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.

O objetivo desta dissertação foi estimar a exigência de lisina (Lis) para frangos de corte machos Cobb x Cobb 500 de 1 a 12 dias de idade (experimento 1), 12 a 28 dias de idade (experimento 2) e 28 a 42 dias de idade (experimento 3). Dietas basais foram formuladas para atingir ou exceder as exigências nutricionais, com exceção da Lis. Cinco níveis de Lis foram suplementados às dietas basais a partir de L-Lis HCl ou sulfato de L-Lis de modo que os níveis variaram de 0,97% a 1,37% de Lis digestível no experimento 1, 0,77% a 1,17% de Lis digestível no experimento 2 e 0,68% a 1,08% de Lis digestível no experimento 3 em incrementos de 0,08%. Os tratamentos foram distribuídos em um delineamento inteiramente casualizado com 8 repetições de 25 aves. Em cada experimento, 2200 aves foram alojadas em 88 unidades experimentais. Nos dias 1 e 12 (experimento 1), 12 e 28 (experimento 2) e 28 e 42 (experimento 3) as aves e a ração foram pesadas para determinar o ganho de peso (GP) e conversão alimentar (CA). No experimento 3, quatro aves por unidade experimental foram abatidas para determinação do rendimento de carcaça e peito. A biodisponibilidade relativa (RBV) das fontes de Lis foi avaliada através de uma regressão multivariada e comparada pelo teste t. A exigência de Lis foi estimada por três modelos de regressão: polinomial quadrática, brokenline linear e broken-line quadrática. A exigência foi representada como 95% do ponto de máxima resposta. Não houve diferença entre a RBV da Lis no sulfato de L-Lis em relação ao L-Lis HCl, portanto ambas as fontes foram utilizadas para estimar as exigências. As exigências encontradas variaram de acordo com o modelo estatístico e a variável analisada. A regressão broken-line quadrática apresentou o melhor ajustamento aos dados de desempenho, enquanto a regressão broken-line linear se ajustou melhor aos dados de rendimento de carcaça e peito. As regressões polinomial quadrática, broken-line linear e broken-line quadrática estimaram, respectivamente, as exigências como 1,190, 1,032 e 1,101% para GP e 1,226, 1,038 e 1,124% para CA no experimento 1; 1,021, 0,900 e 0,961% para GP e 1,064, 0,966 e 1,043% para CA no experimento 2; 0,949, 0,833 e 0,925% para GP, 0,978, 0,851 e 0,960% para CA, 0,933, 0,842 e 0,931% para rendimento de carcaça e 0,952, 0,839 e 0,921% para rendimento de peito no experimento 3. Os resultados demonstraram que as exigências de Lis foram consideravelmente influenciadas pelas diferentes regressões. Portanto, a escolha do modelo estatístico é crítica para a obtenção de estimativas precisas e coerentes. / The objective of this thesis was to estimate lysine (Lys) requirement of male Cobb x Cobb 500 broilers from 1 to 12 days of age (experiment 1), 12 to 28 days of age (experiment 2), and 28 to 42 days of age (experiment 3). Basal diets were formulated to meet or exceed recommendations, except for Lys. Five graded levels of Lys were supplemented from L-Lys HCl or L-Lys sulfate to the basal diets. Dietary treatments ranged from 0.97% to 1.37% digestible Lys in experiment 1, 0.77% to 1.17% digestible Lys in experiment 2, and 0.68% to 1.08% digestible Lys in experiment 3 in 0.08% increments. Treatments were distributed in a completely randomized design with 8 repetitions of 25 birds each. A total of 2,200 birds per experiment were placed in 88 experimental units. At 1 and 12 days (experiment 1), 12 and 28 days (experiment 2), and 28 and 42 days (experiment 3), birds and feed were weighed to determine body weight gain (BWG) and feed conversion ratio (FCR). In experiment 3, four birds per experimental unit were processed for carcass and breast meat yield evaluation. Relative bioavailability (RBV) of Lys sources was assessed by a multivariate regression and compared by