by Cheng Man Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 71-76). / Abstracts in English and Chinese. / Thesis committee --- p.i / Abstract --- p.ii / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vii / List of figures --- p.x / List of tables --- p.xi / Chapter Chapter 1: --- General introduction --- p.1 / Chapter Chapter 2: --- Literature review --- p.3 / Chapter 2.1 --- Nutritional quality of plant proteins --- p.3 / Chapter 2.2 --- Using traditional plant breeding method to enhance amino acid quality of plant proteins --- p.3 / Chapter 2.3 --- Molecular strategies to enhance amino acid quality of plant proteins --- p.4 / Chapter 2.3.1 --- Heterologous gene expression --- p.5 / Chapter 2.3.2 --- Protein sequence modification --- p.8 / Chapter 2.3.3 --- Modification of biosynthesis pathway --- p.10 / Chapter 2.3.4 --- Synthetic gene expression --- p.11 / Chapter 2.3.5 --- Homologous gene overexpression --- p.13 / Chapter 2.4 --- Arabidopsis --- p.14 / Chapter 2.4.1 --- Arabidopsis as a model plant --- p.14 / Chapter 2.4.2 --- Transformation methods --- p.14 / Chapter 2.4.2.1 --- Direct DNA uptake --- p.15 / Chapter 2.4.2.2 --- Agrobacterium-mediated transformation --- p.15 / Chapter 2.5 --- Winged Bean Lysine-Rich protein --- p.17 / Chapter 2.5.1 --- Identification of winged bean polypeptides rich in lysine --- p.17 / Chapter 2.5.2 --- Cloning of the lysine-rich protein gene --- p.17 / Chapter 2.5.3 --- Further characterization of the WBLRP gene --- p.18 / Chapter 2.6 --- Phaseolin --- p.19 / Chapter Chapter 3: --- Expression of LRP in transgenic Arabidopsis --- p.20 / Chapter 3.1 --- Introduction --- p.20 / Chapter 3.2 --- Materials and methods --- p.21 / Chapter 3.2.1 --- Targeting LRP to cytosol --- p.21 / Chapter 3.2.1.1 --- Chemicals --- p.21 / Chapter 3.2.1.2 --- Plant materials --- p.21 / Chapter 3.2.1.3 --- Bacterial strains --- p.22 / Chapter 3.2.1.4 --- Construction of chimeric LRP gene (pBILRP-1) --- p.22 / Chapter 3.2.1.4.1 --- PCR amplification of LRP --- p.22 / Chapter 3.2.1.4.2 --- Cloning of PCR-amplified LRP into vector pD3-8 --- p.26 / Chapter 3.2.1.4.3 --- Cloning of recombinant plasmid pLRP-1 into binary vector --- p.26 / Chapter 3.2.1.5 --- Transformation of Agrobacterium with pBILRP-1 --- p.27 / Chapter 3.2.1.6 --- Vacuum infiltration transformation of Arabidopsis --- p.28 / Chapter 3.2.1.7 --- Selection of transgenic plants --- p.29 / Chapter 3.2.1.8 --- GUS assay --- p.30 / Chapter 3.2.1.9 --- DNA isolation --- p.31 / Chapter 3.2.1.10 --- PCR amplification and detection of transgenes --- p.31 / Chapter 3.2.1.11 --- Southern blot hybridization --- p.31 / Chapter 3.2.1.12 --- RNA isolation --- p.32 / Chapter 3.2.1.13 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.32 / Chapter 3.2.1.14 --- Protein extraction and SDS-PAGE --- p.33 / Chapter 3.2.1.15 --- Protein sequencing --- p.33 / Chapter 3.2.1.16 --- Amino acid analysis --- p.34 / Chapter 3.2.2 --- Targeting LRP to protein bodies --- p.35 / Chapter 3.2.2.1 --- Chemicals --- p.35 / Chapter 3.2.2.2 --- Plant materials --- p.35 / Chapter 3.2.2.3 --- Bacterial strains --- p.35 / Chapter 3.2.2.4 --- Construction of chimeric LRP gene (pBILRP-2) --- p.35 / Chapter 3.2.2.4.1 --- Site-directed mutagenesis --- p.36 / Chapter 3.2.2.4.2 --- Cloning of the mutated phaseolin fragment into pBluescript --- p.36 / Chapter 3.2.2.4.3 --- PCR amplification of LRP --- p.39 / Chapter 3.2.2.4.4 --- Insertion of LRP into plasmid pBK/phas* --- p.39 / Chapter 3.2.2.4.5 --- Insertion of plasmid pLRP-2 into Agrobacterium binary vector --- p.41 / Chapter 3.2.2.5 --- Transformation of Agrobacterium with pBILRP-2 --- p.41 / Chapter 3.2.2.6 --- Vacuum infiltration transformation of Arabidopsis --- p.41 / Chapter 3.2.2.7 --- Selection of transgenic plants --- p.41 / Chapter 3.3 --- Results and discussion --- p.42 / Chapter 3.3.1 --- Targeting LRP to protein bodies --- p.42 / Chapter 3.3.1.1 --- Morphology of transgenic Arabidopsis --- p.42 / Chapter 3.3.1.2 --- Selection of transgenic plants --- p.42 / Chapter 3.3.2 --- Targeting LRP to cytosol --- p.46 / Chapter 3.3.2.1 --- Morphology of transgenic Arabidopsis --- p.46 / Chapter 3.3.2.2 --- Selection of transgenic plants --- p.46 / Chapter 3.3.2.3 --- Detection of GUS activity --- p.49 / Chapter 3.3.2.4 --- Integration of LRP transgene into Arabidopsis genome --- p.54 / Chapter 3.3.2.5 --- LRP transcript in transgenic Arabidopsis --- p.58 / Chapter 3.3.2.6 --- Stable accumulation of LRP in transgenic Arabidopsis --- p.61 / Chapter 3.3.2.7 --- Amino acid analysis of seed protein --- p.64 / Chapter Chapter 4: --- General discussion --- p.67 / Conclusion --- p.70 / References --- p.71

Long, Xiaohang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 146-165). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Diaphorina citri é o inseto vetor das bactérias Candidatus Liberibacter spp., causadoras do huanglongbing (HLB), doença que tem causado sérios danos e grandes perdas econômicas à citricultura mundial, pois acomete todas as variedades comerciais de citros e não existem genótipos comerciais resistentes. Por esta razão, têm-se buscado estratégias que auxiliem no controle do inseto vetor, hoje feito quase que exclusivamente com o uso de excessivas aplicações de agroquímicos que aumentam o custo de produção e potencializam a agressão ao meio ambiente. Tem-se vislumbrado o desenvolvimento de plantas transgênicas que aumentem a resposta de defesa da planta contra Ca. Liberibacter spp. ou o vetor de forma mais efetiva, pelo direcionamento da expressão de genes alvo em tecidos específico, controlados por promotores de floema ou constitutivo. Nosso grupo tem buscado estratégias ainda não utilizadas em citros, mas efetivas para outras culturas, como o uso da bactéria entomopatogênica Bacillus thuringiensis (Bt) para o controle de D. citri. Essa bactéria se destaca devido à produção de proteínas com atividades inseticidas contra diversas fases imaturas de insetos, por isso tem sido amplamente utilizada tanto na produção de bioinseticidas quanto, principalmente, na produção de plantas transgênicas em culturas como algodão, milho e soja. Em trabalhos anteriores, identificamos que o gene cry11 codifica uma toxina capaz de causar elevada mortalidade em ninfas do psilídeo dos citros 120 horas após sua inoculação em seedlings de citros. Este gene foi clonado para a expressão em tecido de plantas de laranja doce sob o direcionamento de dois promotores, um específico de floema e o outro constitutivo. Foram obtidas 19 plantas transgênicas, com a inserção de uma a três cópias do transgene em seus genomas. Algumas delas apresentaram elevados níveis de expressão do transcrito. Todas essas plantas foram propagadas via enxertia em limoeiro Cravo e estão praticamente prontas para serem desafiadas com ninfas de D. citri. Esse é o primeiro esforço para a produção de citrus Bt visando ao manejo do HLB dos citros. / Diaphorina citri is the insect vector of Candidatus Liberibacter spp., the causal agents of huanglongbing (HLB), the most devastating disease of citrus worldwide, due to the severe losses it induces and the lack of commercial resistant genotypes. For this reason, strategies have been sought to assist in the control of the insect vector, nowadays done