It is the study of “Proteome”.
The word "proteome" is a blend of "protein" and "genome”.
Large scale study of Proteins.
Particularly their structures and functions.
Study of full set of proteins in a cell type or tissue, and changes during various conditions
This document provides an overview of plant proteomics techniques, including 2D gel electrophoresis, mass spectrometry, and software analysis. 2D gel electrophoresis separates proteins by isoelectric focusing based on pH in the first dimension, followed by SDS-PAGE based on size in the second dimension. Spots are visualized, excised, digested, and identified using mass spectrometry. Software performs matching, detection, quantitation, and annotation of protein spots across gels to identify differentially expressed proteins.
DNA polymorphisms can be used as genetic markers. Molecular markers include non-PCR based markers like RFLPs and PCR-based markers like microsatellites, minisatellites, and SNPs. RFLPs detect differences in restriction fragment lengths that can be used to identify carriers of genetic diseases or match DNA samples in criminal cases. Microsatellites and minisatellites are variable tandem repeats that provide many alleles for identification. Together these marker types allow DNA fingerprinting for individual identification.
The document provides an overview of the history and techniques of transcriptome analysis. It discusses how RNA was separated from DNA with the formulation of the central dogma in 1958. Key developments include the discoveries of messenger RNA, transfer RNA, and ribosomal RNA in the 1960s. The document outlines techniques such as serial analysis of gene expression (SAGE) and RNA sequencing (RNA-seq) that allow comprehensive analysis of gene expression patterns. It provides details on the basic steps and advantages of SAGE and describes how next generation sequencing revolutionized transcriptome analysis through massive parallel sequencing.
Transcriptome analysis is the study of the set of all RNA molecules, including mRNA, rRNA, tRNA, and non-coding RNAs produced in a population of cells. The transcriptome can vary between different cell types, body parts, and environmental conditions. Transcriptomics aims to catalogue all transcript species and quantify changing expression levels during development and in different conditions. The two main techniques are DNA microarrays and RNA sequencing. Microarrays involve fluorescent labeling and hybridization of samples to probe arrays, while RNA sequencing replaces hybridization with sequencing of individual cDNAs produced from target RNA.
Molecular markers are DNA sequences that can be easily detected and whose inheritance can be monitored. They are based on natural polymorphisms and allow studying the inheritance of genes. Common types of molecular markers include RFLPs, RAPDs, AFLPs, SSRs, and SNPs. RFLPs use restriction enzymes to detect differences in fragment lengths. RAPDs use random primers to detect sequence polymorphisms. AFLPs selectively amplify restriction fragments to detect length differences. SSRs detect variability in simple sequence repeats. Molecular markers are useful for applications like gene mapping, phylogenetic studies, and analyzing genetic diversity.
Arabidopsis thaliana was the very first plant whose genome was sequenced by the Arabidopsis Initiative (AGI) in the year 1966-2000. mouse ear cress has been the plant model ever since 1985.
This document discusses different types of mapping populations used in genetic mapping. It describes F2, backcross, double haploid, recombinant inbred line, and near isogenic line populations. For each type, it provides details on how they are developed and their advantages and disadvantages. It also discusses how marker segregation ratios differ depending on the population type and marker dominance. The document recommends using short-term mapping populations initially for preliminary mapping but developing long-term populations like recombinant inbred lines for global mapping projects.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)Santosh Kumar Sahoo
Two Dimensional Polyacrylamide gel Electrophoresis (2D-PAGE) is a technique used to separate and identify proteins in a biological sample. It involves two sequential steps - isoelectric focusing and SDS-PAGE - to separate proteins based on their isoelectric point and molecular weight. This allows visualization of up to 1,000s of individual protein spots on the gel. The protein spots can then be analyzed through techniques like mass spectrometry to identify specific proteins. While 2D-PAGE provides high resolution of complex protein mixtures, it has limitations such as a narrow dynamic range and difficulties separating some classes of proteins.
This document provides an overview of plant proteomics techniques, including 2D gel electrophoresis, mass spectrometry, and software analysis. 2D gel electrophoresis separates proteins by isoelectric focusing based on pH in the first dimension, followed by SDS-PAGE based on size in the second dimension. Spots are visualized, excised, digested, and identified using mass spectrometry. Software performs matching, detection, quantitation, and annotation of protein spots across gels to identify differentially expressed proteins.