a t-test. Lysine requirement was estimated using three regression models: quadratic polynomial, linear broken-line, and quadratic broken-line. Requirements were represented as 95% of the asymptote. No difference was observed in Lys RBV in L-Lys sulfate compared to L-Lys HCl, thus both sources were used to estimate requirements. Requirement estimates varied according to statistic model and analyzed variable. Quadratic broken-line model presented the best fit to performance data (BWG and FCR), whereas linear broken-line model fitted better to carcass and breast meat yield data. Quadratic polynomial, linear broken-line, and quadratic brokenline estimates were, respectively, 1.190, 1.032, and 1.101% for BW gain and 1.226, 1.038, and 1.124% for FCR in experiment 1; 1.021, 0.900, and 0.961% for BW gain and as 1.064, 0.966, and 1.043% for FCR in experiment 2; and 0.949, 0.833, 0.925% for BW gain, 0.978, 0.851, and 0.960% for FCR, 0.933, 0.842, and 0.931% for carcass yield, and 0.952, 0.839, and 0.921% for breast meat yield in experiment 3. Results demonstrate that Lys requirements were considerably influenced by different regression models. Therefore, the choice of statistical model is crucial to obtain precise, coherent estimates.

A lisina é um dos aminoácidos essenciais e um dos fatores limitantes ao uso de cereais como o milho na alimentação, pois, sem suplementação, não permite a obtenção de uma dieta balanceada. A fim de melhorar a qualidade nutricional dos cereais, várias tentativas têm sido realizadas baseadas em resultados obtidos sobre as rotas de metabolismo da lisina em plantas e o acúmulo de proteínas de reserva no endosperma. Ambrozevicius (2010) com o objetivo de produzir plantas de milho transgênicas com alto teor de lisina utilizou a estratégia de expressão de proteínas de reserva de outras espécies vegetais ricas em lisina, ou seja, através da expressão de uma proteína heteróloga: a zeolina. O presente trabalho teve como objetivo estudar os 6 eventos transformados expressando a zeolina na geração F3, caracterizando as proteínas de reserva, o perfil de aminoácidos e as enzimas envolvidas no metabolismo de lisina em milho geneticamente modificado, para compreender quais as possíveis alterações bioquímicas podem ter sido geradas pela transformação, e que podem ter levado ao incremento dos aminoácidos essenciais neste material transgênico. O perfil de proteínas de reserva dos eventos transformados exibiu redução na proporção das zeínas II e glutelinas em relação ao controle HiII, e ainda aumentos muito discretos da fração globulina, porém não para todos os eventos transformados. Na composição de aminoácidos solúveis totais foram observados incrementos nos teores dos aminoácidos que fazem parte da via metabólica do ácido aspártico: lisina, metionina, treonina e isoleucina. Para os aminoácidos incorporados em proteínas, foram observados incrementos nos teores de lisina nos eventos transformados da fração globulina e da fração glutelina, ambos em relação ao controle HiII. Já a fração zeína I teve o maior conteúdo total de aminoácidos em todos os eventos transformados. A análise das enzimas envolvidas no metabolismo de lisina revelou que ocorreram alterações em duas enzimas, a primeira enzima envolvida na síntese de lisina, aspartato quinase (AK) e a segunda envolvida na degradação de lisina, lisina cetoglutarato redutase (LOR). Embora as outras enzimas envolvidas na síntese e na degradação de lisina também tenham sido alteradas, os resultados foram variáveis para os diferentes eventos. Este trabalho mostrou que a expressão da proteína heteróloga zeolina causou alterações na composição das frações protéicas, no teor dos aminoácidos solúveis totais e aminoácidos incorporados em proteína em consequencia das alterações das enzimas envolvidas na síntese e