ABSTRACT Cassava is currently the second most important source of carbohydrates on the African continent. In the last two decades, cassava crops have been severely affected by outbreaks of cassava mosaic disease (CMD). South African cassava mosaic virus (SACMV) has been associated with CMD outbreaks in the Mpumalanga province. Advances in post-transcriptional gene silencing (PTGS) technology have provided promising new strategies for the engineering of virus resistance in plants. Inverted repeat (IR) constructs are currently the most potent inducers of PTGS, however, these constructs are inherently unstable. The purpose of this study was to develop IR constructs with an improved stability for the efficient induction of PTGS in plants. Two mismatched inverted repeat constructs, one targeting the SACMV BC1 open reading frame, the other targeting the Maize streak virus (MSV) AC1 open reading frame, were successfully created. Sodium bisulfite was used to deaminate cytosine residues on the sense arm of the constructs. The resulting number of GT mismatches was seemingly sufficient to stabilize the linear conformation of the IR constructs, as they were efficiently propagated by E.coli DH5!, and subsequently behaved like linear DNA molecules. Furthermore, it was found that the number of mismatches on the BC1 construct (17.5%) was ideal, as the subsequent stability of the predicted RNA hairpin was not affected. Due to the higher number of mismatches on the AC1 construct (23.5%), it was found that the loop region of the RNA hairpin was marginally destabilized. Despite this, long stretches of stable dsRNA were still produced from the AC1 IR construct, and is likely to induce PTGS. Interestingly, it was observed that the mismatched IR constructs, although still replicated in E.coli, were marginally destabilized in Agrobacterium. Therefore, it was deduced that the stability of a mismatched IR construct may be influenced by the particular intracellular environment of an organism. Due to the recalcitrance of cassava to transformation, a model plant system, Nicotiana benthamiana, was used to screen constructs for toxicity, stability, and efficiency of PTGS induction. Agrobacteriummediated transformation and regeneration of N. benthamiana was optimized, and 86% transformation efficiency was achieved when using leaf disk explants. It was found that the addition of an ethylene scrubber, potassium permanganate, substantially increased the rate of regeneration by reducing the frequency of hyperhydritic plants. Transgene iv integration was confirmed by PCR amplification of the hptII gene in the T-DNA region. Transgene expression was confirmed by screening for GUS and GFP reporter genes. No toxic responses to the transgene have been observed thus far. Studies are currently underway to confirm the stability of the mismatched IR constructs in N. benthamiana. PAGE Northern blotting is being done, as the detection of siRNAs derived from the transgene will confirm that constructs are functional. In addition, infectivity assays are underway to determine the efficacy of BC1 knockdown by a stably integrated construct. Due to the enhanced stability of mismatched IR constructs, they may be an appealing alternative to currently available intron-spliced, or exact matched hairpin systems.