DNA polymorphisms can be used as genetic markers. Molecular markers include non-PCR based markers like RFLPs and PCR-based markers like microsatellites, minisatellites, and SNPs. RFLPs detect differences in restriction fragment lengths that can be used to identify carriers of genetic diseases or match DNA samples in criminal cases. Microsatellites and minisatellites are variable tandem repeats that provide many alleles for identification. Together these marker types allow DNA fingerprinting for individual identification.
The document provides an overview of the history and techniques of transcriptome analysis. It discusses how RNA was separated from DNA with the formulation of the central dogma in 1958. Key developments include the discoveries of messenger RNA, transfer RNA, and ribosomal RNA in the 1960s. The document outlines techniques such as serial analysis of gene expression (SAGE) and RNA sequencing (RNA-seq) that allow comprehensive analysis of gene expression patterns. It provides details on the basic steps and advantages of SAGE and describes how next generation sequencing revolutionized transcriptome analysis through massive parallel sequencing.
Transcriptome analysis is the study of the set of all RNA molecules, including mRNA, rRNA, tRNA, and non-coding RNAs produced in a population of cells. The transcriptome can vary between different cell types, body parts, and environmental conditions. Transcriptomics aims to catalogue all transcript species and quantify changing expression levels during development and in different conditions. The two main techniques are DNA microarrays and RNA sequencing. Microarrays involve fluorescent labeling and hybridization of samples to probe arrays, while RNA sequencing replaces hybridization with sequencing of individual cDNAs produced from target RNA.
Molecular markers are DNA sequences that can be easily detected and whose inheritance can be monitored. They are based on natural polymorphisms and allow studying the inheritance of genes. Common types of molecular markers include RFLPs, RAPDs, AFLPs, SSRs, and SNPs. RFLPs use restriction enzymes to detect differences in fragment lengths. RAPDs use random primers to detect sequence polymorphisms. AFLPs selectively amplify restriction fragments to detect length differences. SSRs detect variability in simple sequence repeats. Molecular markers are useful for applications like gene mapping, phylogenetic studies, and analyzing genetic diversity.
Arabidopsis thaliana was the very first plant whose genome was sequenced by the Arabidopsis Initiative (AGI) in the year 1966-2000. mouse ear cress has been the plant model ever since 1985.
This document discusses different types of mapping populations used in genetic mapping. It describes F2, backcross, double haploid, recombinant inbred line, and near isogenic line populations. For each type, it provides details on how they are developed and their advantages and disadvantages. It also discusses how marker segregation ratios differ depending on the population type and marker dominance. The document recommends using short-term mapping populations initially for preliminary mapping but developing long-term populations like recombinant inbred lines for global mapping projects.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)Santosh Kumar Sahoo
Two Dimensional Polyacrylamide gel Electrophoresis (2D-PAGE) is a technique used to separate and identify proteins in a biological sample. It involves two sequential steps - isoelectric focusing and SDS-PAGE - to separate proteins based on their isoelectric point and molecular weight. This allows visualization of up to 1,000s of individual protein spots on the gel. The protein spots can then be analyzed through techniques like mass spectrometry to identify specific proteins. While 2D-PAGE provides high resolution of complex protein mixtures, it has limitations such as a narrow dynamic range and difficulties separating some classes of proteins.
Anther culture:- the in vitro culturing of anthers containing microspores or immature pollen grains on a nutrient medium for the purpose of generating haploid plantlets.
Culturing anthers for the purpose of obtaining Double Haploid is not easy with many field crop species, particularly with the cereals, cotton, and grain legumes.
Plant metabolomics is the study of small molecule metabolites within plants. It is influenced by both genetic and environmental factors. Metabolomics can help understand plant phenotype, development, physiology, and stress responses. Modern platforms for plant metabolomics use techniques like gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy to detect and quantify metabolites in plant extracts. GC-MS allows for non-targeted profiling while NMR is non-destructive and useful for quantification. Metabolite fingerprinting and profiling are used to identify markers of genetic or environmental disturbances by comparing metabolic states of plants under different conditions. Plant metabolomics has applications in understanding plant diseases, stress responses, and secondary metabolites of therapeutic importance.
The document discusses phenotyping methods for evaluating tolerance to abiotic stresses like drought, heat, and low soil phosphorus in various crops. It describes:
- Screening protocols for evaluating drought tolerance in maize, banana, cowpea and yam through measurements of agronomic traits under irrigated and non-irrigated conditions.