degradação da lisina. Os resultados obtidos sugerem que a expressão da proteína heteróloga zeolina, que tem a necessidade de incorporação de lisina em sua estrutura, pode ter alterado a via metabólica do ácido aspártico para suprir a nova demanda de lisina. Essas alterações podem incluir o aumento na atividade da enzima AK, que é a primeira enzima da via que leva a síntese deste aminoácido e também uma redução na atividade da enzima de degradação LOR, pois o excesso de lisina livre que seria degradada é incorporado à nova proteína. / Lysine is an essential amino acid and one of the limiting factors for the use in cereals such as corn feed therefore, without supplementation; it does not allow obtaining a balanced diet. In order to improve the nutritional quality of cereals, several attempts have been made based on the results about routes of metabolism of lysine in plants and accumulation of storage proteins in endosperm. Ambrozevicius (2010) with the objective of producing transgenic corn plants with high content of lysine used a strategy of expression of storage proteins from other plant species which are rich in lysine, it means, through the expression of a heterologous protein: zeolin. This work aimed to study the 6 events processed expressing zeolina in F3 generation, featuring the storage proteins, the profile of amino acids and enzymes involved in the metabolism of lysine in genetically modified corn, in order to understand the possible biochemical changes which may have been generated by the transformation, and that may have led to the increase of essential amino acids in transgenic material The storage protein profile of transformed events exhibited reduction in the proportion of zein II and glutelins compared to control HiII, and yet very discrete increments of globulin, but not for all events processed. In the composition of soluble amino acids, it was observed increments in concentration of amino acids forming part of the metabolic pathway of aspartic acid: Lysine, methionine, threonine and isoleucine. For the amino acids incorporated into proteins, it was observed increments in the levels of lysine in the transformed events of globulin and glutelin fraction, both in relation to the control HiII. On the other hand, zein fraction I had the highest total amino acid content in all transformed events. The analysis of the enzymes involved in the metabolism of lysine revealed that changes occurred in two enzymes, the first enzyme involved in the synthesis of lysine, aspartate kinase (AK) and the second involved in the degradation of lysine, lysine ketoglutarate reductase (LOR). Although other enzymes involved in the synthesis and degradation of lysine have also been changed, the results were variable for different events. This work showed that the expression of heterologous protein zeolina caused changes in the composition of protein fractions, in the content of soluble amino acids and amino acids incorporated in consequence of changes in enzymes involved in the synthesis and degradation of lysine. The results suggest that the expression of heterologous protein zeolina, which has the need for incorporation of lysine in its structure, may have changed the aspartic acid pathway to meet the new demand for lysine. These changes may include the increase in AK enzyme activity, which is the first enzyme of the pathway leading to the synthesis of this amino acid and also a reduction in the activity of the enzyme degradation LOR, since the excess free lysine would be degraded is incorporated into the new protein.

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.