Phang Tsui-Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 146-162). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vi / Abbreviations --- p.viii / Table of contents --- p.xii / List of figures --- p.xviii / List of tables --- p.xx / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Salinity as a global problem --- p.1 / Chapter 1.2 --- Formation of saline soil --- p.1 / Chapter 1.3 --- Urgent need to reclaim saline lands --- p.2 / Chapter 1.4 --- Cellular routes for Na+ uptake --- p.2 / Chapter 1.4.1 --- Carriers involved in K+ and Na+ uptake --- p.2 / Chapter 1.4.2 --- Channels involved in K+ and Na+ uptake --- p.4 / Chapter 1.5 --- Adverse effects of high salinity --- p.5 / Chapter 1.5.1 --- Hyperosmotic stress --- p.5 / Chapter 1.5.2 --- Ionic stress --- p.6 / Chapter 1.5.2.1 --- Deficiency of K+ --- p.6 / Chapter 1.5.2.2 --- Perturbation of calcium balance --- p.7 / Chapter 1.5.3 --- Toxicity of specific ions --- p.7 / Chapter 1.5.4 --- Oxidative stress --- p.10 / Chapter 1.6 --- Mechanisms of salt stress adaptation in plants --- p.11 / Chapter 1.6.1. --- Maintenance of ion homeostasis --- p.12 / Chapter 1.6.1.1 --- Regulation of cytosolic Na+ concentration --- p.12 / Chapter 1.6.1.2 --- SOS signal transduction pathway --- p.15 / Chapter 1.6.2 --- Dehydration stress adaptation --- p.17 / Chapter 1.6.2.1 --- Aquaporins ´ؤ water channel proteins --- p.17 / Chapter 1.6.2.2 --- Osmotic adjustment --- p.20 / Chapter 1.6.2.2.1 --- Genetic engineering of glycinebetaine biosynthesis --- p.23 / Chapter 1.6.2.2.2 --- Genetic engineering of mannitol biosynthesis --- p.27 / Chapter 1.6.3 --- Morphological and structural adaptation --- p.28 / Chapter 1.6.4 --- Restoration of oxidative balance --- p.29 / Chapter 1.6.5 --- Other metabolic adaptation --- p.31 / Chapter 1.6.5.1 --- Induction of Crassulacean acid (CAM) metabolism --- p.34 / Chapter 1.6.5.2 --- Coenzyme A biosynthesis --- p.34 / Chapter 1.7 --- Soybean as a target for studying salt tolerance --- p.36 / Chapter 1.7.1 --- Economical importance of soybean --- p.36 / Chapter 1.7.2 --- Reasons for studying salt stress physiology in soybeans --- p.38 / Chapter 1.7.3 --- Salt tolerant soybean in China --- p.39 / Chapter 1.7.4 --- Exploring salt tolerant crops by genetic engineering --- p.41 / Chapter 1.8 --- Significance of this project --- p.47 / Chapter 2. --- Materials and methods --- p.48 / Chapter 2.1 --- Materials --- p.48 / Chapter 2.1.1 --- Plant materials used --- p.48 / Chapter 2.1.2 --- Bacteria strains and plasmid vectors --- p.48 / Chapter 2.1.3 --- Growth media for soybean --- p.48 / Chapter 2.1.4 --- Equipment and facilities used --- p.48 / Chapter 2.1.5 --- Primers used --- p.48 / Chapter 2.1.6 --- Chemicals and reagents used --- p.49 / Chapter 2.1.7 --- Solutions used --- p.49 / Chapter 2.1.8 --- Commercial kits used --- p.49 / Chapter 2.1.9 --- Growth and treatment condition --- p.49 / Chapter 2.1.9.1 --- Characterization of salt tolerance of Wenfeng7 --- p.49 / Chapter 2.1.9.2 --- Samples for subtractive library preparations --- p.50 / Chapter 2.1.9.3 --- Samples for slot blot and northern blot analyses --- p.50 / Chapter 2.1.9.4 --- Samples for gene expression pattern analysis --- p.50 / Chapter 2.2. --- Methods --- p.52 / Chapter 2.2.1 --- Total RNA extraction --- p.52 / Chapter 2.2.2 --- Construction of subtractive libraries --- p.53 / Chapter 2.2.3 --- Cloning of salt-stress inducible genes --- p.53 / Chapter 2.2.3.1 --- Preparation of pBluescript II KS(+) T-vector for cloning --- p.53 / Chapter 2.2.3.2 --- Ligation of candidate DNA fragments with T-vector --- p.53 / Chapter 2.2.3.3 --- Transformation --- p.54 / Chapter 2.2.3.4 --- PCR screening --- p.54 / Chapter 2.2.4 --- Preparation of recombinant plasmid for sequencing --- p.55 / Chapter 2.2.5 --- Sequencing