Lu Shanxiang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 122-135). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Figures --- p.xiii / List of Tables --- p.xv / List of Abbreviations --- p.xvi / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter 1.1 --- Lysine-rich protein from winged bean --- p.2 / Chapter 1.1.1 --- Discovery --- p.2 / Chapter 1.1.2 --- Applications in enhancing nutritional values --- p.2 / Chapter 1.2 --- Plant secretory pathway --- p.4 / Chapter 1.2.1 --- Overview of plant secretory pathway --- p.4 / Chapter 1.2.2 --- Three models on protein transportation from ER to Golgi --- p.6 / Chapter 1.2.3 --- Brefeldin A: inhibitor of secretion --- p.9 / Chapter 1.2.4 --- Markers for different organelles --- p.10 / Chapter 1.3 --- Tobacco bright yellow 2 (BY-2) cell system --- p.11 / Chapter 1.3.1 --- Origin of BY-2 cell line --- p.12 / Chapter 1.3.2 --- Characteristics of BY-2 cell line --- p.12 / Chapter 1.4 --- Use of fluorescent proteins as reporters --- p.13 / Chapter 1.4.1 --- GFP and its derivatives --- p.13 / Chapter 1.4.2 --- Reporter system --- p.15 / Chapter 1.4.3 --- Applications of GFP and its derivatives in plants --- p.16 / Chapter 1.5 --- Temperature effects on plants --- p.17 / Chapter 1.6 --- Project objectives --- p.18 / Chapter Chapter 2 --- Subcellular localization of LRP in winged bean (Psophocarpus tetragonolobus)seeds --- p.20 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Chemicals --- p.21 / Chapter 2.2.2 --- Plant materials --- p.21 / Chapter 2.2.3 --- Antibodies --- p.22 / Chapter 2.2.4 --- Western blot --- p.23 / Chapter 2.2.4.1 --- Protein extraction --- p.23 / Chapter 2.2.4.2 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.23 / Chapter 2.2.4.3 --- Immunodetection --- p.24 / Chapter 2.2.5 --- Confocal Immunofluorescence --- p.25 / Chapter 2.2.5.1 --- Preparation of samples for immuno-labeling --- p.25 / Chapter 2.2.5.2 --- Immuno-labeling --- p.26 / Chapter 2.2.5.3 --- Collection and analysis of confocal fluorescent images --- p.27 / Chapter 2.2.6 --- Immuno transmission electron microscope (TEM) study --- p.28 / Chapter 2.2.6.1 --- Preparation of samples --- p.28 / Chapter 2.2.6.2 --- Immuno-labeling --- p.29 / Chapter 2.3 --- Results --- p.31 / Chapter 2.3.1 --- Anti-alpha-TIP and anti-LRP antibodies have good specificity in winged bean seeds --- p.31 / Chapter 2.3.2 --- Anti-a-TIP antibodies could label the PSVs of winged bean seeds specifically --- p.31 / Chapter 2.3.3 --- LRP was localized outside of PSVs --- p.33 / Chapter 2.3.4 --- Immuno-TEM localization of LRP --- p.35 / Chapter 2.4 --- Conclusion and discussion --- p.40 / Chapter Chapter 3 --- Generation of Transgenic Tobacco BY-2 Cell Lines Expressing YFP and LRP Fusions --- p.41 / Chapter 3.1 --- Introduction --- p.42 / Chapter 3.2 --- Materials and methods --- p.42 / Chapter 3.2.1 --- Primers --- p.42 / Chapter 3.2.2 --- Plant materials --- p.43 / Chapter 3.2.3 --- Bacterial strains --- p.44 / Chapter 3.2.4 --- Construction of fusion constructs --- p.44 / Chapter 3.2.4.1 --- Four fusion constructs of LRP and YFP --- p.44 / Chapter 3.2.4.2 --- His-tag-YFP fusion construct --- p.45 / Chapter 3.2.4.3 --- Cloning of the fusion protein genes into Agrobacterium binary vector pBI121 --- p.45 / Chapter 3.2.5 --- Confirmation of the fusion constructs --- p.53 / Chapter 3.2.6 --- Transformation of Agrobacterium by electroporation --- p.53 / Chapter 3.2.7 --- "Transformation, selection and suspension of tobacco BY-2 cells" --- p.54 / Chapter 3.2.8 --- "Transformation, screening and induction of E. coli BL21-DE3 for expression of His-tagged YFP" --- p.55 / Chapter 3.2.9 --- Protein extraction --- p.55 / Chapter 3.2.9.1 --- Protein fractionation from BY-2 cells --- p.55 / Chapter 3.2.9.2 --- protein extraction from E. coli of BL21-DE3 --- p.56 / Chapter 3.2.10 --- Immunolabeling of suspension cultured cells --- p.56 / Chapter 3.2.11 --- Raising anti-GFP antibodies --- p.57 / Chapter 3.2.12 --- Dot blot analysis --- p.58 / Chapter 3.2.13 --- Affinity purification of proteins and antibodies --- p.59 / Chapter 3.2.13.1 --- Metal affinity resin column for protein purification --- p.59 / Chapter 3.2.13.2 --- Cyanogens bromide (CNBr) activated sepharose column for antibody purification --- p.60 / Chapter 3.2.14 --- SDS-PAGE and western blot analysis --- p.61 / Chapter 3.2.15 --- Antibodies --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Two transgenic BY-2 cell lines showed different fluorescent signal patterns --- p.62 / Chapter 3.3.2 --- Two cell lines showed different fluorescent signal stability --- p.63 / Chapter 3.3.3 --- The two fusion proteins were localized in different places in the BY-2 cells --- p.67 / Chapter 3.3.4 --- """Green"" E. coli expressed the recombinant YFP" --- p.69 / Chapter 3.3.5 --- Expressed recombinant YFP could not be affinity purified --- p.69 / Chapter 3.3.6 --- Raised polyclonal anti-GFP antibodies showed good specificity --- p.69 / Chapter 3.4 --- Conclusion and discussion --- p.75 / Chapter Chapter 4 --- Characterization of SpYFP-LRP Fusion in Transgenic BY-2 cells --- p.76 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Materials and Methods --- p.77 / Chapter 4.2.1 --- Plant materials --- p.77 / Chapter 4.2.2 --- BFA and heat treatment --- p.77 / Chapter 4.2.3 --- Confocal immunolabeling --- p.78 / Chapter 4.2.4 --- Conventional TEM study --- p.78 / Chapter 4.2.5 --- Immuno TEM using Lowicryl resin and LR White resin --- p.80 / Chapter 4.3 --- Results --- p.82 / Chapter 4.3.1 --- "BFA induced the SpYFP-LRP-marked organelle to form ""BFA-induced"" compartments" --- p.82 / Chapter 4.3.2 --- Partial recovery from BFA treatment --- p.84 / Chapter 4.3.3 --- SpYFP-LRP was localized in BFA-induced compartments --- p.87 / Chapter 4.3.4 --- BFA treatment induced the formation of various compartmentsin SpYFP-LRP cells --- p.90 / Chapter 4.3.5 --- BFA-induced structures contain SpYFP-LRP --- p.99 / Chapter 4.3.6 --- Elevated temperature affected the signal pattern but not the localization of SpYFP-LRP in transgenic BY-2 cells --- p.100 / Chapter 4.3.7 --- Elevated temperature treatment induced the SPYFP-LRP cells to form new vesicular compartments --- p.105 / Chapter 4.4 --- Conclusions and Discussion --- p.112 / Chapter 4.4.1 --- BFA treatment --- p.112 / Chapter 4.4.2 --- Heat treatment --- p.114 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.116 / Chapter 5.1 --- Summary --- p.117 / Chapter 5.2 --- Future perspectives --- p.119 / Reference --- p.122
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324967 |
| Date | January 2004 |
| Contributors | Lu, Shanxiang., 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, xvii, 135 leaves : ill. (some col.) ; 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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