- Methods for assessing tolerance to low nitrogen and phosphorus availability in maize, cowpea and yam, including establishing low fertility plots and measuring traits like growth, yield and nutrient uptake.
- Techniques for high-throughput phenotyping of root traits and physiological responses that could help mine available nutrients and tap water more efficiently.
OMICS is the comprehensive analysis of the biological system. The technologies which made a revolution such as Genomics, Transcriptomics, Proteomics, Metabolomics and Phenomics, in screening traits and develop novel improved organisms are mentioned here. The presentation gives a brief idea about various OMICS technology used in crop improvement, their steps, techniques used, applications, scope, advantages and disadvantages.
PPT protein separation and purificationKAUSHAL SAHU
This document discusses protein purification techniques. It describes that proteins can be extracellular, secreted from cells, or intracellular, requiring cell disruption. Purification techniques include differential centrifugation to separate components by size and mass, differential salt precipitation to separate based on solubility, and various types of chromatography including size exclusion, ion exchange, and affinity chromatography. Electrophoresis can also separate proteins based on size using electrical current. The overall goal is to purify proteins for uses such as commercial products, research, or analysis of structure and function.
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
1) Germplasm conservation involves preserving genetic material, such as seeds, cells, tissues, and body parts, through in-situ and ex-situ methods to maintain biodiversity and provide resources for breeding programs.
2) Cryopreservation at ultra-low temperatures in liquid nitrogen is an important ex-situ technique that can preserve germplasm long-term without subculturing. It involves preculturing plant materials, treating with cryoprotectants, and either slow-freezing or vitrification prior to storage in liquid nitrogen.
3) A case study demonstrates the successful cryopreservation of mint shoot tips using encapsulation-dehydration and PVS2-vitrification, with
it cover almost all content in cis/intragesis, right from introduction definition, explanation, production of marker free transgenic, intragenic vector construction, regulatory guide lines, current and future status, limitation, advantage over existing technique, swot analysis etc
its very useful for your seminar and presentations. it contain lot of picture, table, figure for your easy understanding
thank you
Mahesh
Omics related approaches for higher productivity and improved quality.pptxAnirudhTV
The document discusses using omics approaches to improve crop productivity and quality. It covers various omics fields including genomics, epigenomics, transcriptomics, proteomics, metabolomics, and phenomics. Examples are provided on applying these approaches in crops like rice, tomato, groundnut, and brassica to traits such as drought tolerance, nutrient enrichment, and reduced anti-nutrients. A case study on analyzing protein abundance changes in wheat cultivars under drought stress using proteomics is also mentioned.
This document describes the process of DNA microarray technology. It discusses:
- How DNA microarrays work by hybridizing DNA or RNA targets to probes arranged on a solid surface.
- The key steps of microarray experiments including array printing, sample preparation, hybridization, and data acquisition and analysis.
- Different types of microarrays like cDNA microarrays and high-density oligonucleotide arrays.
- Details of probe selection, target labeling, hybridization conditions, scanning, and data analysis.
This document discusses different types of reporter genes that are used in plant functional genomics studies. It describes scorable reporter genes like green fluorescent protein (GFP), yellow fluorescent protein (YFP), and β-glucuronidase (GUS) which produce quantifiable phenotypes through enzyme assays. It also describes selectable reporter genes like antibiotic and herbicide resistance genes which allow for selection of transformed cells. Reporter genes are useful for identifying gene expression patterns, performing gene expression assays by fusing the reporter to a gene of interest, and assessing transformation/transfection efficiency. The document provides examples of using GFP fused to the XPR1 gene to study its subcellular localization in tobacco cells.
Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS) is an early high-throughput DNA sequencing technique. It works by attaching cDNA from an mRNA sample to beads, determining short sequence signatures from many beads in parallel, and using the signatures to count the number of individual mRNA molecules from each gene. This digital gene expression data allows MPSS to accurately quantify genes expressed at low levels by analyzing transcripts from virtually all genes simultaneously. The technique involves converting mRNA to cDNA, attaching oligonucleotide tags, PCR amplification on beads, and using fluorescent probes to determine short sequences in increments of four nucleotides from millions of beads in parallel.
Functional genomics uses genome-wide experimental approaches to assess gene function on a large scale. It analyzes gene expression through techniques like transcriptomics and proteomics. Transcriptomics analyzes gene expression profiles through RNA sequencing or microarray analysis. Microarray analysis involves hybridizing fluorescently-labeled cDNA or cRNA to microarrays containing DNA probes to measure gene expression levels across thousands of genes simultaneously. Functional genomics provides a global understanding of gene function and molecular interactions through integrated omics approaches.