of differentially expressed genes --- p.55 / Chapter 2.2.6 --- Homology search of differentially expressed genes --- p.56 / Chapter 2.2.7 --- Expression pattern analysis --- p.56 / Chapter 2.2.7.1 --- Preparation of single-stranded DIG-labeled PCR probes --- p.56 / Chapter 2.2.7.2 --- Preparation of cRNA DIG-labeled probes --- p.57 / Chapter 2.2.7.3 --- Testing the concentration of DIG-labeled probes --- p.58 / Chapter 2.2.7.4 --- Slot blot --- p.58 / Chapter 2.2.7.5 --- Northern blot --- p.59 / Chapter 2.2.7.6 --- Hybridization --- p.60 / Chapter 2.2.7.7 --- Stringency washed --- p.60 / Chapter 2.2.7.8 --- Chemiluminescent detection --- p.60 / Chapter 3. --- Results --- p.61 / Chapter 3.1 --- Characterization of salt tolerance of Wenfeng7 --- p.61 / Chapter 3.2 --- Identification of salt-stress induced genes from Wenfeng7 --- p.70 / Chapter 3.2.1 --- Screening subtractive libraries of Wenfeng 7 for salt inducible genes --- p.70 / Chapter 3.2.1.1 --- Results of homology search for salt inducible genes --- p.71 / Chapter 3.2.1.2 --- Northern blot showing the salt inducibility of selected clones --- p.72 / Chapter 3.3 --- Genes expression pattern of selected salt inducible genes --- p.104 / Chapter 3.3.1 --- Expression of genes related to dehydration adjustment --- p.104 / Chapter 3.3.1.1 --- Dehydration responsive protein RD22 (Clone no.: HML806) --- p.104 / Chapter 3.3.1.2 --- Beta-amylase (Clone no.: HML767) --- p.104 / Chapter 3.3.2 --- Expression of genes related to structural modification --- p.105 / Chapter 3.3.3 --- Expression of genes related to metabolic adaptation --- p.105 / Chapter 3.3.3.1 --- Subgroup 1: Gene related to protein synthesis --- p.105 / Chapter 3.3.3.1.1 --- Translational initiation factor nsp45 (Clone no.: HML1042) --- p.105 / Chapter 3.3.3.1.2 --- Seed maturation protein PM37 (Clone no.: HML931) --- p.106 / Chapter 3.3.3.2 --- Subgroup 2: Genes related to phosphate metabolism (Clone no.: HML1000) --- p.107 / Chapter 3.3.3.3 --- Subgroup 3: Genes related to storage and mobilization of nutrient resources --- p.107 / Chapter 3.3.3.3.1 --- Vegetative storage protein A (Clone no.: HML762) --- p.107 / Chapter 3.3.3.3.2 --- Cysteine proteinase (Clone no.: HML928) --- p.107 / Chapter 3.3.3.4 --- Subgroup 4: Genes related to senescence --- p.109 / Chapter 3.3.4 --- Expression of genes encoding novel protein (Clone no.: HML782) --- p.109 / Chapter 4. --- Discussion --- p.125 / Chapter 4.1 --- Evaluation of salt tolerance of Wenfeng7 --- p.125 / Chapter 4.2 --- Isolation of salt inducible genes in Wenfeng7 --- p.127 / Chapter 4.2.1 --- Genes associated with dehydration adaptation --- p.129 / Chapter 4.2.1.1 --- Dehydration responsive protein RD22 --- p.129 / Chapter 4.2.1.2 --- Beta-amylase --- p.130 / Chapter 4.2.2 --- Genes associated with structural adaptation --- p.132 / Chapter 4.2.3 --- Genes associated with metabolic adaptation --- p.133 / Chapter 4.2.3.1 --- Subgroup 1: Genes related to protein synthesis --- p.133 / Chapter 4.2.3.2 --- Subgroup 2: Genes related to phosphate metabolism --- p.137 / Chapter 4.2.3.3 --- Subgroup 3: Genes related to storage and mobilization of nutrient resources --- p.138 / Chapter 4.2.3.4 --- Subgroup 4: Genes related to senescence --- p.140 / Chapter 4.2.4 --- Novel genes --- p.142 / Chapter 4.3 --- Brief summary --- p.142 / Chapter 5. --- Conclusion and perspectives --- p.144 / References --- p.146 / Appendix I: Screening for salt tolerant soybeans --- p.163 / Appendix II: Major equipment and facilities used --- p.165 / Appendix III: Major chemicals and reagents used in this research --- p.166 / Appendix IV: Major common solutions used in this research --- p.168 / Appendix V: Commercial kits used in this research --- p.170

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.

