guildas tróficas, incluindo pragas não-alvo e insetos benéficos (Carabidae, Coccinellidae, Chrysopidae, Hemerobiidae, Syrphidae, Tachinidae e Apidae). Avaliações de S. frugiperda e D. luteipes nas plantas de milho confirmaram a eficiência de MON810 no controle desta praga e a sua não interferência na dinâmica populacional do predador. E finalmente, diferenças significativas foram observadas na comunidade geral de insetos nas diferentes safras avaliadas. Portanto, nenhum efeito do milho MON810 foi detectado no presente estudo sobre a comunidade de insetos. / The genetically modified corn MON810, which expresses the Cry1Ab protein from Bacillus thuringiensis Berliner, is under evaluation and approval for commercial release in Brazil. Therefore, the objective of this research was to study the effect of MON810 on insect community in Barretos/SP and Ponta Grossa/PR from 1999 to 2001. The evaluations were based on insect sampling with the use of different traps (pitfall, color tray, sticky trap and sweep net) and insect counts on corn plants to evaluate the effect of MON810 on insect community, throphic guilds and population dynamics of predominant species, including beneficial organisms and non-target pests. Tritrophic interaction involving the corn MON810, Spodoptera frugiperda (J.E. Smith) and Doru luteipes (Scudder) was also evaluated in this study. Additionally, a comparative study of general insect community in different corn growing seasons was conducted with the use of a light trap. The following treatments were evaluated: genetically modified corn MON810 (MON), conventional corn with insecticide application (CWI) and conventional corn without insecticide application (CWI). A total of 957,081 specimens were collected, distributed among 409 different species. Based on faunistic analysis and indexes of richness, diversity, evenness and similarity, there were no differences in the insect community among treatments. These results were also confirmed by principal component and Kruskal-Wallis analysis. No statistical differences were found among treatments in the relative proportion of different trophic guilds evaluated (predators, parasitoids, pollinators, decomposers, suckers and chewers). There was also no effect of MON810 on population dynamics of predominant species of spiders and insects of different trophic guilds, including non-target pests and beneficial insects (Carabidae, Coccinellidae, Chrysopidae, Hemerobiidae, Syrphidae, Tachinidae and Apidae). Evaluations of S. frugiperda and D. luteipes on corn plants confirmed the efficacy of MON810 in the control of this pest and its no effect on the population dynamics of D. luteipes. And finally, significant differences were found in the general insect community in different corn growing seasons. Therefore, no effect of the corn MON810 was detected in this study on insect community.

Graduation date: 1994

Plant hormones are responsible for plant growth and adaptation to the environment. Among them the most important are cytokinins. Plants undergo gene silencing processes called homology-dependent gene silencing processes. In Arabidopsis there are two homology-dependent gene silencing genes that were chosen for further study, namely AtHOG1 and AtHOG2. Transgenic plants were generated previously with ten different constructs containing AtHOG1 or AtHOG2 genes and were used in this study. Some of the constructs had GFP attached so that the protein expressed could be visualised in a confocal microscope. Transgenic plants generated were T1 and T2 generations. Their DNA was extracted from leaves. By means of PCR transgenic plants were identified. There were 147 samples. Among them there were 39 positiveswith BAR primers and 32 positives with construct specific primers. The localisation of the HOG2 protein was observed in a confocal microscope. Seeds used were T3 generation and were obtained from the lab. HOG2 protein was found to be localised in cell membrane, root tip and chloroplasts.

As the third most important greenhouse gas, nitrous oxide (N2O) is a stable greenhouse gas and also plays a significant role in stratospheric ozone destruction. The primary anthropogenic source of N2O stems from the use of nitrogen in agriculture, with soils being the major contributors. Currently, the annual N2O emissions from this “soil–microbe-plant” system is more than 2.6 Tg (one Tg equals a million metric tons) of N2O-N globally. My doctoral studies aimed to explore innovative strategies for N2O mitigation, in the context of environmental microbiology’s potential contribution to alleviating global warming. The bacterial enzyme nitrous oxide reductase (N2OR), naturally found in some soils, is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N2O to N2. Therefore, to “scrub” or reduce N2O emissions, bacterial N2OR was heterologously expressed inside the leaves and roots of transgenic plants. Others had previously shown that the functional assembly of the catalytic centres (CuZ) of N2OR is lacking when only nosZ is expressed in other bacterial hosts. There, coexpression of nosZ with nosD, nosF and nosY was found to be necessary for production of the catalytically active holoenzyme. I have generated transgenic tobacco plants expressing the nosZ gene, as well as tobacco plants in which the other four nos genes were coexpressed. More than 100 transgenic tobacco lines, expressing nosZ and nosFLZDY under the control of rolD promoter and d35S promoter, have been analyzed by PCR, RT-PCR and Western blot. The activity of N2OR expressed in transgenic plants, analyzed with the methyl viologen-linked enzyme assay, showed detectable N2O reducing activity. The N2O-reducing patterns observed were similar to that of the positive control purified bacterial N2OR. The data indicated that expressing bacterial N2OR heterologously in plants, without the expression of the accessory Nos proteins, could convert N2O into inert N2. This suggests that atmospheric phytoremediation of N2O by plants harbouring N2OR could be invaluable in efforts to reduce emissions from crop production fields.