Wong Ho Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 140-153). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Acknowledgements --- p.III / Abstract --- p.IV / 摘要 --- p.VI / List of Tables --- p.VIII / List of Figures --- p.IX / List of Abbreviations --- p.XI / Table of Contents --- p.XIII / Chapter 1 --- General introduction --- p.1 / Chapter 2 --- Literature reviews --- p.4 / Chapter 2.1 --- LRP and winged bean --- p.4 / Chapter 2.1.1 --- Nutritional values of crop plants --- p.4 / Chapter 2.1.2 --- Lysine-rich protein (LRP) --- p.7 / Chapter 2.1.2.1 --- Identification of lysine-rich protein (LRP) --- p.7 / Chapter 2.1.2.2 --- Cloning cDNA for WBLRP --- p.7 / Chapter 2.1.2.3 --- Transgenic Expression of LRP in other plants --- p.8 / Chapter 2.1.3 --- Unknowns remained --- p.8 / Chapter 2.2 --- Food allergy and gastro-immunity --- p.10 / Chapter 2.2.1 --- What is allergy? 一 A brief introduction --- p.10 / Chapter 2.2.2 --- Food allergy and its symptoms --- p.12 / Chapter 2.2.3 --- Gastrointestinal immunity --- p.13 / Chapter 2.2.4 --- Possible mechanism of food allergy --- p.16 / Chapter 2.2.5 --- Available tests and limitations --- p.18 / Chapter 2.2.6 --- Radioallergosorbent test (RAST) --- p.19 / Chapter 2.2.7 --- Digestibility test --- p.20 / Chapter 2.2.8 --- Betv-1 Allergen Family --- p.21 / Proteins --- p.23 / Chapter 2.3 --- Pathogenesis-related proteins --- p.23 / Chapter 2.3.1 --- Defense-related proteins and pathogenesis-related proteins (PRs) --- p.23 / Chapter 2.3.2 --- Class 10 PR proteins (PR-10s) --- p.25 / Chapter 2.3.3 --- The expression patterns of PR-10s --- p.27 / Chapter 2.3.3.1 --- Pathogens-induced and signal-induced expression --- p.27 / Chapter 2.3.3.2 --- Spatially- and developmentally-regulated expression --- p.28 / Chapter 2.3.3.3 --- Other induction patterns --- p.29 / Chapter 2.3.4 --- Functions ofPR-10s --- p.30 / Chapter 2.4 --- Development of hypotheses and experiments --- p.32 / Chapter 3 --- Materials and methods --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials --- p.38 / Chapter 3.2.1 --- Chemicals --- p.38 / Chapter 3.2.2 --- Apparatus and commercial kits --- p.39 / Chapter 3.2.3 --- Vectors and bacterial strains --- p.39 / Chapter 3.2.4 --- Plant and animal materials --- p.40 / Chapter 3.2.5 --- Computer software --- p.40 / Chapter 3.3 --- Purification of LRP --- p.41 / Chapter 3.3.1 --- Purification of LRP from winged bean --- p.41 / Chapter 3.3.1.1 --- Extraction of total protein --- p.41 / Chapter 3.3.1.2 --- Differential pI precipitation --- p.41 / Chapter 3.3.1.3 --- Determination of the pI point of LRP --- p.42 / Chapter 3.3.1.4 --- Native tricine-PAGE and gel elution --- p.42 / Chapter 3.3.2 --- Purification from E. coli --- p.45 / Chapter 3.3.2.1 --- Construction of pET vector expressing recombinant LRP (rLRP) --- p.45 / Chapter 3.3.2.2 --- Expression of rLRP --- p.50 / Chapter 3.3.2.3 --- Purification by gel electrophoresis and gel band elution --- p.50 / Chapter 3.4 --- Anti-serum production --- p.52 / Chapter 3.5 --- Allergy tests --- p.53 / Chapter 3.5.1 --- Pepsin digestion --- p.53 / Chapter 3.5.1.1 --- Determination of optimal concentration of pepsin --- p.53 / Chapter 3.5.1.2 --- Pepsin digestion of allergenic and non-allergenic model proteins --- p.55 / Chapter 3.5.1.3 --- Pepsin digestion of LRP and immunodetection --- p.55 / Chapter 3.5.2 --- Trypsin digestion --- p.56 / Chapter 3.5.2.1 --- Determination of optimal trypsin concentration --- p.56 / Chapter 3.5.2.2 --- Trypsin digestion of allergenic and non-allergenic model proteins --- p.57 / Chapter 3.5.2.3 --- Trypsin digestion of LRP and immuno-detection --- p.57 / Chapter 3.5.3 --- Pepsin and trypsin digestion --- p.58 / Chapter 3.5.3.1 --- Digestions of allergenic model proteins --- p.58 / Chapter 3.5.3.2 --- Digestion of LRP --- p.58 / Chapter 3.5.4 --- IgE binding tests --- p.58 / Chapter 3.6 --- Physiology studies --- p.59 / Chapter 3.6.1 --- Preparation for the studies --- p.59 / Chapter 3.6.1.1 --- Growing