No description available.

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

Thesis (Ph. D.)--Oregon State University, 2002. / Typescript (photocopy). Includes bibliographical references (leaves 171-190). Also available on the World Wide Web.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--Washington State University, May 2007. / Includes bibliographical references.

No description available.

Cheung Siu Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 86-91). / 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 Tables --- p.xiii / List of Figures --- p.xiv / Lists of Abbreviations --- p.xvii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- The plant secretory pathways --- p.2 / Chapter 1.1.1 --- Three different protein sorting pathways to plant vacuoles --- p.3 / Chapter 1.1.2 --- VSD and VSR --- p.6 / Chapter 1.2 --- Prevacuolar compartments --- p.7 / Chapter 1.2.1 --- Lytic PVC --- p.7 / Chapter 1.2.2 --- BP-80 reporter as a lytic PVC marker --- p.8 / Chapter 1.2.3 --- PVC of PSV --- p.9 / Chapter 1.2.4 --- α-TIP CT reporter as a PVC of PSV marker --- p.10 / Chapter 1.3 --- Project objectives --- p.11 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Golgi and Prevacuolar Compartments / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.2 --- Materials and Methods --- p.15 / Chapter 2.2.1 --- Chemicals --- p.15 / Chapter 2.2.2 --- Oligonucleotides: Primers and Adapters --- p.15 / Chapter 2.2.3 --- Bacterial Strains --- p.17 / Chapter 2.2.4 --- "Preparation of single-reporter constructs (GONST1 -CFP, CFP-BP-80 and CFP-a-TIP CT reporters)" --- p.17 / Chapter 2.2.4.1 --- "Cloning of pGONSTl-CFPK, a Golgi marker" --- p.17 / Chapter 2.2.4.2 --- "Cloning of pCFP-BP-80K, a lytic PVC marker" --- p.20 / Chapter 2.2.4.3 --- "Cloning of pCFP-α-TIP CTK, a putative marker for PVC of PSV" --- p.22 / Chapter 2.2.5 --- "Preparation of double-reporter constructs (CFP-BP-80-GONST1 - YFP, CFP-α-TIP CT-GONST1-YFP, CFP-BP-80-YFP-α-TIP CT and CFP-α-TIP CT-YFP-BP-80 reporters)" --- p.24 / Chapter 2.2.5.1 --- Insertion ofAdapter-XH to pCFP-BP-80K and pCFP-α-TIP CTK --- p.24 / Chapter 2.2.5.2 --- "Cloning of pCFP-BP-80-GONST 1 -YFPK, pCFP-α-TIP CT- GONST 1-YFPK, pCFP-BP-80-YFP-α-TIP CTK and pCFP- α-TIP CT-YFP-BP-80K" --- p.26 / Chapter 2.2.6 --- Agrobacterium electroporation --- p.30 / Chapter 2.2.7 --- Agrobacterium-mediated transformation of tobacco BY-2 cells --- p.30 / Chapter 2.2.8 --- Selection and screening of transformed BY-2 cells --- p.31 / Chapter 2.2.8.1 --- Antibiotic selection --- p.31 / Chapter 2.2.8.2 --- Fluorescence microscopic screening --- p.31 / Chapter 2.2.9 --- Detection of CFP and YFP reporter genes and their expressions --- p.32 / Chapter 2.2.9.1 --- CTAB genomic DNA extraction --- p.32 / Chapter 2.2.9.2 --- PCR test for CFP (and YFP) transgene in genomic DNA --- p.33 / Chapter 2.2.9.3 --- Subcellular fractionation and protein extraction --- p.33 / Chapter 2.2.9.4 --- Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.2.9.5 --- Confocal