almost exclusively with the use of excessive applications of agrochemicals that increase the cost of production and potentiate the aggression to the environment. It has been envisioned the development of transgenic plants that increase the defense response of the plant against Ca. Liberibacter spp. or the vector more effectively, by targeting the expression of target genes in specific tissues, controlled by phloem or constitutive promoters. Our group has sought strategies not yet used in citrus, but effective for other crops, such as the use of the entomopathogenic bacterium Bacillus thuringiensis (Bt) for the control of D. citri. This bacterium stands out due to the production of proteins with insecticidal activity against several immature phases of insects, so it has been widely used both in the production of bioinsecticides and mainly in the production of transgenic plants in crops such as cotton, corn and soybean. In previous work, we have identified that the cry11 gene encodes a toxin capable of causing high mortality of citrus psyllid nymphs 120 hours after inoculation in citrus seedlings. This gene was cloned for expression in sweet orange plant tissue under the guidance of two promoters, one phloem specific and the other constitutive. Nineteen transgenic plants were obtained, with the insertion of one to three copies of the transgene into their genomes. Some of them had high levels of transcript expression. All these plants were propagated via grafting in Clove lemon tree and are practically ready to be challenged with D. citri nymphs. This is the first effort to produce citrus Bt for the management of citrus HLB.

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

by Tse Yu Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 133-138). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The Plant secretory pathway --- p.2 / An overview on the secretory pathway --- p.2 / Vesicular pathways and transport vesicles --- p.4 / Chapter 2. --- Vacuolar sorting receptors --- p.6 / BP-80 and its homologues --- p.6 / RMR proteins --- p.7 / Chapter 3. --- Prevacuolar compartments --- p.8 / PVCs in mammalian and yeast cells --- p.8 / PVCs for seed protein storage vacuoles --- p.9 / PVCs for lytic vacuoles --- p.11 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Markers for Golgi and Prevacuolar Compartments --- p.15 / Chapter 1. --- Introduction --- p.16 / Chapter 1.1 --- Fluorescent proteins are useful tools in studying protein trafficking and subcellular localization in living cells --- p.16 / Chapter 1.2 --- Tobacco BY-2 cells --- p.18 / Chapter 1.3 --- Plant prevacuolar compartments --- p.19 / Chapter 2. --- Materials and Methods --- p.21 / Chapter 2.1 --- Construction of RFP-BP-80 and RFP-α-TIP reporters --- p.21 / Chapter 2.2 --- Construction of YFP-BP-80 and YFP-α-TIP reporters --- p.27 / Chapter 2.3 --- Construction of YFP markers for Golgi organelles --- p.32 / Chapter 2.4 --- Agrobacterium electroporation --- p.33 / Chapter 2.5 --- Transformation of tobacco BY-2 cells --- p.34 / Chapter 2.6 --- Screening of transgenic BY-2 cells expressing RFP markers --- p.35 / Chapter 2.8 --- Production of anti-BP-80 CT antibody --- p.43 / Chapter 2.9 --- Chemicals --- p.45 / Chapter 2.10 --- Primers --- p.45 / Chapter 2.11 --- Bacterial strain --- p.46 / Chapter 3. --- Results --- p.47 / Chapter 3.1 --- Generation and characterization of transgenic BY-2 cell lines expressing RFP reporters --- p.47 / Chapter 3.2 --- Generation and preliminary characterization of transgenic BY-2 cell lines expressing YFP reporters --- p.55 / Chapter 3.3 --- Confocal detection ofYFP reporters in