winged bean in the field --- p.59 / Chapter 3.6.1.2 --- Growing winged bean in sterile conditions --- p.60 / Chapter 3.6.1.3 --- Production ofLRP-cDNA probe --- p.60 / Chapter 3.6.2 --- Detecting the expression of LRP in winged bean --- p.61 / Chapter 3.6.2.1 --- RNA extraction --- p.61 / Chapter 3.6.2.2 --- RT-PCR and DNA sequencing --- p.62 / Chapter 3.6.2.3 --- RNA electrophoresis and northern blot analysis --- p.63 / Chapter 3.6.2.4 --- Protein extraction --- p.63 / Chapter 3.6.2.5 --- Western blot and immuno-detection --- p.63 / Chapter 3.6.3 --- Expression of LRP in germinating winged bean seeds --- p.64 / Chapter 3.6.3.1 --- Seed germination --- p.64 / Chapter 3.6.3.2 --- Detection of LRP in germinating seeds --- p.64 / Chapter 3.6.4 --- RNase activity test --- p.65 / Chapter 4 --- Results --- p.67 / Chapter 4.1 --- Purification of LRP --- p.67 / Chapter 4.1.1 --- Purification from winged bean --- p.67 / Chapter 4.1.1.1 --- Identification of pI point of LRP --- p.67 / Chapter 4.1.1.2 --- Native tricine PAGE and gel elution --- p.70 / Chapter 4.1.2 --- Purification from E. coli --- p.71 / Chapter 4.1.2.1 --- Construction of pET-LRP vector --- p.71 / Chapter 4.1.2.2 --- Expression of rLRP and gel purification --- p.74 / Chapter 4.2 --- Antiserum production --- p.76 / Chapter 4.3 --- Allergy tests --- p.81 / Chapter 4.3.1 --- Pepsin digestion --- p.81 / Chapter 4.3.2 --- Trypsin digestion --- p.89 / Chapter 4.3.3 --- Pepsin and trypsin digestion --- p.96 / Chapter 4.3.4 --- Human serum IgE binding test --- p.104 / Chapter 4.4 --- Physiological studies --- p.105 / Chapter 4.4.1 --- Samples preparation --- p.105 / Chapter 4.4.2 --- RT-PCR and DNA sequencing --- p.105 / Chapter 4.4.3 --- Expression profile of WBLRP in winged bean somatic organs --- p.108 / Chapter 4.4.4 --- Expression profile ofWBLRP in winged bean flower --- p.111 / Chapter 4.4.5 --- Expression profile ofWBLRP in winged bean maturing seeds --- p.114 / Chapter 4.4.6 --- Expression profile of WBLRP gene in winged bean germinating seeds --- p.117 / Chapter 4.4.7 --- Functional assay of LRP --- p.121 / Chapter 5 --- Discussion --- p.124 / Chapter 5.1 --- LRP purification and antibody production --- p.124 / Chapter 5.2 --- Allergy tests --- p.125 / Chapter 5.3 --- Expression of LRP in WB --- p.131 / Chapter 5.4 --- Functional assay of LRP --- p.134 / Chapter 5.5 --- Hypothesis Testing --- p.135 / Chapter 5.6 --- Future prospective， --- p.136 / Chapter 6 --- Conclusion --- p.138 / Chapter 7 --- References --- p.140

Yu, Chun Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 115-124). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / LIST OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xvi / LIST OF TABLES --- p.xx / LIST OF ABBREVIATIONS --- p.xxi / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.20 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.22 / Chapter 2.1 --- Major storage proteins in rice --- p.22 / Chapter 2.1.1 --- Structure and composition of glutelin --- p.22 / Chapter 2.1.2 --- Structure and composition of prolamin --- p.22 / Chapter 2.2 --- Biosynthesis pathway --- p.23 / Chapter 2.2.1 --- "The Biosynthesis, processing & compartmentalization of glutelin" --- p.23 / Chapter 2.2.1.1 --- Endoplasmic reticulum as the site of protein folding and compartmentalization --- p.23 / Chapter 2.2.1.2 --- COP-coated vesicles for protien trafficking between ER and Golgi --- p.25 / Chapter 2.2.1.3 --- Glutelin trafficking beyond ER --- p.26 / Chapter 2.2.1.3.1 --- Golgi as the site of post-translational modification of glutelin / Chapter 2.2.1.3.1.1 --- """Sorting for entry"" and ""sorting by retention"" models: mechanism of dense vesicle formation" --- p.26 / Chapter 2.2.1.3.1.2 --- "“Classical ligand-receptor"" and ""aggregation-mediated"" as the model describing protein