Liu, Yang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 99-104). / Abstracts in English and Chinese. / Table of Contents / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The plant secretory pathway --- p.2 / Chapter 2. --- Vesicular pathways in plant cells --- p.3 / Chapter 3. --- Prevacuolar Compartments --- p.6 / Chapter 4. --- Vacuolar sorting receptors (VSRs) --- p.7 / Chapter 5. --- BP-80 & Arabidopsis VSR Proteins --- p.8 / Chapter 6. --- Research Objectives --- p.9 / Chapter Chapter 2 --- Development and Expression of GFP-OsVSRs Fusion Reporters in Tobacco BY-2 and Rice Suspension Cultured Cells --- p.11 / Chapter 1. --- Introduction --- p.12 / Chapter 2. --- Materials and methods --- p.14 / Chapter 2.1 --- Construction of GFP-OsVSR chimeric reporters --- p.14 / Chapter 2.2 --- Construction of Golgi Marker and PVC marker --- p.18 / Chapter 2.3 --- Agrobacterium electroporation --- p.27 / Chapter 2.4 --- Transformation of tobacco BY-2 cells --- p.27 / Chapter 2.5 --- Transient expression of GFP-OsVSRs in protoplasts of tobacco BY-2 cells and rice suspension cultured cells --- p.28 / Chapter 2.6 --- Screening of transgenic BY-2 cells expressing GFP-OsVSR reporters --- p.30 / Chapter 2.7 --- Chemicals --- p.33 / Chapter 3. --- Results --- p.34 / Chapter 3.1 --- Study subcellular localization of OsVSR proteins with chimeric GFP-OsVSR reporters --- p.34 / Chapter 3.2 --- Generation of transgenic tobacco BY-2 cell lines expressing GFP-OsVSR reporter constructs --- p.38 / Chapter 3.3 --- Transient expression of GFP-OsVSR reporters in tobacco BY-2 and rice cell protoplasts --- p.40 / Chapter 4. --- Conclusion --- p.42 / Chapter Chapter 3 --- Subcellular Localization of GFP-OsVSR Fusion Reporters in Tobacco BY-2 and Rice Suspension Cultured Cells --- p.43 / Chapter 1. --- Introduction --- p.44 / Chapter 2. --- Materials and methods --- p.45 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.45 / Chapter 2.2 --- Antibodies --- p.46 / Chapter 2.3 --- Wortmannin and BFA drug treatment --- p.46 / Chapter 2.4 --- Electron microscopy of resin-embedded cells --- p.47 / Chapter 2.5 --- Two-dimensional (2-D) gel analysis --- p.47 / Chapter 3 --- Results --- p.49 / Chapter 3.1 --- "Distinct subcellular localizations of GFP-OsVSRl, GFP-OsVSR2 and GFP-OsVSR4 reporters in transgenic BY-2 cell lines" --- p.49 / Chapter 3.2 --- "Subcellular localizations of GFP-OsVSRl, GFP-OsVSR2 and GFP-OsVSR4 in protoplasts of rice suspension cultured cells" --- p.58 / Chapter 3.3 --- Distinct localizations of GFP-OsVSR3 and GFP-OsVSR5 --- p.62 / Chapter 3.4 --- Immunogold EM localization of VSR proteins in rice suspension cultured cells --- p.65 / Chapter 3.5 --- 2-D western blot detection of VSR proteins in various plants --- p.68 / Chapter 4. --- Conclusions --- p.70 / Chapter Chapter 4 --- Summary and Discussion --- p.71 / Chapter 1. --- The significance of this study --- p.72 / Chapter 2. --- The hypothesis in this study --- p.73 / Chapter 3. --- A reporter system to study subcellular localization of OsVSR proteins in both tobacco BY-2 cells and rice suspension cultured cells --- p.75 / Chapter 4. --- Transiently expression of GFP-OsVSR reporters in BY-2 and rice protoplasts ..… --- p.76 / Chapter 5. --- Distinct PVC and Golgi localizations of GFP-OsVSR fusions --- p.77 / Chapter 6. --- Summary and future perspective --- p.78 / Appendix --- p.79 / Characterization of A Novel Rice Protein --- p.79 / Chapter 1. --- Introduction --- p.80 / Chapter 2. --- Materials and methods --- p.83 / Chapter 2.1 --- Antibodies --- p.83 / Chapter 2.2 --- Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and western blot analysis of proteins from different plant species --- p.84 / Chapter 2.3 --- Sucrose gradient fractionation with protein F antibody --- p.84 / Chapter 2.4 --- Confocal immunofluorescence studies --- p.85 / Chapter 2.5 --- Affinity purification of Protein F by its antibody --- p.85 / Chapter 3. --- Results --- p.87 / Chapter 3.1 --- Protein F is presented in different plant species --- p.87 / Chapter 3.2 --- Protein F is an integral membrane protein --- p.89 / Chapter 3.3 --- Subcelluar localization of Protein F --- p.91 / Chapter 3.4 --- Affinity purification of Protein F for identification --- p.95 / Chapter 4. --- Summary and future perspectives --- p.97 / Chapter 4.1 --- Summary --- p.97 / Chapter 4.2 --- Future Perspectives --- p.97 / References --- p.99