transgenic cell lines --- p.64 / Chapter 3.4 --- Characterization of anti-BP-80 CT antibody --- p.66 / Chapter 4. --- Discussion --- p.68 / Chapter Chapter 3 --- Dynamic of Plant Prevacuolar Compartments in Transgenic Tobacco BY-2 Cells --- p.72 / Chapter 1. --- Introduction --- p.73 / Chapter 1.1 --- The plant secretory pathway --- p.73 / Chapter 1.2 --- Organelle markers in plant secretory pathway --- p.74 / Chapter 1.3 --- Markers for Lytic PVCs --- p.75 / Chapter 2. --- Materials and Methods --- p.77 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.77 / Chapter 2.2 --- FM4-64 uptake study --- p.79 / Chapter 2.3 --- Brefeldin A treatment --- p.79 / Chapter 2.4 --- Wortmannin treatment --- p.80 / Chapter 2.5 --- Movement study of YFP-marked PVC --- p.82 / Chapter 3. --- Results --- p.83 / Chapter 3.1 --- Different internal organelles were labeled by two different YFP reporters --- p.83 / Chapter 3.2 --- The YFP-BP-80 reporter localized with endogenous VSR proteins --- p.86 / Chapter 3.3 --- Brefeldin A enlarged PVC organelles --- p.89 / Chapter 3.4 --- Identity of PVC-derived BFA-induced compartments --- p.99 / Chapter 3.5 --- Wortmannin induced PVCs to form small vacuoles --- p.102 / Chapter 3.6 --- PVCs are mobile organelles in living cells --- p.112 / Chapter 4. --- Discussion --- p.114 / Chapter Chapter 4 --- Summary and Future Perspectives --- p.123 / Chapter 1. --- Summary --- p.124 / The hypothesis --- p.124 / Development of three transgenic cell lines --- p.125 / Distinct organelles were marked by two different YFP reporters --- p.126 / The YFP-BP-80 reporter defined the lytic PVCs --- p.126 / Response of YFP-marked PVCs to Brefeldin A treatment --- p.127 / Response of YFP-marked PVCs to Wortmannin treatment --- p.127 / PVCs are mobile organelles in living cells --- p.129 / Chapter 2. --- Future perspectives --- p.130 / References --- p.133

by Yuan Dingyang. / "September 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 206-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Chen Li. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 136-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Cell cycle progression is the series of steps a cell has to take in order to duplicate its DNA and produce two daughter cells. Correct spatial and temporal coordination of the cell cycle is key for the normal development of any organ or tissue and is stringently controlled during embryogenesis and homeostasis. Misregulation of cell cycle progression is causal in many developmental disorders and diseases such as microcephaly and cancer. Fucci (Fluorescent Ubiquitination based Cell Cycle Indicator) is a system that allows for the visualisation of cell cycle progression by the use of two differently coloured fluorescent probes whose abundance is regulated reciprocally during the cell cycle. The probes contain the E3 ligase recognition domains of Cdt1 and Geminin fused to the fluorophores mCherry (red fluorescence) and mVenus (yellow fluorescence) respectively. Cells are therefore labelled red during G1, yellow in the G1/S transition and green during late S/G2 and M phases of the cell cycle. In order to study development and tissue homoeostasis a Fucci expressing mouse line was developed however this has several key limitations: First, the two Fucci probes are expressed from separate loci complicating mouse colony maintenance. Second, the constructs were not inducible, making it impossible to follow cell cycle progression in specific cell lineages and third the mice were generated by random transgenesis which is prone to silencing and can exhibit variation in expression between