sorting in Golgi" --- p.27 / Chapter 2.2.1.3.2 --- Pathway bypassing Golgi apparatus --- p.30 / Chapter 2.2.1.4 --- Prevacuolar compartment and protein body --- p.30 / Chapter 2.2.2 --- "The Biosynthesis, processing and compartmentalization of prolamin" --- p.31 / Chapter 2.3 --- Protein processing enzymes --- p.31 / Chapter 2.3.1 --- Luminal chaperone binding protein (BiP) --- p.31 / Chapter 2.3.2 --- Protein disulfide isomerase (PDI) --- p.33 / Chapter 2.4 --- ER quality control： unfolded protein response --- p.34 / Chapter 2.4.1 --- The importance of quality control in ER --- p.34 / Chapter 2.4.2 --- The target of ER quality control: misfolded protein --- p.35 / Chapter 2.4.3 --- Unfolded protein response --- p.36 / Chapter 2.4.3.1 --- IRE1 --- p.37 / Chapter 2.4.3.2 --- PERK --- p.37 / Chapter 2.4.3.3 --- ATF6 --- p.38 / Chapter 2.4.3.4 --- BiP as the master regulator of three transducers --- p.38 / Chapter 2.5 --- The cause of chalkiness --- p.41 / Chapter 2.5.1 --- "The relationship between ER stress, unfolded protein response and chalkiness" --- p.42 / Chapter 2.6 --- Organelle separation： sucrose density gradient centrifugation --- p.43 / Chapter 2.6.1 --- General introduction --- p.43 / Chapter 2.6.2 --- Plant organelle separation --- p.43 / Chapter 2.6.3 --- Organelle marker enzyme as a mean to elucidate the homogeneity of isolated organelle fraction --- p.44 / Chapter 2.7 --- Rice grain quality improvement by genetic engineering --- p.45 / Chapter 2.7.1 --- Increase in lysine content of rice endosperm --- p.45 / Chapter 2.7.2 --- Physiological and phenotypic changes in GT and LRP-fusion lines --- p.46 / Chapter 2.8 --- Hypotheses and objectives --- p.48 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.49 / Chapter 3.1 --- Materials --- p.49 / Chapter 3.1.1 --- Chemicals and commercial kits --- p.49 / Chapter 3.1.2 --- Instruments --- p.49 / Chapter 3.1.3 --- Plant materials --- p.49 / Chapter 3.1.3.1 --- Glutelin-enriched line (GT) --- p.50 / Chapter 3.1.3.2 --- Gtl-LRP-fusion line (LRP fusion) --- p.50 / Chapter 3.2 --- RNA extraction and northern-blot analysis --- p.50 / Chapter 3.2.1 --- Seed harvesting and RNA extraction --- p.50 / Chapter 3.2.2 --- Northern-blot analysis --- p.51 / Chapter 3.3 --- SDS-PAGE and western-blot analysis --- p.52 / Chapter 3.3.1 --- Seed harvesting and protein extraction --- p.52 / Chapter 3.3.2 --- SDS-PAGE and western-blot analysis s --- p.52 / Chapter 3.4 --- Purification of cellular organelles by SDG centrifugation --- p.53 / Chapter 3.4.1 --- Purification of ER by SDG centrifugation --- p.53 / Chapter 3.4.2 --- Purification of protein body by SDG centrifugation --- p.54 / Chapter 3.4.3 --- Protein body isolation by pepsin treatment --- p.54 / Chapter 3.5 --- Electron-microscopic observation --- p.55 / Chapter 3.5.1 --- Sample preparation for immuno-localization analysis --- p.55 / Chapter 3.5.1.1 --- Sample preparation --- p.55 / Chapter 3.5.1.2 --- Immunocytochemical observation --- p.55 / Chapter 3.5.2 --- Sample preparation for structural analysis --- p.56 / Chapter 3.6 --- Antibodies --- p.56 / Chapter 3.6.1 --- KLH conjugation of synthetic peptide --- p.57 / Chapter 3.6.2 --- Immunization of rabbits --- p.57 / Chapter 3.6.3 --- Antibody purification by affinity column --- p.57 / Chapter 3.6.3.1 --- Preparation of column for coupling --- p.57 / Chapter 3.6.3.2 --- Affinity purification of antibody by prepared column --- p.58 / Chapter 3.6.4 --- Testing of antibody specificity --- p.58 / Chapter CHAPTER 4. --- RESULTS --- p.60 / Chapter 4.1 --- Pro-glutelin accumulation in GT and LRP fusion transgenic lines --- p.60 / Chapter 4.2 --- General morphology and glutelin localization in rice seed --- p.61 / Chapter 4.3 --- "Studies on glutelin, BiP and pdi expression profiles of GT, LRP fusion lines and wild