As the third most important greenhouse gas, nitrous oxide (N2O) is a stable greenhouse gas and also plays a significant role in stratospheric ozone destruction. The primary anthropogenic source of N2O stems from the use of nitrogen in agriculture, with soils being the major contributors. Currently, the annual N2O emissions from this “soil–microbe-plant” system is more than 2.6 Tg (one Tg equals a million metric tons) of N2O-N globally. My doctoral studies aimed to explore innovative strategies for N2O mitigation, in the context of environmental microbiology’s potential contribution to alleviating global warming. The bacterial enzyme nitrous oxide reductase (N2OR), naturally found in some soils, is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N2O to N2. Therefore, to “scrub” or reduce N2O emissions, bacterial N2OR was heterologously expressed inside the leaves and roots of transgenic plants. Others had previously shown that the functional assembly of the catalytic centres (CuZ) of N2OR is lacking when only nosZ is expressed in other bacterial hosts. There, coexpression of nosZ with nosD, nosF and nosY was found to be necessary for production of the catalytically active holoenzyme. I have generated transgenic tobacco plants expressing the nosZ gene, as well as tobacco plants in which the other four nos genes were coexpressed. More than 100 transgenic tobacco lines, expressing nosZ and nosFLZDY under the control of rolD promoter and d35S promoter, have been analyzed by PCR, RT-PCR and Western blot. The activity of N2OR expressed in transgenic plants, analyzed with the methyl viologen-linked enzyme assay, showed detectable N2O reducing activity. The N2O-reducing patterns observed were similar to that of the positive control purified bacterial N2OR. The data indicated that expressing bacterial N2OR heterologously in plants, without the expression of the accessory Nos proteins, could convert N2O into inert N2. This suggests that atmospheric phytoremediation of N2O by plants harbouring N2OR could be invaluable in efforts to reduce emissions from crop production fields.

Thesis (M. Sc.)--University of Stellenbosch, 2002. / Includes bibliographical references. Also available via the Internet.

Five cultivars, all belonging to the family Cucurbitaceae, have been tested for the ability to regenerate shoots or somatic embryos from cotyledonary explants. The influence of several combinations of growth regulators on regeneration from cotyledonary and other explants was tested. No regeneration was obtained from the two cultivars Cucurbita maxima Duch. cv A-Line and Cucurbitapepo L. cv Rolet. Somatic embryos developed on Cucurbita maxima Duch. cv Chicago Waited, a Hubbard squash. A shoot regeneration response was observed for the cultivar Cucumis sativus L. cv Ashley, but the frequency was low and results could not be repeated in subsequent experiments. A reliable shoot regeneration protocol was developed for Cucumis melo L. cv Hales Best 36. The influence of the antibiotics kanamycin sulphate and cefotaxime on shoot regeneration from cotyledonary explants of Cucumis melo L. cv Hales Best 36 was tested. The plasmid pBI121 was transferred from Escherichia coli strain HB101 into Agrobacterium tumefaciens strain LBA4404 via a triparental mating. The plasmid pBI121, contains the screenable marker gene β-glucuronidase (GUS) and the selectable neomycin phosphotransferase-II gene (NPT-II) that confers kanamycin resistance. Cotyledonary tissue was transformed using this Agrobacterium tumefaciens transformation system. The influence of co-cultivation time, inoculation time and the wound factor acetosyringone on transformation was established. Rooted plantlets were regenerated from transformed cotyledonary tissue placed on kanamycin supplemented regeneration media. Plantlets tested positive for the presence of the GUS gene, using fluorometric and histochemical assays. The developed protocol was used to transform Cucumis melo cv Hales best 36 with the pat gene that provides resistance to the herbicide Ignite®. A selection medium was developed containing phosphinothricin, the active ingredient of the herbicide Transformants were selected on this medium and five lines were recovered. These plants were acclimatized and the herbicide resistance was confirmed in greenhouse spray tests. The ploidy level of these plants was deduced from indirect evidence of micro- and macroscopic characteristics that have been shown to have a correlation with the chromosome number of melon plants. The five lines were subjected to molecular analysis. The polymerase chain reaction was used to give an indication of the transformed nature of the selected plants. Agarose gel electrophoresis confirmed that the correct size band could be obtained from the putative transformants and the presence of pat in the product was verified using a non-radioactive system for nucleic acid analysis. Stable gene insertion into the genome of the plant was verified with a Southern blot of the total genomic DNA. This was achieved by hybridising a radioactively labelled ³²P probe specific for the pat gene to a blot of restriction digested plant DNA. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.