different tissues. Here I have characterised an improved version of the original Fucci system known as Fucci2a designed by Dr Richard Mort (University of Edinburgh) to overcome these limitations. The Fucci2a genetic construct contains both Fucci probes fused with the Thosea asigna virus self-cleaving peptide sequence T2A. This allows expression of both probes as a single bicistronic mRNA with subsequent cleavage by ribosomal ‘skipping’ during translation to yield separate proteins. A Fucci2a mouse (R26Fucc2aR) was generated by homologous recombination into the ROSA26 locus using the strong, ubiquitous CAG promoter to drive expression and incorporating a floxed-Neo stop cassette. This allows tissue specific activation by Cre recombinase when combined with a second Cre expressing mouse line. Building on the bicistronic Fucci2a technology I have gone on to develop and characterise four new tricistronic reporter constructs which allow for the dual visualisation of cell cycle progression with apoptosis, cytokinesis and ciliogenesis. In each case an additional fluorescent probe was added to the original Fucci2a construct separated by the self-cleaving peptide P2A and the construct characterised in 3T3 stable cell lines. The combination of a dual cilia and cell cycle reporter construct proved fruitful and I have gone on to investigate the relationship between cell cycle progression and ciliogenesis in 3T3 cells and have generated and characterised the R26Arl13b-Fucci2aR mouse line. I have also illustrated the utility of the R26Fucci2aR mouse for generating quantitative data in development research in two development situations; melanocyte development and lung branching morphogenesis. Melanocytes are specialised melanin producing cells responsible for the pigmentation of the hair, skin and eyes. Their precursors, melanoblasts, are derived from the neural crest where they migrate and proliferate before becoming localised to hair follicles and their study provides a good model for understanding the development of other neural crest derived lineages such as the peripheral nervous system. Using time-lapse imaging of ex vivo skin cultures in which melanoblasts are labelled with the Fucci probes I have characterised melanoblast migration and proliferation. In addition, I have shown that Kit signalling, which is necessary for melanoblast migration and survival, controls melanoblast proliferation in a density dependent manner and that melanoblast migration is more persistent in S/G2/M phases of the cell cycle. Lung branching morphogenesis requires constant proliferation at the apical tip of a growing epithelial branch. Loss of epithelial symmetry through an unidentified mechanism (requiring BMP, FgF10, Shh and Wnt signalling) within a branch is required to initiate branching either latterly from the side of a elongating branch by domain branching or by bifurcation of the tip. In the final section of this thesis I performed a comparative analysis of the behaviour of the developing lung epithelium using proliferative status (Fucci2a expression) to categorise each cell. Using a combination of live imaging and immunohistochemistry I have identified a transition zone 100-150μm from the tip of the branching lung epithelium where epithelial cells become stationary and drop out of the cell cycle corresponding with the onset of proximal bronchial progenitor marker Sox2. A comparative gene expression analysis of the proliferating and non-proliferating regions using Fucci2a to distinguish them has eluded to several interesting genes which could influence branching morphogenesis during lung development.