The essence of metabolic engineering is the modification of microbes for the overproduction of useful compounds. These cellular factories are increasingly recognized as an environmentally-friendly and cost-effective way to convert inexpensive and renewable feedstocks into products, compared to traditional chemical synthesis from petrochemicals. The products span the spectrum of specialty, fine or bulk chemicals, with uses such as pharmaceuticals, nutraceuticals, flavors and fragrances, agrochemicals, biofuels and building blocks for other compounds. However, the process of metabolic engineering can be long and expensive, primarily due to technological hurdles, our incomplete understanding of biology, as well as redundancies and limitations built into the natural program of living cells. Combinatorial or directed evolution approaches can enable us to make progress even without a full understanding of the cell, and can also lead to the discovery of new knowledge. This thesis is focused on addressing the technological bottlenecks in the directed evolution cycle, specifically de novo DNA assembly to generate strain libraries and small molecule product screens and selections.

Mechanistic understanding of gene regulation is crucial for rational engineering of new genetic systems through synthetic biology. Genetic engineering efforts in new organisms are often hampered by a lack of knowledge about how regulatory components function in new host contexts. This dissertation focuses on efforts to overcome these challenges through the development of generalizable experimental methods for studying the behavior of DNA regulatory sequences in diverse species at large-scale. Chapter 2 describes experimental approaches for quantitatively assessing the functions of thousands of diverse natural regulatory sequences through a combination of metagenomic mining, high-throughput DNA synthesis and deep sequencing. By employing these methods in three distinct bacterial species, we revealed striking functional differences in gene regulatory capacity. We identified regulatory sequences with activity levels with activity levels spanning several orders of magnitude, which will aid in efforts to engineer diverse bacterial species. We also demonstrate functional species-selective gene circuits with programmable host behaviors that may be useful for microbial community engineering. In Chapter 3 we provide evidence for the evolution of altered stringency in σ70-mediated transcriptional activation based on patterns of initiation and activity from promoters of diverse compositions. We show that the contrast in GC content between a regulatory element and the host genome dictates both the likelihood and the magnitude of expression. We also discuss the potential implications of this proposed mechanism on horizontal gene transfer. The next two chapters focus on efforts aimed at extending the high-throughput methods described in earlier chapters to new organisms. Chapter 4 presents an in vitro approach for multiplexed gene expression profiling. Through the development and use of cell-free expression systems made from diverse bacteria, it was possible to rapidly acquire thousands of transcriptional measurements in small volume reactions, enabling functional comparisons of regulatory sequence function across multiple species. In Chapter 5 we characterize the restriction-modification system repertoires of several commensal bacterial species. We also describe ongoing efforts to develop methods for bypassing these systems in order to increase transformation efficiencies in species that are difficult or impossible to transform using current approaches.