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 86-100). Also available on the World Wide Web.

abstract: The green fluorescent protein (GFP)-like fluorescent proteins play an important role for the color of reef-building corals. Different colors of extant coral fluorescent proteins (FPs) have evolved from a green ancestral protein. Interestingly, green-to-red photoconversion FPs (Kaede-type Red FPs) are only found in clade D from Scleractinia (Faviina suborder). Therefore, I focus on the evolution of Kaede-type FPs from Faviina suborder ancestral FP. A total of 13 mutations have been identified previously that recapitulate the evolution of Kaede-type red FPs from the ancestral green FP. To examine the effect of each mutation, total ten reconstructed FPs were analyzed and six x-ray crystal structures were solved. These substitutions created a more hydrophilic environment around the carbonyl group of Phe61. Also, they increased the flexibility of the c-terminal chain, which keeps it from interacting with the entrance of the putative solvent channel. The photoconversion reaction shows a twophase kinetics. After the rapid initial phase, the overall reaction followed the firstorder kinetics. Based on the crystal structure analysis, I propose a new mechanism for Kaede-type FP photoconversion process, which a proton transfers via Gln38 to the carbonyl group of Phe61. / Dissertation/Thesis / Ph.D. Chemistry 2012

abstract: Proteins function as molecular machines which perform a diverse set of essential jobs. To use these proteins as tools and manipulate them with directed engineering, a deeper understanding of their function and regulation is needed. In the studies presented here, the chemical mechanism of a fluorescent protein and the assembly behavior of a chemo-mechanical protein were explored to better understand their operation. In the first study a photoconvertible fluorescent protein (pcFP) was examined which undergoes a photochemical transformation upon irradiation with blue light resulting in an emission wavelength change from green to red. Photo-transformable proteins have been used in high resolution, subcellular biological imaging techniques, and desires to engineer them have prompted investigations into the mechanism of catalysis in pcFPs. Here, a Kinetic Isotope Effect was measured to determine the rate-limiting step of green-to-red photoconversion in the reconstructed Least Evolved Ancestor (LEA) protein. The results provide insight on the process of photoconversion and evidence for the formation of a long-lived intermediate. The second study presented here focuses on the AAA+ protein Rubisco activase (Rca), which plays a critical role in the removal of inhibitors from the carbon-dioxide fixing enzyme Rubisco. Efforts to engineer Rubisco and Rca can be guided by a deeper understanding of their structure and interactions. The structure of higher plant Rca from spinach, and its interactions with its cognate Rubisco, were investigated through negative-stain electron microscopy (EM) and cryo-EM experiments. Multiple types of higher-order oligomers of plant Rca were imaged which have never been structurally characterized, and the AAA+ core of plant Rca was shown to bind Rubisco side-on, similar to bacterial Rca’s. Higher resolution structures of these aggregates and complexes are needed to make definitive observations on protein-protein interactions. However, the results presented here provide evidence for the formation of regulatory structures and an experimental foundation for future exploration of plant Rca through cryo-EM. / Dissertation/Thesis / Masters Thesis Biochemistry 2020