Wong Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 152-169). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.v / Abbreviations --- p.vi / List of Figures --- p.vii / List of Tables --- p.ix / Table of Contents --- p.xi / Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- GS-GOGAT cycle in plants and bacteria --- p.2 / Chapter 1.2 --- Roles of PII in regulation of glutamine synthetase in E. coli --- p.4 / Chapter 1.2.1 --- Regulation of GS in E. col --- p.4 / Chapter 1.2.2 --- Transcriptional regulation --- p.5 / Chapter 1.2.2.1 --- The glnALG operon / Chapter 1.2.2.2 --- Intracellular signal through PII and Utase-UR / Chapter 1.2.2.3 --- NRI/NRII as two-component system / Chapter 1.2.3 --- Post-translational regulation by adenylylation and deadenylylation --- p.11 / Chapter 1.2.3.1 --- Role of PII in adenylylation/deadenylylation / Chapter 1.2.4 --- Cumulative Feedback Inhibition --- p.15 / Chapter 1.3 --- PII in other bacteria --- p.15 / Chapter 1.4 --- PII in other higher organisms --- p.20 / Chapter 1.5 --- "PII protein is conserved in enteric bacteria, cyanobacteria, archaea, algae and higher plants" --- p.23 / Chapter 1.6 --- Nitrogen assimilation in higher plants --- p.25 / Chapter 1.6.1 --- Nitrogen uptake --- p.25 / Chapter 1.6.2 --- Primary nitrogen assimilation --- p.28 / Chapter 1.6.3 --- Nitrogen transport and interconversions --- p.28 / Chapter 1.6.4 --- Nitrogen flow --- p.29 / Chapter 1.6.5 --- Molecular regulation of nitrogen assimilation and possible roles of PII in plants --- p.30 / Chapter 1.7 --- Hypothesis of this study --- p.33 / Chapter 2. --- Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Plant materials --- p.35 / Chapter 2.1.2 --- Equipment and facilities used --- p.35 / Chapter 2.1.3 --- Growth media --- p.37 / Chapter 2.1.4 --- Buffers and solutions used in RNA extraction --- p.38 / Chapter 2.1.5 --- Buffers and solutions used in Northern blot analysis --- p.41 / Chapter 2.1.6 --- Molecular reagents and synthetic oligonucleotides used in the preparation of DIG-labeled probes --- p.45 / Chapter 2.1.7 --- Chemicals used in BioRad Protein Assay --- p.48 / Chapter 2.1.8 --- Chemicals and apparatus used in chlorophylls extraction and quantitation --- p.49 / Chapter 2.1.9 --- Buffers and solutions used in the glutamine synthetase enzyme extraction and assay --- p.49 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Plant growth --- p.50 / Chapter 2.2.2 --- RNA extraction --- p.52 / Chapter 2.2.3 --- Northern blot analysis --- p.54 / Chapter 2.2.4 --- Chlorophyll extraction and quantitation --- p.61 / Chapter 2.2.5 --- Root length measurement --- p.61 / Chapter 2.2.6 --- Total glutamine synthetase enzyme assay --- p.61 / Chapter 2.2.7 --- Measurement of total nitrogen in seeds --- p.64 / Chapter 2.2.8 --- Recording growth and development --- p.64 / Chapter 3. --- Results --- p.65 / Chapter 3.1 --- Overexpression ofPII and truncated PII mRNA in transgenic plants --- p.65 / Chapter 3.2 --- General growth characteristics of PII transgenic plants when grown on soil --- p.70 / Chapter 3.3 --- Physiological changes in the PII and truncated PII transgenic lines --- p.72 / Chapter 3.3.1 --- Fresh weight of the young seedlings --- p.73 / Chapter 3.3.2 --- Chlorophyll contents of shoots --- p.75 / Chapter 3.3.3 --- Root lengths --- p.88 / Chapter 3.3.4 --- Carbon and nitrogen status of seeds --- p.94 / Chapter 3.4 --- Expression of nitrogen assimilatory genes in PII and truncated PII transgenic lines --- p.96 / Chapter 3.4.1 --- Nitrate reductases --- p.96 / Chapter 3.4.2 --- Glutamine synthetases --- p.99 / Chapter 3.4.3 --- Asparagine synthetases --- p.107 / Chapter 3.5 --- Total glutamine synthetase enzyme activity --- p.117 / Chapter 4. --- Discussion --- p.126 / Chapter 4.1 --- Overexpressing PII and truncated PII in the transgenic plants --- p.126 / Chapter 4.2 --- The overall growth and development --- p.135 / Chapter 4.3 --- Chlorophyll --- p.135 / Chapter 4.4 --- Root length --- p.137 / Chapter 4.5 --- Expression of nitrogen assimilatory genes --- p.138 / Chapter 4.5.1 --- Genes encoding nitrate reductase --- p.138 / Chapter 4.5.2 --- Genes encoding glutamine synthetase --- p.140 / Chapter 4.5.3 --- Genes encoding asparagine synthetase --- p.141 / Chapter 4.6 --- Overall GS enzyme levels in the rosette leaves --- p.144 / Chapter 4.7 --- N/C ratio of the seed storage --- p.146 / Chapter 4.8 --- Proposed model for the roles of PII --- p.147 / Chapter 4.9 --- Conclusions --- p.149 / Chapter 4.10 --- Further studies --- p.150 / References --- p.152

