Proteomic study on the developing high-lysine rice seeds.

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.iSTATEMENT FROM AUTHOR --- p.iiACKNOWLEDGEMENTS --- p.iiiABSTRACT --- p.vTABLE OF CONTENTS --- p.xiLIST OF FIGURES --- p.xviLIST OF TABLES --- p.xviiiLIST OF ABBREVIATIONS --- p.xixChapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.4Chapter 2.1 --- Nutritional quality of rice --- p.4Chapter 2.1.1 --- Classification of seed proteins --- p.4Chapter 2.1.2 --- Amino acid composition of rice proteins --- p.5Chapter 2.1.3 --- Other nutritional components of rice --- p.6Chapter 2.2 --- Rice seed storage proteins --- p.7Chapter 2.2.1 --- Properties and classification of seed storage proteins --- p.7Chapter 2.2.2 --- Composition and stucture --- p.9Chapter 2.2.2.1 --- Glutelin --- p.9Chapter 2.2.2.2 --- Prolamin --- p.10Chapter 2.2.2.3 --- Albumin and globulin --- p.12Chapter 2.2.3 --- "Synthsis, assembly and deposition of rice seed storage proteins" --- p.13Chapter 2.2.3.1 --- Storage protein folding and assembly in the ER --- p.14Chapter 2.2.3.2 --- Storage protein transport and protein body formation --- p.16Chapter 2.2.3.3 --- Protein bodies and their distribution in endosperm --- p.18Chapter 2.3 --- Transgenic approaches to improve the nutritional quality of rice seed proteins --- p.19Chapter 2.3.1 --- General introduction --- p.19Chapter 2.3.2 --- Attempts to improve the nutritional quality of seed proteins --- p.20Chapter 2.3.3 --- Rice grain quality improvement by genetic engineering --- p.22Chapter 2.3.3.1 --- Increase in the lysine content of rice endosperm --- p.22Chapter 2.2.3.2 --- Other examples of rice nutritional quality improvement --- p.25Chapter 2.3.4 --- Expression of recombinant protein in transgenic plants --- p.26Chapter 2.3.5 --- Effects of recombinant proteins on the high-lysine rice --- p.27Chapter 2.4 --- Proteomics --- p.28Chapter 2.4.1 --- General overview --- p.28Chapter 2.4.1.1 --- Two-dimensional polyacrylamide gel electrophoresis for proteome analysis --- p.29Chapter 2.4.1.2 --- Protein visualization --- p.32Chapter 2.4.1.3 --- Computer-aided image analysis --- p.34Chapter 2.4.1.4 --- Mass spectrometry-based methods for protein identification --- p.35Chapter 2.4.1.5 --- Database search --- p.36Chapter 2.4.1.6 --- Protein sequence database --- p.37Chapter 2.4.2 --- Plant proteomics --- p.40Chapter 2.4.2.1 --- Rice proteomics --- p.41Chapter 2.4.2.2 --- Comparative proteomics --- p.43Chapter 2.5 --- Hypothesis and objectives --- p.45Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.47Chapter 3.1 --- Materials --- p.47Chapter 3.1.1 --- Chemicals and commercial kits --- p.47Chapter 3.1.2 --- Instruments --- p.47Chapter 3.1.3 --- Softwares --- p.48Chapter 3.1.4 --- Plant materials --- p.48Chapter 3.2 --- Methods --- p.49Chapter 3.2.1 --- Collection of developing rice seeds --- p.49Chapter 3.2.2 --- Extraction of rice seed proteins --- p.51Chapter 3.2.2.1 --- Extraction of total protein --- p.51Chapter 3.2.3.2 --- Extraction of four fractions of rice seed proteins --- p.51Chapter 3.2.3 --- 2D gel electrophoresis --- p.53Chapter 3.2.3.1 --- Protein precipitation and quantification --- p.53Chapter 3.2.3.2 --- Isoelectric