This presentation discusses strategies for developing transgenic plants without selectable marker genes. Marker genes are commonly used to identify transformed cells but can be problematic for public acceptance and future transformations. Methods described for producing marker-free transgenics include the MAT system which uses oncogenes for selection instead of antibiotics, site-specific recombination systems which flank the marker gene for later excision, and transposon-based systems which separate the gene of interest from the marker gene. While several viable methods exist, more work is still needed before marker-free crops can be commercialized. Removing marker genes supports multiple-gene stacking and improves public acceptance of transgenic technologies.
Map-based cloning is a technique used to identify the genetic cause of a mutant phenotype by isolating overlapping DNA segments that progress along the chromosome toward a candidate gene. The process involves initially identifying a marker close to the gene of interest and then saturating the region with additional markers. Large populations are screened to find markers that rarely recombine with the gene. Genomic libraries are screened to find clones containing the markers, and chromosomal walking is used to obtain flanking markers on a single clone. DNA fragments between the markers are tested to rescue the wild-type phenotype and identify the candidate gene.
Proteomics, definatio , general concept, signficanceKAUSHAL SAHU
INTRODUCTION
GENERAL CONCEPT
WHY PROTEIOMIC NECESERY?
WHAT PROTEOMIC CAN ANSWER?
PRTEOMICS- ANALYSIS AND IDENTIFICATION OF PROTEIN
TWO-DIMENSIONAL SDS-PAGE
MASS SPECTROMETERS
SIGNIFICANCE OF STUDY AN ITS IMPORTANCE
APPLICATIONS
CHALLENGES
CONCLUSIONS
REFERENCES
The document describes the development of a QTL map for Egyptian durum wheat using an F2 mapping population derived from a cross between two parental varieties, Baniswif-1 and Souhag-2. A variety of DNA markers including SSRs, RAPDs, AFLPs, ESTs and SCoTs were used to genotype the mapping population. Traits related to yield and drought tolerance such as root length, plant height, spike characteristics, and biomass were measured. Linkage analysis was performed to construct a genetic linkage map, which was then used to detect QTLs associated with the traits of interest.
Gene pyramiding is a plant breeding technique that uses molecular markers to select plants with multiple pest-resistance and yield-enhancing genes through iterative hybridization. It aims to enhance trait performance, remedy genetic deficits, and increase durability against pests. Strategies for gene stacking include iterative hybridization between plants containing different transgenes, re-transformation of a plant with additional transgenes, and co-transformation of a plant with multiple transgenes simultaneously. Gene pyramiding is an important strategy for improving germplasm and developing durable pest resistance using multiple genes.
This document provides an overview of molecular markers that can be used for crop improvement. It discusses different types of markers such as morphological, cytological, biochemical, DNA-based markers. DNA-based markers are further classified into hybridization-based markers like RFLP and PCR-based markers like RAPD, AFLP, SSR, ISSR, SNP. The document compares various marker techniques and provides their principles, strengths, weaknesses and applications in crop breeding programs. Molecular markers can be useful for tasks like hybrid purity testing, genetic diversity analysis, linkage mapping and marker-assisted selection.
This document discusses different types of mapping populations that can be used for genetic mapping and molecular breeding programs. It describes F2, F2:F3, backcross, doubled haploid (DH), recombinant inbred line (RIL), and near-isogenic line (NIL) populations. For each type, it provides details on how they are developed, their characteristics, and merits and demerits relative to mapping objectives. The document emphasizes that the choice of mapping population depends on the research goals, availability of markers, and existing molecular maps. RILs and DHs allow for replication over environments but require more time and resources to develop.
This study analyzed G proteins, proteomics, glycomics and metabolomics in plants grown under protected agriculture. Protein profiles showed variation between phenological stages, and western blot detected G protein subunits of 37, 46, and 57 kDa. Two-dimensional electrophoresis identified a 57 kDa protein with a pI of 5.9. Phloem sap proteins also detected G protein subunits of 28, 67 kDa between stages. Sugar analysis found glucose, fructose and sucrose varied between stages, with neutral sugars dominated by glucose, galactose and mannose. The Lightbourn Biochemical Model was applied to integrate results and propose bionanotechnology and biodynamic nutrition for high agricultural competitiveness and sustainability
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology.
Anther culture:- the in vitro culturing of anthers containing microspores or immature pollen grains on a nutrient medium for the purpose of generating haploid plantlets.