microscopic study --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Establishment of kanamycin-resistant BY-2 cells expressing CFP (and YFP) reporters --- p.36 / Chapter 2.3.2 --- Fluorescence microscopic screening of transgenic BY-2 cell lines --- p.37 / Chapter 2.3.3 --- CFP (and YFP) reporter was successfully integrated into transgenic BY-2 cell genome --- p.41 / Chapter 2.3.4 --- CFP (and YFP) reporter was expressed in transgenic BY-2 cell lines --- p.44 / Chapter 2.3.5 --- Punctate CFP (and YFP) signals were detected in transgenic BY-2 cell lines expressing single (or double) reporter --- p.48 / Chapter 2.4 --- Discussion --- p.53 / Chapter 2.4.1 --- "Transgenic BY-2 cell lines expressing single reporter marking Golgi, lytic PVC and putative PVC of PSV have been developed" --- p.53 / Chapter 2.4.2 --- "Golgi, lytic PVC and putative PVC of PSV were separate and distinct organelles" --- p.53 / Chapter 2.4.3 --- Transgenic BY-2 cell lines expressing double reporter were not yet suitable for subsequent study --- p.55 / Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Prevacuolar Compartments / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.60 / Chapter 3.2.1 --- Confocal immunofluorescence study --- p.60 / Chapter 3.2.2 --- Drug treatment study (for single-reporter transgenic tobacco BY-2 cell line) --- p.62 / Chapter 3.2.2.1 --- Wortmannin treatment --- p.62 / Chapter 3.2.2.1.1 --- Dosage effect --- p.62 / Chapter 3.2.2.1.2 --- Time-course study --- p.62 / Chapter 3.2.2.2 --- Brefeldin A treatment --- p.63 / Chapter 3.2.2.1.1 --- Dosage effect --- p.63 / Chapter 3.2.2.1.2 --- Time-course study --- p.63 / Chapter 3.2.3 --- Drug treatment study (for double-reporter transgenic tobacco BY-2 cell line) --- p.64 / Chapter 3.2.3.1 --- Wortmannin treatment --- p.64 / Chapter 3.2.3.2 --- Brefeldin A treatment --- p.64 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.65 / Chapter 3.3.2 --- Wortmannin induced CFP-α-TIP CT reporter-marked compartment to vacuolate --- p.69 / Chapter 3.3.3 --- BFA induced CFP-α-TIP CT reporter-marked compartment to form aggregates --- p.72 / Chapter 3.3.4 --- Wortmannin and BFA treatment caused lytic PVC to form small vacuole and Golgi to form aggregate respectively in transgenic BY-2 cell lines expressing double-reporter --- p.75 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.77 / Chapter 3.4.2 --- CFP-α-TIP CT reporter-marked compartment was not lytic PVC --- p.77 / Chapter 3.4.3 --- Transgenic BY-2 cell lines expressing double reporter could successfully mark two compartments simultaneously in the same cell --- p.78 / Chapter Chapter 4 --- Summary and Future Prospects / Chapter 4.1 --- Summary --- p.80 / Chapter 4.1.1 --- Hypothesis --- p.80 / Chapter 4.1.2 --- Development of transgenic tobacco BY-2 cell lines --- p.81 / Chapter 4.1.3 --- Characterization of α-TIP CT reporter-marked PVC-like compartment --- p.82 / Chapter 4.2 --- Conclusions --- p.84 / Chapter 4.3 --- Future prospects --- p.85 / References --- p.86