focusing (IEF) --- p.54Chapter 3.2.3.3 --- IPG strips equilibration --- p.54Chapter 3.2.3.4 --- Second-dimension SDS-PAGE --- p.55Chapter 3.2.3.5 --- Silver staining of 2D gel --- p.55Chapter 3.2.3.6 --- Image and data analysis --- p.56Chapter 3.2.4 --- MALDI-ToF mass spectrometry (Matrix Assisted Laser Desorption Ionization-Time of Flight) --- p.56Chapter 3.2.4.1 --- Sample destaining --- p.56Chapter 3.2.4.2 --- In-gel digestion with trypsin --- p.57Chapter 3.2.4.3 --- Desalination of the digested sample with Zip Tip --- p.58Chapter 3.2.4.4 --- Protein identification by mass spectrometry and database searching --- p.58Chapter 3.2.5 --- Detection of LRP fusion protein in 2D PAGE --- p.59Chapter 3.2.5.1 --- 2D gel electrophoresis --- p.59Chapter 3.2.5.2 --- Western blotting using anti-LRP antibody --- p.60Chapter 3.2.6 --- Antiserum production --- p.61Chapter 3.2.6.1 --- Purification of glutelin and prolamin proteins --- p.61Chapter 3.2.6.2 --- Immunization of rabbits and mice --- p.62Chapter 3.2.6.3 --- Testing of antibody specificity --- p.62Chapter 3.2.7 --- Transmission electron microscopy (TEM) --- p.63Chapter 3.2.7.1 --- Sample fixation and section preparation --- p.63Chapter 3.2.7.2 --- TEM observation --- p.64Chapter 3.2.7.3 --- Immunocytochemical observation --- p.64Chapter CHAPTER 4. --- RESULTS --- p.66Chapter 4.1 --- Proteomic analysis of high-lysine rice --- p.66Chapter 4.1.1 --- Extraction of proteins --- p.66Chapter 4.1.2 --- The proteomic profiles of different storage proteins in developing high-lysine rice seeds --- p.67Chapter 4.1.3 --- Quantitative analysis of protein spots --- p.76Chapter 4.1.4 --- Proteomic analysis of salt-soluble proteins --- p.79Chapter 4.1.5 --- Proteomic analysis of alcohol-soluble proteins --- p.81Chapter 4.1.6 --- Proteomic analysis of salt-soluble proteins --- p.82Chapter 4.1.7 --- Proteomic analysis of water-soluble proteins --- p.89Chapter 4.1.8 --- Comparison of changes in expression patterns of specific proteins in the high lysine rice --- p.89Chapter 4.2 --- Antibody production --- p.92Chapter 4.2.1 --- The production of anti-prolamin and anti-glutelin antibodies --- p.92Chapter 4.2.2 --- The specificity of anti-prolamin and anti-glutelin antibodies --- p.93Chapter 4.3 --- Transmission electron microscopy observation of rice protein bodies --- p.95Chapter 4.3.1 --- Morphology of protein bodies in high-lysine rice --- p.95Chapter 4.3.2 --- Subcellular localization of storage proteins and LRP --- p.98Chapter CHAPTER 5. --- DISCUSSION --- p.100Chapter 5.1 --- Protein profiling of LRP fusion protein and its effects on the expression of other proteins --- p.100Chapter 5.2 --- Over-expression of glutelin and its effects on the expression of other proteins --- p.102Chapter 5.3 --- Formation of malformed protein bodies and deposition of storage proteins --- p.103Chapter 5.4 --- Relationship between changes in protein expression and the Unfolded Protein Response --- p.105Chapter 5.5 --- Effects of transgenes on rice grain quality --- p.108Chapter 5.6 --- Allergenic effects of transgenic rice --- p.109Chapter 5.7 --- Future perspectives --- p.110Chapter CHAPTER 6. --- CONCLUSIONS --- p.112REFERENCES --- p.11