Nanomedicine has emerged in the past decades, and a variety of designs for drug/gene delivery have been reported since the concept of nanomedicine was first demonstrated. However, with the exception of a few notable successes, the clinical translation of nanomedicine has been slow. Specificity and delivery efficiency are the major obstacles; only a few nanomedicine systems can effectively reach and release the therapeutic payload at the target site, thereby limiting the therapeutic efficacy. To tackle these issues, this work aims to design new strategies to improve nanomedicine systems at the gene-, protein- and tissue- levels. We applied CRISPR/Cas9 technology for gene targeting. Delivering CRISPR/Cas9 elements, including Cas9 endonuclease and a corresponding guide RNA, allows for specific gene mutagenesis. A conventional gene delivery carrier often has a highly positive charge density for higher transgene expression, but this may result in unfavorable effects on the Cas9 plasmid transfection. As a large plasmid, strong interaction between the Cas9 plasmid and the polycation with high charge density may hinder the plasmid’s intracellular release. Moreover, high Cas9 expression usually leads to undesirable off-target effects. We addressed these two major obstacles by designing a low-charged density micelle, composed of quaternary ammonium‐terminated poly(propylene oxide) and amphiphilic Pluronic F127. We tested this design on a human papillomavirus (HPV)-induced cervical cancer model to target the HPV oncogene, E7. Our micellar carrier enabled effective Cas9 transfection with a transient Cas9 expression, which offered enhanced Cas9 on-target specificity. This nonviral Cas9‐mediated E7 mutagenesis resulted in significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo. Although CRISPR/Cas9 technology is a powerful toolkit for gene manipulation, gene editing might not be practical for therapeutics in the cancers that develop from endogenous mutations, which may vary among patients and disease stages. Protein-targeting, therefore, may be a more efficient approach. Aptamer and its selection technology, namely SELEX, offer direct evolution to obtain a nucleic acid ligand that specifically recognizes the protein target. Yet, aptamer screening remains unsatisfactory, and the success rate of SELEX is limited. We designed two approaches to improve the aptamer screening. We first employed a microarray platform to deconvolute the aptamer sequence and identified the aptamer functional motif. The resulted protein-targeting motif with an optimal length and showed enhanced structural and functional characteristics compared with its parental sequence. In addition to sequence optimization, conjunction of two distinct aptamers that recognize different epitopes of the protein target is another approach to improve the aptamer’s affinity. In looking for a rapid way to screen this bivalent aptamer pair, we designed a quantum dot (QD)/ Förster resonance energy transfer (FRET) sensor. Using a thrombin aptamer as a model system, we conjugated an anti-thrombin aptamer with QD and stained the other one with the intercalation dye, YOYO-3. If the two aptamers recognized different epitopes of thrombin, the conformational change of the two aptamers would take place when interacting with thrombin, and this would induce YOYO-3 dye’s translocation. YOYO-3 would be transferred from the aptamer to QD surface, resulting in a strong FRET signal. In contrast, if they recognized the same epitope, binding competition between two aptamers would inhibit dye translocation, thereby giving a minimal FRET signal. By measuring the FRET signal, we can verify if the two aptamers may form a bivalent pair. Lastly, we integrated mesenchymal stem cell (MSC) with a nanomedicine system to achieve active tissue-targeting. MSC is known to migrate toward certain types of cancer cells by chasing the chemotaxis release from the cancer cells, but the therapeutic payload that MSC can carry is limited. Forming an MSC spheroid allowed the loading of the nanomedicine system with another type of anti-cancer drug. We therefore designed a hybrid MSC/nanomedicine spheroid, which functioned as an active tumor-targeting platform, enabling effective delivery for both cytotoxic protein and chemotherapeutic drugs. In a heterotopic glioblastoma model, the hybrid spheroid significantly improved the retention of the nanomedicine system at the tumor site, leading to enhanced tumor inhibition in vivo. Collectively, this work demonstrated the effective approaches for gene, protein and tissue targeting by addressing the issues of low specificity and limited delivery efficiency that many current nanomedicine systems face. Particularly, the results may add to the armamentarium of cancer therapeutics, which remains largely challenging and intractable.