Culturing anthers for the purpose of obtaining Double Haploid is not easy with many field crop species, particularly with the cereals, cotton, and grain legumes.
Plant metabolomics is the study of small molecule metabolites within plants. It is influenced by both genetic and environmental factors. Metabolomics can help understand plant phenotype, development, physiology, and stress responses. Modern platforms for plant metabolomics use techniques like gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy to detect and quantify metabolites in plant extracts. GC-MS allows for non-targeted profiling while NMR is non-destructive and useful for quantification. Metabolite fingerprinting and profiling are used to identify markers of genetic or environmental disturbances by comparing metabolic states of plants under different conditions. Plant metabolomics has applications in understanding plant diseases, stress responses, and secondary metabolites of therapeutic importance.
The document discusses phenotyping methods for evaluating tolerance to abiotic stresses like drought, heat, and low soil phosphorus in various crops. It describes:
- Screening protocols for evaluating drought tolerance in maize, banana, cowpea and yam through measurements of agronomic traits under irrigated and non-irrigated conditions.
- Methods for assessing tolerance to low nitrogen and phosphorus availability in maize, cowpea and yam, including establishing low fertility plots and measuring traits like growth, yield and nutrient uptake.
- Techniques for high-throughput phenotyping of root traits and physiological responses that could help mine available nutrients and tap water more efficiently.
OMICS is the comprehensive analysis of the biological system. The technologies which made a revolution such as Genomics, Transcriptomics, Proteomics, Metabolomics and Phenomics, in screening traits and develop novel improved organisms are mentioned here. The presentation gives a brief idea about various OMICS technology used in crop improvement, their steps, techniques used, applications, scope, advantages and disadvantages.
PPT protein separation and purificationKAUSHAL SAHU
This document discusses protein purification techniques. It describes that proteins can be extracellular, secreted from cells, or intracellular, requiring cell disruption. Purification techniques include differential centrifugation to separate components by size and mass, differential salt precipitation to separate based on solubility, and various types of chromatography including size exclusion, ion exchange, and affinity chromatography. Electrophoresis can also separate proteins based on size using electrical current. The overall goal is to purify proteins for uses such as commercial products, research, or analysis of structure and function.
The isolation, culture and fusion of protoplasts is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells (comparable to microbial cells) for a variety of studies.
1) Germplasm conservation involves preserving genetic material, such as seeds, cells, tissues, and body parts, through in-situ and ex-situ methods to maintain biodiversity and provide resources for breeding programs.
2) Cryopreservation at ultra-low temperatures in liquid nitrogen is an important ex-situ technique that can preserve germplasm long-term without subculturing. It involves preculturing plant materials, treating with cryoprotectants, and either slow-freezing or vitrification prior to storage in liquid nitrogen.
3) A case study demonstrates the successful cryopreservation of mint shoot tips using encapsulation-dehydration and PVS2-vitrification, with
it cover almost all content in cis/intragesis, right from introduction definition, explanation, production of marker free transgenic, intragenic vector construction, regulatory guide lines, current and future status, limitation, advantage over existing technique, swot analysis etc
its very useful for your seminar and presentations. it contain lot of picture, table, figure for your easy understanding
thank you
Mahesh
Omics related approaches for higher productivity and improved quality.pptxAnirudhTV
The document discusses using omics approaches to improve crop productivity and quality. It covers various omics fields including genomics, epigenomics, transcriptomics, proteomics, metabolomics, and phenomics. Examples are provided on applying these approaches in crops like rice, tomato, groundnut, and brassica to traits such as drought tolerance, nutrient enrichment, and reduced anti-nutrients. A case study on analyzing protein abundance changes in wheat cultivars under drought stress using proteomics is also mentioned.
This document describes the process of DNA microarray technology. It discusses:
- How DNA microarrays work by hybridizing DNA or RNA targets to probes arranged on a solid surface.
- The key steps of microarray experiments including array printing, sample preparation, hybridization, and data acquisition and analysis.
- Different types of microarrays like cDNA microarrays and high-density oligonucleotide arrays.
- Details of probe selection, target labeling, hybridization conditions, scanning, and data analysis.