Proteomic study on the starch synthesis and regulation in developing hybrid rice seeds.

Long Xiaohang.Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.Includes bibliographical references (leaves 132-155).Abstracts in English and Chinese.Thesis/Assessment Committee --- p.IStatement from Author --- p.IIAcknowledgements --- p.IIIAbstract --- p.V摘要 --- p.VIITable of Contents --- p.IXList of Tables --- p.XVList of Figures --- p.XVIList of Abbreviations --- p.XVIIIChapter Chapter 1 --- General Introduction and Literature Review --- p.1Chapter 1.1 --- General introduction --- p.1Chapter 1.2 --- Literature review --- p.5Chapter 1.2.1 --- Rice --- p.5Chapter 1.2.1.1 --- Classification of rice --- p.5Chapter 1.2.1.2 --- Rice grain quality --- p.5Chapter 1.2.2 --- Overview of current information on the starch biosynthesis and regulation during seed development --- p.7Chapter 1.2.2.1 --- Starch property --- p.7Chapter 1.2.2.1.1 --- Structure of rice starch granules --- p.7Chapter 1.2.2.1.2 --- Properties of rice starch --- p.7Chapter 1.2.2.2 --- Starch synthesis related proteins --- p.8Chapter 1.2.2.2.1 --- The formation of ADP-glucose through AGPase --- p.10Chapter 1.2.2.2.2 --- The synthesis of starch by starch synthases --- p.10Chapter 1.2.2.2.2.1 --- Amylose biosynthesis --- p.10Chapter 1.2.2.2.2.2 --- Amylopectin biosynthesis --- p.11Chapter 1.2.2.2.3 --- Branching of the glucan chain by starch branching enzymes --- p.12Chapter 1.2.2.2.4 --- The role of debranching enzymes in polymer synthesis --- p.13Chapter 1.2.2.2.5 --- Starch degradation in plastids --- p.13Chapter 1.2.2.2.6 --- Other enzymes involved in starch synthesis pathway --- p.13Chapter 1.2.2.3 --- Starch biosynthesis regulation --- p.14Chapter 1.2.2.3.1 --- Developmental regulation --- p.14Chapter 1.2.2.3.2. --- Diurnal regulation --- p.15Chapter 1.2.2.3.3 --- 3-PGA/Pi regulation --- p.16Chapter 1.2.2.3.4. --- Sugar signaling --- p.17Chapter 1.2.2.3.5. --- Hormonal signaling --- p.18Chapter 1.2.2.3.6 --- Post translational modification regulation --- p.18Chapter 1.2.2.3.6.1 --- Post translational redox modulation --- p.18Chapter 1.2.2.3.6.2 --- Protein phosphorylation --- p.19Chapter 1.2.2.4 --- Rice grain quality improvement by genetic engineering --- p.20Chapter 1.2.2.4.1 --- Cooking and eating quality improvement --- p.20Chapter 1.2.2.4.1.1 --- Manipulation of starch content --- p.20Chapter 1.2.2.4.1.2 --- Manipulation of amylose/ amylopectin ratio --- p.20Chapter 1.2.2.4.2 --- Other targets for manipulating starch quality and quantity --- p.21Chapter 1.2.3 --- Proteomics --- p.23Chapter 1.2.3.1 --- General introduction --- p.23Chapter 1.2.3.2 --- Current technologies of proteomics --- p.25Chapter 1.2.3.2.1 --- Protein separation by 2D or non-2D method --- p.25Chapter 1.2.3.2.2 --- Protein visualization --- p.26Chapter 1.2.3.2.3 --- Computer-assisted image analysis --- p.27Chapter 1.2.3.2.4 --- Protein identification by mass spectrometry --- p.28Chapter 1.2.3.2.5 --- Database search --- p.28Chapter 1.2.3.2.5.1 --- Database searching software --- p.29Chapter 1.2.3.2.5.2 --- Protein sequence database --- p.29Chapter 1.2.3.2.5.3 --- Evaluating database hits --- p.30Chapter 