Leung, Hoi Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 114-128). / Abstracts in English and Chinese. / THESIS/ASSESSMENT COMMITTEE --- p.i / STATEMENT FROM AUTHOR --- p.ii / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.xi / LIST OF FIGURES --- p.xvi / LIST OF TABLES --- p.xviii / LIST OF ABBREVIATIONS --- p.xix / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.4 / Chapter 2.1 --- Nutritional quality of rice --- p.4 / Chapter 2.1.1 --- Classification of seed proteins --- p.4 / Chapter 2.1.2 --- Amino acid composition of rice proteins --- p.5 / Chapter 2.1.3 --- Other nutritional components of rice --- p.6 / Chapter 2.2 --- Rice seed storage proteins --- p.7 / Chapter 2.2.1 --- Properties and classification of seed storage proteins --- p.7 / Chapter 2.2.2 --- Composition and stucture --- p.9 / Chapter 2.2.2.1 --- Glutelin --- p.9 / Chapter 2.2.2.2 --- Prolamin --- p.10 / Chapter 2.2.2.3 --- Albumin and globulin --- p.12 / Chapter 2.2.3 --- "Synthsis, assembly and deposition of rice seed storage proteins" --- p.13 / Chapter 2.2.3.1 --- Storage protein folding and assembly in the ER --- p.14 / Chapter 2.2.3.2 --- Storage protein transport and protein body formation --- p.16 / Chapter 2.2.3.3 --- Protein bodies and their distribution in endosperm --- p.18 / Chapter 2.3 --- Transgenic approaches to improve the nutritional quality of rice seed proteins --- p.19 / Chapter 2.3.1 --- General introduction --- p.19 / Chapter 2.3.2 --- Attempts to improve the nutritional quality of seed proteins --- p.20 / Chapter 2.3.3 --- Rice grain quality improvement by genetic engineering --- p.22 / Chapter 2.3.3.1 --- Increase in the lysine content of rice endosperm --- p.22 / Chapter 2.2.3.2 --- Other examples of rice nutritional quality improvement --- p.25 / Chapter 2.3.4 --- Expression of recombinant protein in transgenic plants --- p.26 / Chapter 2.3.5 --- Effects of recombinant proteins on the high-lysine rice --- p.27 / Chapter 2.4 --- Proteomics --- p.28 / Chapter 2.4.1 --- General overview --- p.28 / Chapter 2.4.1.1 --- Two-dimensional polyacrylamide gel electrophoresis for proteome analysis --- p.29 / Chapter 2.4.1.2 --- Protein visualization --- p.32 / Chapter 2.4.1.3 --- Computer-aided image analysis --- p.34 / Chapter 2.4.1.4 --- Mass spectrometry-based methods for protein identification --- p.35 / Chapter 2.4.1.5 --- Database search --- p.36 / Chapter 2.4.1.6 --- Protein sequence database --- p.37 / Chapter 2.4.2 --- Plant proteomics --- p.40 / Chapter 2.4.2.1 --- Rice proteomics --- p.41 / Chapter 2.4.2.2 --- Comparative proteomics --- p.43 / Chapter 2.5 --- Hypothesis and objectives --- p.45 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.47 / Chapter 3.1 --- Materials --- p.47 / Chapter 3.1.1 --- Chemicals and commercial kits --- p.47 / Chapter 3.1.2 --- Instruments --- p.47 / Chapter 3.1.3 --- Softwares --- p.48 / Chapter 3.1.4 --- Plant materials --- p.48 / Chapter 3.2 --- Methods --- p.49 / Chapter 3.2.1 --- Collection of developing rice seeds --- p.49 / Chapter 3.2.2 --- Extraction of rice seed proteins --- p.51 / Chapter 3.2.2.1 --- Extraction of total protein --- p.51 / Chapter 3.2.3.2 --- Extraction of four fractions of rice seed proteins --- p.51 / Chapter 3.2.3 --- 2D gel electrophoresis --- p.53 / Chapter 3.2.3.1 --- Protein precipitation and quantification --- p.53 / Chapter 3.2.3.2 --- Isoelectric focusing (IEF) --- p.54 / Chapter 