Transgenic techniques have rapidly evolved in recent years. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. The purpose of this study was to assess in vitro development, transgene expression, and integration following pronuclear or cytoplasmic microinjection of condensed or linear green fluorescent protein DNA into murine embryos using electroporation. In experiment 1, the effect of embryo orientation (group or linear) within the electroporation chamber on development was evaluated using zygotes which received one pulse duration (10 msec), and one of two voltages (250 or 400 V). Zygotes that received 400 V had the lowest development score (Group, 2.06 ? 0.12; Linear, 1.97 ? 0.13), irrespective of orientation. Embryos that received 250 V had the highest development of the voltage treated groups (Group 3.42 ? 0.12; Linear 3.32 ? 0.12), irrespective of orientation, and development was lower than the control embryos (Control 4.28 ? 0.12; Mannitol control 4.36 ? 0.18). In experiment 2, the efficiency of utilization of the prepared enhanced green fluorescent protein (EGFP) construct as a visual marker of protein expression was evaluated using pronuclear microinjection. Embryo development and fluorescence were evaluated following pronuclear injection of EGFP at a concentration of 3 μg/ml and compared to an uninjected control. Embryos injected with the EGFP had lower development scores (3.85 ± 0.15) than uninjected control embryos (5.72 ± 0.2). Of the embryos injected, 32.4% fluoresced due to expression of EGFP. Experiment 3 evaluated the effect of combining cytoplasmic injection of EGFP (425 μg/ml) with electroporation at 250 V on EGFP expression. The non-manipulated control embryos had significantly higher (P < 0.01) 4 d development scores (5.57 ± 0.11) than manipulated control embryos (4.6 ± 0.18), where the injection needle was inserted into the cytoplasm and no DNA was injected. Combining cytoplasmic DNA injection and electroporation caused a significant (P < 0.01) decrease in development scores, irrespective of DNA construct, when compared to embryos injected with a DNA construct alone. The mechanical effects of needle insertion combined with electroporation were not significantly different (P > 0.05) from embryos injected with DNA alone, irrespective of construct injected. Cytoplasmic injection of condensed DNA (0.38%), linear DNA (0.38%), and condensed DNA combined with electroporation (0.36%) resulted in one fluorescent embryo respectively. Cytoplasmic injection of linear DNA when combined with electroporation (3.57%) resulted in 13 fluorescent embryos. Pronuclear injection of the prepared EGFP construct results in lower development than control embryos. Electrical stimulation of zygotes reduces early embryo development. However, low amounts of electrical stimulation may allow for enhancement of gene integration in transgenic embryos. / Master of Science

Yes / We report a simple temperature-responsive bioconjugate system comprising superfolder green fluorescent protein (sfGFP) decorated with poly[(oligo ethylene glycol) methyl ether methacrylate] (PEGMA) polymers. We used amber suppression to site-specifically incorporate the non-canonical azide-functional amino acid p-azidophenylalanine (pAzF) into sfGFP at different positions. The azide moiety on modified sfGFP was then coupled using copper-catalyzed “click” chemistry with the alkyne terminus of a PEGMA synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization. The protein in the resulting bioconjugate was found to remain functionally active (i.e., fluorescent) after conjugation. Turbidity measurements revealed that the point of attachment of the polymer onto the protein scaffold has an impact on the thermoresponsive behavior of the resultant bioconjugate. Furthermore, small-angle X-ray scattering analysis showed the wrapping of the polymer around the protein in a temperature-dependent fashion. Our work demonstrates that standard genetic manipulation combined with an expanded genetic code provides an easy way to construct functional hybrid biomaterials where the location of the conjugation site on the protein plays an important role in determining material properties. We anticipate that our approach could be generalized for the synthesis of complex functional materials with precisely defined domain orientation, connectivity, and composition. / Engineering and Physical Sciences Research Council (EPSRC), University of Warwick, National Science Foundation (U.S.) (NSF), United States. Defense Advanced Research Projects Agency (DARPA), Seventh Framework Programme (European Commission) (FP7), European Research Council (ERC)

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 143-151). Also available for download via the World Wide Web; free to University of Oregon users.

