Yu, Wai Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 87-101). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / 摘要 --- p.vi / LIST OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xiv / LIST OF ABBREVIATIONS --- p.xv / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.3 / Chapter 2.1 --- The importance of rice --- p.3 / Chapter 2.2 --- Limitation of essential amino acids in rice --- p.4 / Chapter 2.3 --- Lysine biosynthetic pathway --- p.6 / Chapter 2.3.1 --- The biosynthesis of aspartate --- p.6 / Chapter 2.3.2 --- Aspartate family pathway --- p.3 / Chapter 2.3.2.1 --- Aspartate kinase (AK) --- p.10 / Chapter 2.3.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.12 / Chapter 2.3.2.3 --- Other enzymes --- p.14 / Chapter 2.4 --- Regulation of lysine content in plant --- p.15 / Chapter 2.5 --- Enhancement of lysine content in plants --- p.16 / Chapter 2.5.1 --- "Breeding, selection and naturally occuring muatnts" --- p.17 / Chapter 2.5.2 --- Induced biochemical mutants --- p.18 / Chapter 2.5.3 --- Transgenic plants --- p.19 / Chapter 2.6 --- Hypothesis --- p.24 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.25 / Chapter 3.1 --- Introduction --- p.25 / Chapter 3.2 --- Chemicals --- p.25 / Chapter 3.3 --- Bacterial strains --- p.25 / Chapter 3.4 --- Cloning of AK and DHPS cDNAs --- p.25 / Chapter 3.4.1 --- Plant materials --- p.25 / Chapter 3.4.2 --- RNA extraction --- p.26 / Chapter 3.4.3 --- RT-PCR amplification of AK and DHPS cDNAs --- p.26 / Chapter 3.4.4 --- Sequence modification of AK and DHPS cDNAs --- p.27 / Chapter 3.4.5 --- DNA sequencing of AK and DHPS cDNAs --- p.32 / Chapter 3.5 --- Chimeric gene construction for rice transformation --- p.32 / Chapter 3.5.1 --- Plasmid and genetic material --- p.32 / Chapter 3.5.2 --- Construction of chimeric genes with seed-specific promoter --- p.35 / Chapter 3.5.3 --- Sequence fidelity of chimeric genes --- p.37 / Chapter 3.6 --- AEC resistance of E.coli expressing modified AK and DHPS --- p.37 / Chapter 3.7 --- Rice transformation --- p.38 / Chapter 3.7.1 --- Plant materials --- p.38 / Chapter 3.7.2 --- Preparation of agrobacterium --- p.33 / Chapter 3.7.3 --- Agrobacterium-mediated rice transformation --- p.39 / Chapter 3.7.3.1 --- Callus induction from mature rice seed embryos --- p.39 / Chapter 7.3.2 --- "Co-cultivation, selection and regeneration of transgenic rice" --- p.39 / Chapter 3.8 --- Analysis of transgenic expression --- p.41 / Chapter 3.8.1 --- Genomic DNA extraction --- p.41 / Chapter 3.8.2 --- Total RNA extraction --- p.41 / Chapter 3.8.3 --- Synthesis of DIG-labeled DNA probe --- p.42 / Chapter 3.8.4 --- Southern blot analysis --- p.43 / Chapter 3.8.5 --- Northern blot analysis --- p.43 / Chapter 3.8.6 --- Extraction of rice seed protein --- p.43 / Chapter 3.8.7 --- Tricine SDS-PAGE --- p.44 / Chapter 3.8.8 --- Raising AK and DHPS antibody --- p.44 / Chapter 3.8.9 --- Western blot analysis --- p.46 / Chapter 3.9 --- Free amino acid analysis --- p.46 / Chapter CHAPTER 4. --- RESULTS --- p.48 / Chapter 4.1 --- Cloning of AK and DHPS cDNAs from rice --- p.48 / Chapter 4.1.1 --- RNA extraction and cDNAs amplification --- p.43 / Chapter 4.1.2 --- Sequencing of AK and DHPS cDNAs --- p.50 / Chapter 4.2 --- Sequence modification of AK and DHPS cDNAs --- p.50 / Chapter 4.3 --- Construction of chimeric genes --- p.50 / Chapter 4.4 --- AEC resistance of E.coli expressing modified AK and DHPS --- p.56 / Chapter 4.5 --- Rice transformation --- p.58 / Chapter 4.6 --- Detection of target genes in transgenic rice lines --- p.60 / Chapter 4.6.1 --- PCR of genomic DNA --- p.60 / Chapter 4.6.2 --- Southern blot analysis --- p.63 / Chapter 4.7 --- Northern blot analysis --- p.65 / Chapter 4.8 --- Western blot analysis of AK and DHPS proteins --- p.66 / Chapter 4.9 --- Free amino acid analysis --- p.68 / Chapter 4.9.1 --- Free lysine content --- p.68 / Chapter 4.9.2 --- Changes in other amino acids --- p.69 / Chapter CHAPTER 5. --- DISCUSSION --- p.82 / Chapter 5.1 --- Cloning and modification of AK and DHPS cDNAs --- p.82 / Chapter 5.2 --- Seed-specific expression of modified AK and DHPS in rice --- p.82 / Chapter 5.3 --- Free amino acid changes in transgenic rice lines --- p.83 / Chapter 5.4 --- Future perspectives --- p.85 / Chapter CHAPTER 6. --- CONCLUSION --- p.86 / REFERENCES --- p.87 / APPENDIX --- p.102