3.2.3.3 --- IPG strips equilibration --- p.54 / Chapter 3.2.3.4 --- Second-dimension SDS-PAGE --- p.55 / Chapter 3.2.3.5 --- Silver staining of 2D gel --- p.55 / Chapter 3.2.3.6 --- Image and data analysis --- p.56 / Chapter 3.2.4 --- MALDI-ToF mass spectrometry (Matrix Assisted Laser Desorption Ionization-Time of Flight) --- p.56 / Chapter 3.2.4.1 --- Sample destaining --- p.56 / Chapter 3.2.4.2 --- In-gel digestion with trypsin --- p.57 / Chapter 3.2.4.3 --- Desalination of the digested sample with Zip Tip --- p.58 / Chapter 3.2.4.4 --- Protein identification by mass spectrometry and database searching --- p.58 / Chapter 3.2.5 --- Detection of LRP fusion protein in 2D PAGE --- p.59 / Chapter 3.2.5.1 --- 2D gel electrophoresis --- p.59 / Chapter 3.2.5.2 --- Western blotting using anti-LRP antibody --- p.60 / Chapter 3.2.6 --- Antiserum production --- p.61 / Chapter 3.2.6.1 --- Purification of glutelin and prolamin proteins --- p.61 / Chapter 3.2.6.2 --- Immunization of rabbits and mice --- p.62 / Chapter 3.2.6.3 --- Testing of antibody specificity --- p.62 / Chapter 3.2.7 --- Transmission electron microscopy (TEM) --- p.63 / Chapter 3.2.7.1 --- Sample fixation and section preparation --- p.63 / Chapter 3.2.7.2 --- TEM observation --- p.64 / Chapter 3.2.7.3 --- Immunocytochemical observation --- p.64 / Chapter CHAPTER 4. --- RESULTS --- p.66 / Chapter 4.1 --- Proteomic analysis of high-lysine rice --- p.66 / Chapter 4.1.1 --- Extraction of proteins --- p.66 / Chapter 4.1.2 --- The proteomic profiles of different storage proteins in developing high-lysine rice seeds --- p.67 / Chapter 4.1.3 --- Quantitative analysis of protein spots --- p.76 / Chapter 4.1.4 --- Proteomic analysis of salt-soluble proteins --- p.79 / Chapter 4.1.5 --- Proteomic analysis of alcohol-soluble proteins --- p.81 / Chapter 4.1.6 --- Proteomic analysis of salt-soluble proteins --- p.82 / Chapter 4.1.7 --- Proteomic analysis of water-soluble proteins --- p.89 / Chapter 4.1.8 --- Comparison of changes in expression patterns of specific proteins in the high lysine rice --- p.89 / Chapter 4.2 --- Antibody production --- p.92 / Chapter 4.2.1 --- The production of anti-prolamin and anti-glutelin antibodies --- p.92 / Chapter 4.2.2 --- The specificity of anti-prolamin and anti-glutelin antibodies --- p.93 / Chapter 4.3 --- Transmission electron microscopy observation of rice protein bodies --- p.95 / Chapter 4.3.1 --- Morphology of protein bodies in high-lysine rice --- p.95 / Chapter 4.3.2 --- Subcellular localization of storage proteins and LRP --- p.98 / Chapter CHAPTER 5. --- DISCUSSION --- p.100 / Chapter 5.1 --- Protein profiling of LRP fusion protein and its effects on the expression of other proteins --- p.100 / Chapter 5.2 --- Over-expression of glutelin and its effects on the expression of other proteins --- p.102 / Chapter 5.3 --- Formation of malformed protein bodies and deposition of storage proteins --- p.103 / Chapter 5.4 --- Relationship between changes in protein expression and the Unfolded Protein Response --- p.105 / Chapter 5.5 --- Effects of transgenes on rice grain quality --- p.108 / Chapter 5.6 --- Allergenic effects of transgenic rice --- p.109 / Chapter 5.7 --- Future perspectives --- p.110 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.112 / REFERENCES --- p.114

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.

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