Fung Ka Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 83-93). / 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 Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- An overview of the secretory pathway in eukaryotic cells --- p.2 / Chapter 1.2 --- The secretory pathway in plants --- p.4 / Chapter 1.2.1 --- Plant cells contain two functionally distinct vacuoles --- p.4 / Chapter 1.2.2 --- Three vesicular pathways to two vacuole --- p.6 / Chapter 1.2.3 --- Transport vesicles in the three vesicular pathways --- p.9 / Chapter 1.2.4 --- Vacuolar sorting determinants (VSDs) --- p.10 / Chapter 1.2.5 --- Vacuolar sorting receptors (VSRs) --- p.12 / Chapter 1.3 --- The PSVs in mature seeds --- p.15 / Chapter 1.3.1 --- Biogenesis of PSV --- p.15 / Chapter 1.3.2 --- The two chimeric integral membrane reporters --- p.16 / Chapter 1.3.3 --- Subcellular localization of the two chimeric integral membrane reporters in PSVs of mature tobacco seeds --- p.17 / Chapter 1.4 --- Project objectives --- p.19 / Chapter Chapter 2 --- Materials and Methods --- p.20 / Chapter 2.1 --- Construction of the YFP-BP-80 and the YFP- a -TIP reporters --- p.21 / Chapter 2.1.1 --- The pYFP-BP-80-K construct --- p.21 / Chapter 2.1.2 --- The pYFP- a -TIP-K construct --- p.22 / Chapter 2.2 --- Construction of GFP-RMR reporter --- p.23 / Chapter 2.2.1 --- Cloning of pGFP-RMR --- p.23 / Chapter 2.2.2 --- Cloning of pGFP-RMR-K --- p.23 / Chapter 2.3 --- Construction of pGONST1-YFP construct --- p.26 / Chapter 2.3.1 --- The pGONSTl-YFP construct --- p.26 / Chapter 2.4 --- Transformation of Agrobacterium by electroporation --- p.27 / Chapter 2.5 --- Tobacco transformation and selection --- p.28 / Chapter 2.5.1 --- Plant materials --- p.28 / Chapter 2.5.2 --- Tobacco transformation --- p.28 / Chapter 2.6 --- Screening of transgenic tobacco plants expressing YFP fusion proteins --- p.30 / Chapter 2.6.1 --- Kanamycin screening --- p.30 / Chapter 2.6.2 --- Extraction of genomic DNA from leaves --- p.30 / Chapter 2.6.3 --- PCR of genomic DNA --- p.31 / Chapter 2.7 --- Southern blot analysis of genomic DNA --- p.32 / Chapter 2.8 --- Western blot analysis of transgenic tobacco plants --- p.33 / Chapter 2.8.1 --- Extraction of total protein from tobacco leaves or seeds --- p.33 / Chapter 2.8.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.9 --- Confocal immunofluorescence studies --- p.35 / Chapter 2.9.1 --- Preparation of sections --- p.35 / Chapter 2.9.2 --- Single labeling --- p.35 / Chapter 2.9.3 --- Double labeling with one polyclonal and one monoclonal antibodies --- p.36 / Chapter 2.9.4 --- Double labeling with two polyclonal antibodies --- p.36 / Chapter 2.9.5 --- Collection of images --- p.37 / Chapter 2.10 --- Chemicals --- p.38 / Chapter 2.11 --- Primers --- p.38 / Chapter 2.12 --- Bacterial strain --- p.38 / Chapter 2.13 --- Antibodies --- p.39 / Chapter 2.14 --- Growing condition of transgenic plants and determining the developmental stage of tobacco flowers --- p.39 / Chapter Chapter 3 --- Results --- p.41 / Chapter 3.1 --- Generation of transgenic tobacco plants --- p.42 / Chapter 3.2 --- PCR screening of transgenic tobacco plants --- p.46 / Chapter 3.3 --- Southern blot analysis --- p.48 / Chapter 3.4 --- Detection of the YFP fusion proteins in transgenic tobacco plants by western blot analysis --- p.50 / Chapter 3.4.1 --- Detection of the YFP fusion proteins in leaves --- p.50 / Chapter 3.4.2 --- Western blot analysis of vegetative tissues --- p.57 / Chapter 3.4.3 --- Western blot analysis of mature seeds --- p.59 / Chapter 3.5 --- Confocal immunofluorescence studies --- p.61 / Chapter 3.5.1 --- Detection of YFP signals in root tip cells --- p.61 / Chapter 3.5.2 --- Detection of YFP signals in developing seeds --- p.65 / Chapter 3.5.3 --- Subcellular localization of the YFP fusion proteins in mature seeds --- p.67 / Chapter Chapter 4 --- Discussion --- p.72 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.77 / Chapter 5.1 --- Summary --- p.78 / Chapter 5.1.1 --- Generation of transgenic tobacco plants expressing the YFP fusion proteins --- p.78 / Chapter 5.1.2 --- Full-length fusion proteins and cleaved soluble YFP were detected in vegetative tissues --- p.79 / Chapter 5.1.3 --- Only cleaved soluble YFP was detected in mature seeds --- p.79 / Chapter 5.1.4 --- The two fusion proteins might localized in different compartments in developing seeds --- p.79 / Chapter 5.1.5 --- Both fusion proteins were localized within the PSVs of mature seeds --- p.80 / Chapter 5.2 --- Future perspectives --- p.81 / References --- p.83