1.2.3.2.6 --- Bioinformatics involved in proteomics --- p.31Chapter 1.2.3.2.7 --- Post translational modification --- p.32Chapter 1.2.3.2.7.1 --- Glycosylation --- p.32Chapter 1.2.3.2.7.1.1 --- N-linked glycosylation --- p.33Chapter 1.2.3.2.7.1.2 --- O-linked glycosylation --- p.33Chapter 1.2.3.2.7.2 --- Phosphorylation --- p.33Chapter 1.2.3.2.7.3 --- Strategies for studying PTMs --- p.34Chapter 1.2.3.2.8 --- Other aspects of proteomics --- p.36Chapter 1.2.3.2.8.1 --- 2D DIGE --- p.36Chapter 1.2.3.2.8.2 --- LC/LC-MS/MS --- p.36Chapter 1.2.3.2.8.2.1 --- MudPIT --- p.36Chapter 1.2.3.2.8.2.2 --- ICAT --- p.37Chapter 1.2.3.3 --- Plant proteomics --- p.37Chapter 1.2.3.3.1 --- Proteome analysis of plant tissues and organs --- p.38Chapter 1.2.3.3.2 --- Plant organelle proteomics --- p.39Chapter 1.2.3.3.3 --- Post translational modifications in plant --- p.41Chapter 1.2.3.4 --- Recent progress in rice proteomics --- p.42Chapter 1.2.3.4.1 --- General introduction of rice proteomics --- p.42Chapter 1.2.3.4.2 --- Rice proteome database construction --- p.43Chapter 1.2.3.4.3 --- Comparative proteomics --- p.43Chapter 1.2.3.4.4 --- Post translational modification study of rice proteome --- p.44Chapter Chapter 2 --- Materials and methods --- p.45Chapter 2.1 --- Materials --- p.45Chapter 2.1.1 --- Plant materials --- p.45Chapter 2.1.2 --- Chemical reagents and commercial kits --- p.46Chapter 2.1.3 --- Instruments --- p.46Chapter 2.1.4 --- Software --- p.46Chapter 2.2 --- Methods --- p.47Chapter 2.2.1 --- Fractionation of amyloplast and amyloplast membrane proteins --- p.47Chapter 2.2.2 --- Marker enzyme assays --- p.47Chapter 2.2.3 --- 2D gel electrophoresis --- p.48Chapter 2.2.4 --- Silver staining of 2D gel --- p.49Chapter 2.2.5 --- In-gel digestion of protein spots --- p.49Chapter 2.2.6 --- Desalination of the digested sample with ZipTip --- p.49Chapter 2.2.7 --- Protein identification by mass spectrometry and database searching --- p.50Chapter 2.2.8 --- Image and data analysis --- p.50Chapter 2.2.9 --- Extraction of starch granule associated proteins --- p.51Chapter 2.2.10 --- Western blot analysis --- p.51Chapter 2.2.11 --- Sample preparation for N terminal sequencing --- p.51Chapter 2.2.12 --- Phosphorylation and glycosylation assays --- p.52Chapter Chapter 3 --- Results --- p.53Chapter 3.1 --- Protein identification by ID and 2D PAGE --- p.53Chapter 3.1.1 --- Isolation and purification of amyloplasts from rice seeds --- p.53Chapter 3.1.2 --- Identification of amyloplast and amyloplast membrane proteins by MS/MS --- p.54Chapter 3.1.2.1 --- Sample preparation --- p.54Chapter 3.1.2.2 --- 2D and ID gel electrophoresis --- p.55Chapter 3.1.2.3 --- Protein identification by MS and MS/MS --- p.56Chapter 3.1.3 --- Functional classification of identified proteins --- p.69Chapter 3.1.3.1 --- Metabolism proteins --- p.71Chapter 3.1.3.2 --- Non starch synthesis metabolism proteins --- p.73Chapter 3.1.3.3 --- Protein destination --- p.73Chapter 3.1.3.4 --- Proteins with other functions --- p.74Chapter 3.1.4 --- Cross-correlation of experimental and calculated Mw of proteins --- p.74Chapter 3.1.5 --- Granule bound starch synthase (GBSS) --- p.75Chapter 3.1.5 --- N-terminal sequencing --- p.77Chapter 3.2 --- Protein profiling --- p.80Chapter 3.2.1 --- Seed collection and stages chosen --- p.80Chapter 3.2.2 --- The proteomic profiles of rice amyloplasts at different developing stages --- p.81Chapter 3.2.4 --- Comparing the proteome of three rice lines --- p.85Chapter 3.2.4.1 --- Spot number analysis --- p.85Chapter 3.2.4.2 --- Functional distribution analysis --- p.86Chapter 3.2.4.3 --- Protein amount analysis --- p.87Chapter 3.2.5 --- Comparison of expression pattern: cluster analysis (SOM) --- p.88Chapter 3.2.5.1 --- Cluster analysis of rice amyloplast proteome --- p.88Chapter 3.2.5.2 --- Three major categories of rice amyloplast proteome expression patterns --- p.91Chapter 3.2.6 --- Scatter plots analysis --- p.94Chapter 3.2.7 --- Comparison of changes in proteins related to starch synthesis --- p.96Chapter 3.2.7.1 --- GBSS --- p.96Chapter 3.2.7.2 --- AGPase --- p.98Chapter 3.2.7.3 --- SSS --- p.98Chapter 3.2.7.4 --- SBE --- p.98Chapter 3.2.7.5 --- SP --- p.98Chapter 3.3 --- Study on protein post translational modifications --- p.102Chapter 3.3.1 --- Post translational modifications that potentially regulate starch synthesis --- p.102Chapter 3.3.2 --- Post translational modifications at different developing stages --- p.104Chapter 3.3.2.1 --- Profiling of post translational modifications of rice amyloplast proteome --- p.104Chapter 3.3.2.2 --- Starch synthesis related proteins --- p.106Chapter 3.3.2.2.1 --- GBSS --- p.106Chapter 3.3.2.2.2 --- SSS --- p.108Chapter Chapter 4 --- Discussion --- p.111Chapter 4.1 --- Methodology --- p.111Chapter 4.1.1 --- Amyloplast isolation --- p.111Chapter 4.1.2 --- Protein extraction from amyloplasts --- p.111Chapter 4.1.3 --- Protein identification by PMF and MS/MS --- p.112Chapter 4.1.4 --- Methods used to study protein expression patterns --- p.113Chapter 4.1.5 --- New methods introduced to study post translational modifications --- p.114Chapter 4.2 --- Characteristics of rice amyloplast proteins --- p.115Chapter 4.2.1 --- Amyloplast proteins associated with starch granules --- p.116Chapter 4.2.2 --- Most proteins in amyloplast proteome contain the transit peptide --- p.116Chapter 4.2.3 --- Multiple isoforms of starch synthesis related proteins --- p.117Chapter 4.2.3.1 --- Multiple spots of GBSS --- p.118Chapter 4.2.4 --- Expression patterns of amyloplast proteome --- p.120Chapter 4.2.5 --- Post translational modifications potentially regulate starch synthesis --- p.122Chapter 4.3 --- Other characteristic aspects of amyloplast proteome --- p.123Chapter 4.3.1 --- Comparison between the rice and wheat amyloplast proteomes --- p.123Chapter 4.3.2 --- Proteomic comparisons among the three rice lines --- p.124Chapter 4.3.3 --- Comparison of starch synthesis enzymes at protein and transcript levels --- p.124Chapter 4.3.4 --- Comparison of the starch synthesis related proteins among the three rice lines --- p.126Chapter 4.4 --- Limitations of proteomic approach in directly answering the question on how to improve eating and cooling quality --- p.126Chapter Chapter 5 --- Conclusion --- p.128Chapter Chapter 6 --- Future perspectives --- p.130References --- p.13