Miao Yansong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 78-83). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / 摘要 --- p.vi / Acknowledgements --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The plant secretory pathway --- p.2 / Chapter 2. --- Two different types of vacuoles in plant cells --- p.2 / Chapter 3. --- Vacuolar sorting receptor (VSR) proteins --- p.3 / Chapter 4. --- BP-80 and prevacuolar compartment --- p.6 / Chapter 5. --- The Arabidopsis VSR proteins --- p.7 / Chapter 6. --- Research objectives --- p.8 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing GFP-AtVSR Fusions --- p.10 / Chapter 1. --- Introduction --- p.11 / Chapter 2. --- Materials and Methods --- p.12 / Chapter 2.1 --- Structure of Golgi marker and PVC marker --- p.12 / Chapter 2.2 --- Construction of GFP-VSR reporters --- p.14 / Chapter 2.3 --- Agrobacterium electroporation --- p.24 / Chapter 2.4 --- Transformation of tobacco BY-2 cells --- p.24 / Chapter 2.5 --- Screening of transgenic BY-2 cells expressing GFP-VSR fusions --- p.25 / Chapter 2.6 --- Chemicals --- p.27 / Chapter 3. --- Result.s --- p.28 / Chapter 3.1 --- Chimeric GFP reporters as tools to study subcellular localization of Arabidopsis vacuolar sorting receptor proteins in transgenic BY-2 cells --- p.28 / Chapter 3.2 --- Establishment of transgenic tobacco (Nicotiana tabacum) BY-2 cell lines stably expressing seven GFP-AtVSR reporters --- p.29 / Chapter 4. --- Conclusion --- p.38 / Chapter Chapter 3 --- Subcellular Localization of the Seven GFP-AtVSR Fusions to Prevacuolar Compartments in Transgenic Tobacco BY-2 Cells --- p.39 / Chapter 1. --- Introduction --- p.40 / Chapter 2. --- Materials and Methods --- p.41 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.41 / Chapter 2.2 --- Antibodies for immunolabeling --- p.42 / Chapter 2.3 --- Wortmannin and brefeldin A treatment --- p.42 / Chapter 2.4 --- Electron microscopy of resin-embedded cells --- p.43 / Chapter 3. --- Results --- p.44 / Chapter 3.1 --- Vacuolar sorting receptor proteins in plants --- p.44 / Chapter 3.2 --- PVC localization of GFP-AtVSR fusions in transgenic tobacco BY-2 cells --- p.47 / Chapter 3.3 --- The spacer sequences did not affect PVC localization of GFP-AtVSR7 --- p.56 / Chapter 3.4 --- Wortmannin-induced vacuolated PVCs contained VSRs in tobacco BY-2 cells --- p.58 / Chapter 3.5 --- Wortmannin-induced vacuolation of PVCs is a general response in plant cells --- p.62 / Chapter 4. --- Conclusion --- p.65 / Chapter Chapter 4 --- Discussion and Future Perspectives --- p.66 / Chapter 1. --- The hypothesis in this study --- p.67 / Chapter 2. --- GFP and BY-2 cells --- p.67 / Chapter 3. --- A reporter system to study subcellular localization of VSR proteins in transgenic tobacco BY-2 cells --- p.68 / Chapter 4. --- PVC localization of the seven GFP-AtVSR fusions in transgenic BY-2 cells --- p.69 / Chapter 5. --- VSR spacer sequences did not affect PVC localization of GFP-AtVSR fusions in transgenic tobacco BY-2 cells --- p.71 / Chapter 6. --- PVC localization of GFP-PV72 and GFP-AtVSR 1 fusions in transgenic tobacco BY-2 cells --- p.73 / Chapter 7. --- Wortmannin-induced vacuolation of PVC is a general response in plant cells --- p.75 / Chapter 8. --- Future perspectives --- p.75 / References --- p.78

<i>Pseudomonas fluorescens</i> BRG100 produces secondary metabolites with herbicidal activity to the grass weeds wild oat, Avena fatua, and green foxtail, Setaria viridis. The green fluorescence protein (gfp) gene was introduced into P. fluorescens BRG100 from Escherichia coli S17-1¥ë via a Tn5 mini transposon suicide vector system. Colony morphology, growth rate in liquid media, weed biocontrol efficacy (plant growth pouch), carbon utilization (Biolog GN) and root colonization of green foxtail by several P. fluorescens BRG100gfp transformants were determined to be the same as the wild type. <i>Pseudomonas fluorescens</i> BRGgfp-15 was found to be most similar to the wild-type in all of the above characteristics and was thus used in subsequent experiments. Note: all strains of Pseudomonas fluorescens will be referred to by only their strain throughout (ie. BRGgfp-15 and BRG100). <p>It was determined by population dynamics per section of root with spiral plating on culture medium, epi-fluorescence and confocal microscopy that BRGgfp-15 colonized all areas of the root, but showed a preference for the proximal 1/3 section and the seed. In the proximal section the mean number of viable cells per gram dry weight was log109.06 and log109.31, when applied as liquid inoculum and as the pesta granular formulation, respectively. With liquid inoculum there was only log107.53 viable cells/g in the middle 1/3 section and log107.01 viable cells/g in the distal 1/3 section. The number of viable cells/g with pesta granules was log107.61 and log107.34, for the middle and distal sections, respectively. The root hairs, root tip, and ventral portion of the seed were all areas of heavy colonization relative to the other areas of the root. <p>Survival of BRGgfp-15 in the pesta formulation was examined in 2 soil types, clay and clay loam, in a thermogradient plate apparatus by a factorial randomized design complete block experiment. The experiment included: 3-12 hour diurnal temperature regimes: 5-15¨¬C, 15-25¨¬C, and 25-35¨¬C and 3 moisture levels: 25, 50 and 75% of soil moisture holding capacity. Sampling was carried out after 0, 14, 28 and 42 days. The highest numbers of viable BRGgfp-15 cells/g were found in the pesta granules in soil subjected to the lowest diurnal temperature regime and moisture content. The lowest numbers of viable cells/g were found in the pesta granules incubated in the highest diurnal temperature and moisture. This suggests lower soil temperature and moisture enhances survival of BRGgfp-15 in pesta and/ or higher soil temperature and moisture enhances the release and dissemination of BRGgfp-15 from pesta granules. When subjected to a 5-15¨¬C-temperature regime the number of viable cells/g was log109.80. When subjected to 15-25¨¬C the viable cells/g was log108.96 and with 25-35¨¬C it was log107.33. The mean number of viable cells/g was log109.36, log108.86, and log107.87, for 25, 50, and 75% soil moisture holding capacity, respectively. There was also a significantly higher number of viable cells/g in the clay soil collected from Saskatoon, log109.00, as compared to the clay loam soil collected from Scott, which was log108.40. <p>These results suggest that Pseudomonas fluorescens BRG100 has considerable potential as a bioherbicide because of its successful root colonization of green foxtail and wheat. <i>Pseudomonas fluorescens</i> BRGgfp-15 survived well under various environmental conditions when formulated into pesta granules, proving the pesta formulation was an excellent formulation. In addition, gfp was shown to be an excellent conservative marker for monitoring the root colonization and survival of <i>P. fluorescens</i> BRG100.

