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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326492 |
| Date | January 2008 |
| Contributors | Yu, Chun Wai., Chinese University of Hong Kong Graduate School. Division of Biology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, 129 leaves : ill. ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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