type rice" --- p.63 / Chapter 4.3.1 --- Comparison of the protein and RNA profiles of BiP between wild type and FH transgenic rice lines --- p.64 / Chapter 4.3.2 --- Comparison of the protein and RNA profiles of PDI between wild type and FH transgenic rice lines --- p.66 / Chapter 4.3.3 --- "Comparison of the RNA and protein profiles of BiP between wild type, GH and GL transgenic rice lines" --- p.68 / Chapter 4.3.4 --- "Comparison of the RNA and protein expression profiles of PDI between wild type, GH and GL transgenic lines" --- p.70 / Chapter 4.3.5 --- Summary of RNA and protein level comparison of different transgenic lines with wild type --- p.72 / Chapter 4.4 --- Electron microscopic studies of morphological changes in GLUTELIN OVER-EXPRESSED AND GT1-LRP-FUSION TRANSGENIC LINES AND WILD type rice --- p.73 / Chapter 4.5 --- Isolation of ER-enriched fractions by sucrose density gradient centrifugation --- p.76 / Chapter 4.5.1 --- Cross-contamination assessment by organelle specific marker proteins --- p.77 / Chapter 4.5.2 --- Identification of ER enriched fractions of different transgenic lines --- p.78 / Chapter 4.5.3 --- Studies on ER enriched fraction --- p.85 / Chapter 4.6 --- Isolation and studies on PB enriched fractions of different transgenic lines --- p.91 / Chapter 4.7 --- TEM studies on immuno-localization of ER chaperones (BlP and pdI) in immature rice seeds of different transgenic lines --- p.94 / Chapter CHAPTER 5. --- DISCUSSIONS --- p.101 / Chapter 5.1 --- Distortion of glutelin processing and translocation pathway --- p.101 / Chapter 5.1.1 --- The relationship between proglutelin localization and novel protein body in Gt1-LRP-fusion lines --- p.101 / Chapter 5.1.2 --- The presence of BiP and PDI in novel protein body in Gt1-LR-fusion lines --- p.103 / Chapter 5.1.2.1 --- Glutelin translocation pathway bypassing Golgi --- p.105 / Chapter 5.1.2.2 --- Glutelin translocation pathway through Golgi --- p.105 / Chapter 5.1.2.3 --- Gt1-LRP-fusion protein and proglutelin are trapped in ER --- p.107 / Chapter 5.2 --- "The relationship between novel protein body formation, ER stress, unfolded protein response and chalkiness" --- p.108 / Chapter 5.2.1 --- Relationship between novel protein body formation and unfolded protein response --- p.108 / Chapter 5.2.2 --- Repressing the expression of other storage proteins: consequence of unfold protein response or protein nutrients regulation --- p.109 / Chapter 5.2.3 --- Relationship between novel protein body formation and chalkiness --- p.110 / Chapter 5.3 --- The causes of ER dilation --- p.110 / Chapter 5.4 --- The relationship between different physiological changes in transgenic glutelin lines --- p.111 / Chapter 5.5 --- Future perspectives --- p.112 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.114 / REFERENCES --- p.115 / APPENDIX --- p.125

Simultaneous measurements of energy and protein metabolism can provide valuable information about their interactions. Dietary lysine is limiting in typical feedstuffs fed to swine and, therefore, limits protein synthesis. Current recommendations for dietary amino acid and energy intakes may not be reflective of the requirements for modern, highly productive sows and, therefore, invalidate requirement estimates determined according to the factorial approach. Current feeding recommendations suggest a constant amino acid intake throughout gestation. However, the demands for amino acids changes from maternal tissue accretion in early-gestation to fetal, conceptus, and mammary tissue development in late-gestation. This thesis reports the method development associated with simultaneous measurements of energy and protein metabolism and its application to determine dietary lysine requirements in non-pregnant and pregnant sows using the indicator amino acid