This document discusses different types of reporter genes that are used in plant functional genomics studies. It describes scorable reporter genes like green fluorescent protein (GFP), yellow fluorescent protein (YFP), and β-glucuronidase (GUS) which produce quantifiable phenotypes through enzyme assays. It also describes selectable reporter genes like antibiotic and herbicide resistance genes which allow for selection of transformed cells. Reporter genes are useful for identifying gene expression patterns, performing gene expression assays by fusing the reporter to a gene of interest, and assessing transformation/transfection efficiency. The document provides examples of using GFP fused to the XPR1 gene to study its subcellular localization in tobacco cells.
Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS) is an early high-throughput DNA sequencing technique. It works by attaching cDNA from an mRNA sample to beads, determining short sequence signatures from many beads in parallel, and using the signatures to count the number of individual mRNA molecules from each gene. This digital gene expression data allows MPSS to accurately quantify genes expressed at low levels by analyzing transcripts from virtually all genes simultaneously. The technique involves converting mRNA to cDNA, attaching oligonucleotide tags, PCR amplification on beads, and using fluorescent probes to determine short sequences in increments of four nucleotides from millions of beads in parallel.
Functional genomics uses genome-wide experimental approaches to assess gene function on a large scale. It analyzes gene expression through techniques like transcriptomics and proteomics. Transcriptomics analyzes gene expression profiles through RNA sequencing or microarray analysis. Microarray analysis involves hybridizing fluorescently-labeled cDNA or cRNA to microarrays containing DNA probes to measure gene expression levels across thousands of genes simultaneously. Functional genomics provides a global understanding of gene function and molecular interactions through integrated omics approaches.
This presentation discusses strategies for developing transgenic plants without selectable marker genes. Marker genes are commonly used to identify transformed cells but can be problematic for public acceptance and future transformations. Methods described for producing marker-free transgenics include the MAT system which uses oncogenes for selection instead of antibiotics, site-specific recombination systems which flank the marker gene for later excision, and transposon-based systems which separate the gene of interest from the marker gene. While several viable methods exist, more work is still needed before marker-free crops can be commercialized. Removing marker genes supports multiple-gene stacking and improves public acceptance of transgenic technologies.
Map-based cloning is a technique used to identify the genetic cause of a mutant phenotype by isolating overlapping DNA segments that progress along the chromosome toward a candidate gene. The process involves initially identifying a marker close to the gene of interest and then saturating the region with additional markers. Large populations are screened to find markers that rarely recombine with the gene. Genomic libraries are screened to find clones containing the markers, and chromosomal walking is used to obtain flanking markers on a single clone. DNA fragments between the markers are tested to rescue the wild-type phenotype and identify the candidate gene.
Proteomics, definatio , general concept, signficanceKAUSHAL SAHU
INTRODUCTION
GENERAL CONCEPT
WHY PROTEIOMIC NECESERY?
WHAT PROTEOMIC CAN ANSWER?
PRTEOMICS- ANALYSIS AND IDENTIFICATION OF PROTEIN
TWO-DIMENSIONAL SDS-PAGE
MASS SPECTROMETERS
SIGNIFICANCE OF STUDY AN ITS IMPORTANCE
APPLICATIONS
CHALLENGES
CONCLUSIONS
REFERENCES
The document describes the development of a QTL map for Egyptian durum wheat using an F2 mapping population derived from a cross between two parental varieties, Baniswif-1 and Souhag-2. A variety of DNA markers including SSRs, RAPDs, AFLPs, ESTs and SCoTs were used to genotype the mapping population. Traits related to yield and drought tolerance such as root length, plant height, spike characteristics, and biomass were measured. Linkage analysis was performed to construct a genetic linkage map, which was then used to detect QTLs associated with the traits of interest.
Gene pyramiding is a plant breeding technique that uses molecular markers to select plants with multiple pest-resistance and yield-enhancing genes through iterative hybridization. It aims to enhance trait performance, remedy genetic deficits, and increase durability against pests. Strategies for gene stacking include iterative hybridization between plants containing different transgenes, re-transformation of a plant with additional transgenes, and co-transformation of a plant with multiple transgenes simultaneously. Gene pyramiding is an important strategy for improving germplasm and developing durable pest resistance using multiple genes.
This document provides an overview of molecular markers that can be used for crop improvement. It discusses different types of markers such as morphological, cytological, biochemical, DNA-based markers. DNA-based markers are further classified into hybridization-based markers like RFLP and PCR-based markers like RAPD, AFLP, SSR, ISSR, SNP. The document compares various marker techniques and provides their principles, strengths, weaknesses and applications in crop breeding programs. Molecular markers can be useful for tasks like hybrid purity testing, genetic diversity analysis, linkage mapping and marker-assisted selection.