Atualmente o Brasil ocupa lugar de destaque entre os produtores de citros sendo o maior produtor de laranja doce do mundo. Entretanto, essa produção vem sendo gravemente afetada, por doenças como o huanglongbing (HLB), que tem causado perdas significativas para toda cadeia citrícola. O HLB está associado a bactérias Gram-negativas, restritas ao floema das plantas, denominadas Candidatus Liberibacter spp., que além de reduzir a produção de frutos, podem, em casos mais severos da doença, ocasionar a morte da planta. Uma importante estratégia para controle desta doença é a produção de plantas transgênicas expressando genes, especificamente no local de colonização do patógeno. O objetivo deste trabalho foi a obtenção de plantas transgênicas de Citrus sinensis cvs. \'Pera\' e \'Natal\', contendo o gene atacina A (attA) que codifica um peptídeo antibacteriano, dirigido por promotores preferencialmente expressos no floema: AtSuc2 (transportador de sacarose) ou AtPP2 (proteína de floema 2), clonados de Arabidopsis thaliana, ou CsPP2 (proteína de floema 2) clonado de Citrus sinensis. A identificação de 65 plantas transgênicas foi realizada, por meio da análise de PCR. Foi verificado um número menor de eventos transgênicos, utilizando-se a construção gênica pCAtSuc2/attA em relação ao número de eventos transgênicos obtidos com as construções gênicas pCCsPP2/attA e pCAtPP2/attA. Plantas identificadas como transgênicas pela análise de PCR, foram aclimatizadas e transferidas para casa-devegetação certificada para o cultivo de plantas transgênicas. Análises de Southern blot foram realizadas em plantas aclimatizadas que apresentaram desenvolvimento suficiente, confirmando-se a integração do gene attA. A expressão do gene attA foi confirmada pela análise de RT-qPCR. As plantas transgênicas obtidas neste trabalho, contendo o gene attA dirigido por promotores preferencialmente expressos no floema, serão avaliadas em uma etapa futura para resistência a Candidatus Liberibacter spp. / Currently, Brazil is a major citrus producer and the world\'s largest producer of sweet oranges. However, diseases, such as huanglongbing (HLB) have seriously affected this production, causing significant losses in citrus production chain. HLB is associated with Gram-negative bacterias, restricted to the phloem of plants, called Candidatus Liberibacter spp., which besides reducing fruit production, can lead to plant death. An important strategy to control this disease is the production of transgenic plants expressing genes, specifically at the region of pathogen colonization. The aim of this study was to obtain transgenic plants of Citrus sinensis cv. \'Natal\' and \'Pera\', containing the gene attacin A (attA) that encodes an antibacterial peptide, driven by preferentially expressed in phloem promoters: AtSuc2 (sucrose transporter) or AtPP2 (phloem protein 2), cloned from Arabidopsis thaliana, or CsPP2 (phloem protein 2) cloned from Citrus sinensis. The identification of 65 transgenic plants was performed by PCR analysis. A lower number of transgenic events were verified using the gene construct pCAtSuc2/attA in relation events obtained with the gene constructs pCCsPP2/attA and pCAtPP2/attA. Plants identified as transgenic by PCR analysis were acclimatized and transferred to a greenhouse certified for growing transgenic plants. The Southern blot analyses were performed in acclimatized plants, which had sufficiently developed, confirming the integration of attA gene. The expression of attA gene was confirmed by RT-qPCR analysis. The transgenic plants obtained in this work, containing the gene attA directed by preferentially expressed in phloem promoters, will be further evaluated for resistance to Candidatus Liberibacter spp.