Proteomics Study of Pre-Harvest Sprouting in Wheat

Pre-harvest sprouting (PHS) of wheat (Triticum aestivum L.) is a condition characterized by the early germination of spikes during moist environmental conditions. PHS lowers yield, degrades the quality of grain and thus limits the profits of wheat producers groups. During this investigation, proteomics studies of PHS-resistant and PHS-susceptible wheat embryos were conducted at different imbibition time periods via a cutting-edge technology called iTRAQ. Proteomic analysis revealed that 190 differentially expressed proteins might be involved in various cellular functions, such as carbohydrate metabolism, nitrogen metabolism, stress response, redox regulation, ATP synthesis, and protein translation, during this untimely germination of the wheat embryo. Hierarchical clustering analysis revealed the expression pattern of proteins in each of the resistant and susceptible germplasm and relative abundance of respective proteins between the two germplasm. Expression of stress-related and inhibitors proteins was found to be important in maintaining seed dormancy in resistant germplasm; whereas over-expression of energy metabolism related proteins was observed in PHS-susceptible germplasm for the production of energy required for seedling growth. ABA appeared to be involved in seed dormancy, directly or indirectly by controlling the expression of several LEA and EMB-1 proteins. A higher level of ROS production was observed in PHS-susceptible germplasm. Through bioinformatics analysis, a Thioredoxin h protei

Tissue-specific gene expression and promoter characterization in triticale

xxiii, 425 leaves : col. ill. ; 29 cmTriticale (x Triticosecale Whitm.) is a cereal with favorable agronomic traits for a Canadian bioproduction platform crop. Appropriate tissue sampling times were determined and gene expression profiles were evaluated in five triticale seed tissues and eleven vegetative tissues using the Affymetrix Wheat GeneChip®. Genes that were expressed, not expressed, tissue-specific, tissue-enriched and developmentally regulated were identified. The percentage of probe sets on the wheat GeneChip with gene ontology annotations was improved from less than 3% to over 76% using homologous sequence identification and annotation transfer. This information was used to determine functions and processes over-represented within the identified gene lists and provide biological meaning to the results. Expression of candidate genes was further evaluated using qRT-PCR, RNA in situ hybridization and promoter characterization. This study has provided a comprehensive triticale gene expression atlas; knowledge regarding triticale development, gene function, expression and regulation; and tools enabling further triticale research and development

Biotechnologies for Plant Mutation Breeding: Protocols

Plant Breeding/Biotechnology; Agriculture; Genetic Engineering; Plant Genetics & Genomic

Accessing genetic variability in Spanish barleys through high-throughput sequencing

193 Pags.- Tabls.- Grafcs. Research memory presented by Carlos Pérez Cantalapiedra to obtain the title of Doctor in Plant Biology and Biotechnology from Universidad Autónoma de Barcelona (UAB). This work has been done at Estación Experimental de Aula Dei (EEAD), belonging to Consejo Superior de Investigaciones Científicas (CSIC), in Zaragoza[EN] High-throughput sequencing (HTS) has revolutionized plant research. It has made it possible to sequence the genomes of multiple organisms. The sequence-enriched physical map of barley was published in late 2012. A first step to exploit barley genomics, for practical purposes, was facilitating geneticists and breeders access to the barley physical map. This was the aim which led us to the development of Barleymap, a software tool which allows locating genetic markers in the barley physical-genetic map. This application effectively integrates and maps markers from different widely used barley genotyping platforms, and, in general, any marker with sequence information.[ES] La secuenciación de alto rendimiento (HTS, por sus siglas en inglés) ha revolucionado la investigación, haciendo posible secuenciar los genomas de múltiples organismos. El mapa físico de cebada y sus secuencias asociadas fueron publicados a finales de 2012. Para sacar partido de estos recursos, había que facilitar el acceso a ellos a genetistas y mejoradores. Este fue el objetivo que nos llevó a desarrollar Barleymap, una herramienta informática que permite localizar marcadores genéticos en el genoma de cebada. La aplicación integra y localiza marcadores de distintas plataformas de genotipado de cebada ampliamente utilizadas.Peer reviewe