This document discusses different types of mapping populations that can be used for genetic mapping and molecular breeding programs. It describes F2, F2:F3, backcross, doubled haploid (DH), recombinant inbred line (RIL), and near-isogenic line (NIL) populations. For each type, it provides details on how they are developed, their characteristics, and merits and demerits relative to mapping objectives. The document emphasizes that the choice of mapping population depends on the research goals, availability of markers, and existing molecular maps. RILs and DHs allow for replication over environments but require more time and resources to develop.
This study analyzed G proteins, proteomics, glycomics and metabolomics in plants grown under protected agriculture. Protein profiles showed variation between phenological stages, and western blot detected G protein subunits of 37, 46, and 57 kDa. Two-dimensional electrophoresis identified a 57 kDa protein with a pI of 5.9. Phloem sap proteins also detected G protein subunits of 28, 67 kDa between stages. Sugar analysis found glucose, fructose and sucrose varied between stages, with neutral sugars dominated by glucose, galactose and mannose. The Lightbourn Biochemical Model was applied to integrate results and propose bionanotechnology and biodynamic nutrition for high agricultural competitiveness and sustainability
IJRET : International Journal of Research in Engineering and Technology is an international peer reviewed, online journal published by eSAT Publishing House for the enhancement of research in various disciplines of Engineering and Technology. The aim and scope of the journal is to provide an academic medium and an important reference for the advancement and dissemination of research results that support high-level learning, teaching and research in the fields of Engineering and Technology. We bring together Scientists, Academician, Field Engineers, Scholars and Students of related fields of Engineering and Technology.
Mitochondria are organelles found in plant cells that provide energy and regulate important metabolic processes. Plant mitochondrial genomes vary significantly in size but generally encode proteins involved in oxidative phosphorylation as well as rRNAs and tRNAs. These genomes often contain introns, open reading frames, and chloroplast DNA sequences. Mutations in mitochondrial DNA can impact plant development and cause cytoplasmic male sterility. Expression of chimeric mitochondrial genes is associated with some cases of male sterility. Studies examine the role of mitochondrial proteins like uncoupling proteins and alternative oxidases in conferring stress tolerance in plants. The WA352 mitochondrial gene is implicated in cytoplasmic male sterility in rice through interaction with the nuclear-encoded protein COX11.
The document describes a research study aimed at developing biomarkers for detecting potential allergenicity of novel foods, including genetically modified foods. The researcher conducted experiments challenging mice with known food allergens (egg ovomucoid protein and peanut protein) and analyzed gene expression profiles in the mice spleens. Several hundred genes were found to be differentially expressed. After validating some genes, the researcher identified potential biomarker genes that could help detect allergenicity of GM foods. The study provides insights into transcriptomic responses to food allergens and biomarkers that may help evaluate allergenicity of novel foods like GM crops.
An attempt was made to study the Cytogenetical effects of gamma rays and ethyl methane sulphonate on meiotic chromosomal abnormalities in two cultivars viz., PKV-1 and JS-335. The most frequently observed aberrations in meiosis were univalents, trivalent, multivalents chromosomal fragments, desynapsis of chromosome, laggards, and clumping of chromosomes etc. The physical mutagens were more effective than chemical mutagens. The effect of gamma-rays and ethyl methane sulphonate shows chlorophyll mutations such as Chlorina, Xantha, Albina, and Alboviridis in an M2 generation in both the cultivars. Cultivar JS-335 shows more pronounced effect than cultivar PKV-1. Gamma-rays recorded maximum macro mutations as compared to chemical mutagens (EMS). The frequency and spectrum of morphological mutation indicated that variety JS-335 was more sensitive than PKV-1. Different response of the two varieties to various mutagens was noticed. Key-words- Gamma radiation, EMS, Chromosomal aberrations, Mutagens, Chlorophyll mutation
Post genomic tools for genetic enhancement of germplasmVishu1234567
This document discusses how post-genomic tools like transcriptomics, proteomics, and metabolomics can be used for genetic enhancement of germplasm. It provides an introduction to each omics technique, examples of technologies used, and applications in understanding biological processes and identifying genes/proteins involved in traits. The conclusion states that omics expression analysis of germplasm will help characterize genome function and restore traits from wild varieties, aiding development of more sustainable crop varieties.