<i>Pseudomonas fluorescens</i> BRG100 produces secondary metabolites with herbicidal activity to the grass weeds wild oat, Avena fatua, and green foxtail, Setaria viridis. The green fluorescence protein (gfp) gene was introduced into P. fluorescens BRG100 from Escherichia coli S17-1¥ë via a Tn5 mini transposon suicide vector system. Colony morphology, growth rate in liquid media, weed biocontrol efficacy (plant growth pouch), carbon utilization (Biolog GN) and root colonization of green foxtail by several P. fluorescens BRG100gfp transformants were determined to be the same as the wild type. <i>Pseudomonas fluorescens</i> BRGgfp-15 was found to be most similar to the wild-type in all of the above characteristics and was thus used in subsequent experiments. Note: all strains of Pseudomonas fluorescens will be referred to by only their strain throughout (ie. BRGgfp-15 and BRG100). <p>It was determined by population dynamics per section of root with spiral plating on culture medium, epi-fluorescence and confocal microscopy that BRGgfp-15 colonized all areas of the root, but showed a preference for the proximal 1/3 section and the seed. In the proximal section the mean number of viable cells per gram dry weight was log109.06 and log109.31, when applied as liquid inoculum and as the pesta granular formulation, respectively. With liquid inoculum there was only log107.53 viable cells/g in the middle 1/3 section and log107.01 viable cells/g in the distal 1/3 section. The number of viable cells/g with pesta granules was log107.61 and log107.34, for the middle and distal sections, respectively. The root hairs, root tip, and ventral portion of the seed were all areas of heavy colonization relative to the other areas of the root. <p>Survival of BRGgfp-15 in the pesta formulation was examined in 2 soil types, clay and clay loam, in a thermogradient plate apparatus by a factorial randomized design complete block experiment. The experiment included: 3-12 hour diurnal temperature regimes: 5-15¨¬C, 15-25¨¬C, and 25-35¨¬C and 3 moisture levels: 25, 50 and 75% of soil moisture holding capacity. Sampling was carried out after 0, 14, 28 and 42 days. The highest numbers of viable BRGgfp-15 cells/g were found in the pesta granules in soil subjected to the lowest diurnal temperature regime and moisture content. The lowest numbers of viable cells/g were found in the pesta granules incubated in the highest diurnal temperature and moisture. This suggests lower soil temperature and moisture enhances survival of BRGgfp-15 in pesta and/ or higher soil temperature and moisture enhances the release and dissemination of BRGgfp-15 from pesta granules. When subjected to a 5-15¨¬C-temperature regime the number of viable cells/g was log109.80. When subjected to 15-25¨¬C the viable cells/g was log108.96 and with 25-35¨¬C it was log107.33. The mean number of viable cells/g was log109.36, log108.86, and log107.87, for 25, 50, and 75% soil moisture holding capacity, respectively. There was also a significantly higher number of viable cells/g in the clay soil collected from Saskatoon, log109.00, as compared to the clay loam soil collected from Scott, which was log108.40. <p>These results suggest that Pseudomonas fluorescens BRG100 has considerable potential as a bioherbicide because of its successful root colonization of green foxtail and wheat. <i>Pseudomonas fluorescens</i> BRGgfp-15 survived well under various environmental conditions when formulated into pesta granules, proving the pesta formulation was an excellent formulation. In addition, gfp was shown to be an excellent conservative marker for monitoring the root colonization and survival of <i>P. fluorescens</i> BRG100.