oxidation method. Two indirect calorimetry systems and an experimental feeding regimen were tested and validated for use in studies of amino acid requirements by stable isotope dilution. Protein and energy balance studies were performed in non-pregnant sows fed two distinct levels of energy and protein intake. The systems reacted appropriately to changes in gas concentrations induced by sow respiration. Protein and energy balance studies were also performed in pregnant and lactating sows fed typical diets. Sows appeared more anabolic during mid-gestation and were catabolic by late-gestation and through lactation, where additional energy intake provided by ad libitum feed intake increased milk energy output. The dietary lysine requirement in non-pregnant sows at maintenance was determined as 49 mg/kg0.75, 30% greater than current recommendations. The dietary lysine requirement was determined to be 10.1 g/d and 16.5 g/d, in early- and late-gestation, respectively. These results suggest that a constant diet formulation for the entirety of gestation is not appropriate. In conclusion, simultaneous measurements of energy and protein metabolism combining indirect calorimetry and stable isotope techniques may be used to define requirements for dietary amino acids in sows. Basic assumptions of the factorial approach to estimate requirements require further investigation, including the dietary lysine requirement. Application of phase feeding for sows during gestation can more correctly meet the demands for amino acids and energy, improving sow longevity. / Animal Science

In eukaryotic organisms, polyubiquitination is the modification of a protein with polymerized ubiquitin (Ub) chain. This process is well known for its function in targeting proteins for degradation by the 26S proteasome. However, a polyUb chain assembled through the lysine 63 residue of the Ub moiety (Lys63-linked polyubiquitination) has been shown to play a signaling role rather than targeting proteins for degradation. In plants, the functions of Lys63-linked polyubiquitination are currently not well understood. Ub-protein ligase (E3) catalyzes the last step in the ubiquitination reactions, and to a large extent it also determines the substrate specificity of protein ubiquitination. In order to study the roles of Lys63-linked polyubiquitination in plants, two E3s of Arabidopsis thaliana, proteins encoded by AtCHIP and At1g74370 (tentatively named E3-A1), were chosen for functional studies, since they interacted with AtUbc13A protein. Sequence analysis showed that AtCHIP is the only member in the family. A T-DNA insertion mutant line (Atchip-1) was obtained to study the AtCHIP gene knock-out effect. The mutant line was grown in normal conditions and further tested in a variety of conditions: hormonal treatments, osmotic stress, seed deterioration, high temperature stress, high-intensity light stress, oxidative stress and DNA damaging stress. However, no clear difference was observed between the mutant and wild type plants based on the several parameters measured. Sequence analysis of E3-A1 indicated two closely related proteins, tentatively named E3-A2 and E3-A3. As E3-A1 and E3-A2 appeared to share more sequence similarity, RNA interference (RNAi) transformants, with the level of transcripts for either of the two E3-A genes reduced by over 90% were generated. Selected RNAi mutant lines for E3-A1 and E3-A2 were crossed with each other, and double RNAi mutants were obtained. However, no distinct phenotype was detected under normal, high-sucrose or hormonal conditions for either single or double RNAi lines. Although various assays did not reveal a significant phenotype in the mutants in this study, the materials generated and the assays used will benefit a wider range of phenotypic survey in the future.