The availability and assimilation of NH₄⁺ plays an integral role in the growth of microorganisms and the production of amino acids by these organisms. This study investigated the dissociation of NH₄⁺in aqueous solution, its availability and effect on the enzymes of NH₄⁺ assimilation and its influence on lysine production by Corynebacterium glutamicum.In aqueous solution the extent of dissociation of NH₄C1, {NH₄)₂S0₄ and (NH₄)₂HP0₄ increases with decreasing concentration. A model is proposed for the dissociation of these molecules. It is believed that at very low concentrations, dissociation to NH₃ plus the respective counter-ions occurs. At these low concentrations the NH₃ acts as the substrate for glutamine synthetase. At the higher concentrations dissociation is to NH₄⁺ which is the substrate for glutamate dehydrogenase. At these higher concentrations the enzyme activities obtained for glutamate dehydrogenase, at equivalent concentrations of the above ammonium salts, were different when based on the total concentration of NH₄⁺, and similar when based on the concentration of free NH₄⁺. L-Iysine occurs in the +1 ionic form, at pH 7,2. The lysine which is produced during fermentation associates with the anionic counter-ion of the ammonium salt used. The concentration of the free NH₄⁺ in the media appears to affect both the rate of lysine synthesis as well as the yield. The lysine fermentation occurs in two stages; a growth (or replicative) phase, during which very little lysine is produced, and a lysine synthesis (or maturation) phase. During the lysine synthesis phase there is no cell replication, however an increase in the mass of the biomass produced is apparent. Evidence is provided for the possible concomitant synthesis of the the cell wall polymer, glycerol teichoic acid, and lysine. On the basis of this evidence, a nucleotide balance is proposed for lysine and teichoic acid synthesis. The replicative phase and the maturation phase have to be effectively separated to obtain optimal lysine yields and titres. It is believed that teichoic acid synthesis during the replicative phase must be kept to a minimum for optimal yields and titres to be obtained, and on completion of the cell wall and therefore teichoic acid synthesis, lysine synthesis ceases. As the production of lysine appears to be affected by the NH₄⁺ concentration in the culture media, it is proposed that a futile cycle may exist around the transport and assimilation of the NH₄⁺. If the fermentations are run at low free NH₄⁺ concentrations, it was shown that lysine yields of 0,66, on the glucose utilised, are attainable during the fermentation.

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

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

The Amadumbe crop [Colocasia esculenta (L.) Schott] is a traditional Southern African tuber crop which is rich in starch, mucilage and micronutrients. Amadumbe tubers have limited amount of proteins and as a result, amadumbe-processed foods lack adequate protein. The purpose of this study was therefore to develop protein-rich amadumbe-soya composite biscuits, which would be acceptable to consumers. Biscuits were prepared by combining amadumbe and soya flours at ratios: 90:10, 70:30 and 50:50. Functional properties of composite flours and the physical properties of composite biscuits were determined. The proximate composition, amino acid composition and protein digestibility of composite biscuits were determined. Consumer acceptability test of biscuits was performed using nine-point hedonic scale. The results indicated that the 90% amadumbe and 10% soya composite biscuits had high significant values of moisture, ash, carbohydrates contents and energy values. The 50% amadumbe and 50% soya composite biscuits had significantly high values of fat, crude protein contents and acid detergent fibre (ADF). The protein digestibility, amino acid contents, especially the lysine contents of composite biscuits increased significantly (p ≤ 0.05) with an increase in the percentage of soya. The mineral contents of composite biscuits; Ca, Mg, P, Zn, Cu, Mn and Fe increased significantly (p ≤ 0.05) with the increase of soya in the composite biscuits. There was a significant difference in the mean taste acceptability and mean overall acceptability when the soya concentration was increased to 50%. Soya was successfully used to produce amadumbe composite biscuits with better nutritional quality with respect to protein content, amino acid profile and selected mineral contents and which were acceptable to consumers. / National Research Foundation (South Africa) / Life and Consumer Science / M.Sc. (Agriculture)

Indiana University-Purdue University Indianapolis (IUPUI) / Histones are highly basic proteins which when digested by trypsin are hard to analyze using mass spectrometry. Because histones are basic nuclear proteins, a nuclei prep followed by acid extraction is the best purification strategy to increase overall abundance of purified histones. Blocking the lysine residues and cleaving with trypsin is a useful technique to increase detection of histone peptides using MudPIT. In particular, carbamylation and propionylation are the best two methods to block lysine residues. Using both propionylation and carbamylation along with no treatment has been shown to increase the identification of unmodified and modified histone peptides when coupled with MudPIT analysis.