Native and recombinant sources can be used to produce enzymes. Preparation of crude extracts involves homogenizing or lysing the source cells or tissues and preventing proteolysis. Various techniques can then be used to separate and purify proteins based on exploiting differences in their solubility, charge, size, or ability to bind specifically. Key steps include precipitation with ammonium sulfate or organic solvents, ion exchange chromatography, size exclusion chromatography, and affinity chromatography. Fractional precipitation provides a simple initial concentration and enrichment of proteins.
Access to large-scale omics datasets i.e. genomics, transcriptomics, proteomics, metabolomics, phenomics, etc. has revolutionized biology and led to the emergence of systems approaches to advance our understanding of biological processes. With decreasing time and cost to generate these datasets, omics data integration has created both exciting opportunities and immense challenges for biologists, computational biologists, biostatisticians and biomathematicians. Genomics, transcriptomics, proteomics, and metabolomics together they help to bring out the best of characters in plants.
Creating a detailed map of the wheat flour proteome: a critical step in under...CIMMYT
The document discusses creating a detailed proteomic map of wheat flour to understand how environmental conditions affect flour quality and immunogenic potential. It outlines challenges in mapping wheat's complex gluten proteins. The researchers used multiple proteases and mass spectrometry to identify 168 distinct protein sequences in wheat flour. They found environmental factors like fertilizer can significantly change the levels of specific gluten proteins, providing insights into how the wheat grain responds to its growth environment.
The document discusses an overview of livestock metabolomics. It defines metabolomics as the large-scale study of small molecules present in cells, biofluids, tissues and organs. Various techniques for metabolomic analysis are described including mass spectrometry, NMR spectroscopy, GC-MS and LC-MS. Applications of metabolomics in livestock include disease diagnosis, biomarker identification, monitoring drug and surgical impacts, and understanding gene-environment interactions. Specific examples include identifying metabolic biomarkers for mastitis resistance in dairy cows and detection of milk fever in cattle. The challenges and future prospects of metabolomics research are also outlined.
Meta-genomics is the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species”
Environmental Stresses on Listeria monocytogenesMalvi Prakash
The document summarizes research on how environmental stresses impact the pathogenicity of Listeria monocytogenes. It discusses how L. monocytogenes is able to withstand stresses like salt, low temperatures, and pH levels. The research found that in response to salt stress, L. monocytogenes both represses and induces various proteins. General stress proteins and osmolyte transporters were among those induced to help the bacteria adapt. Signal transduction and surface proteins also help L. monocytogenes interact with and survive in changing environmental conditions. The conclusion discusses how a better understanding of these response mechanisms could help develop techniques to control the pathogen in food processing.
Purification of G-Protein Coupled Receptor from Membrane Cell of Local Strain...iosrjce
The aim of this study to purify GPCR from a local strain of S. cerevisiae using gel filtration
chromatography techniques , by packing materials for columns which will be chosen of low cost comparing to
the already used in published researches, which depend on the costly affinity chromatography and other
expensive methods of purification. Local strain of S. cerevisiae chosen for extraction and purification of Gprotein
coupled receptor (GPCR) .The strains were obtained from biology department in Al- Mosul University,
Iraq. The isolated colony was activated on Yeast Extract Pepton Dextrose Broth (YEPDB) and incubated at 30
C˚ for 24 h .Loop fully of the yeast culture was transferred to (10ml) of yeast extract peptone glucose agar
(YEPGA) slant , then incubated at 30C˚for 24h , after that it was stored at 4C˚ ,the yeast cultures were
reactivated and persevered after each two weeks period. S.cerevisiae was identified by morphological,
microscopic characterization and biochemical test . The GPCR that extract from membrane of S.cerevisiae was
purified by gel filtration chromatography in two steps using Sepharose 6B. The optical density for each fraction
was measured at 280 nm by UV-VS spectrophotometer then the GPCR concentration was determined by using
ELISA Kit . The fractions which gave the highest absorbance and concentration of GPCR were collected .The
molecular weight of GPCR was determined by gel filtration chromatography using blue dextrin solution.
Standard curve was plotted between log of molecular weight for standard protein and the ratio of Ve/Vo of
GPCR . The purity of the GPCR that extracted and purified from whole cell of S, cerevisiae were carried out by
using SDS-PAGE electrophoresis In the first step 5ml of crude extract was applied on sepharose 6B column
(1.6x 96 cm) which previously equilibrated with 50 mM phosphate buffer saline pH= 7.4 . Multiple proteins
peaks appeared after elution with elution buffer (PBS PH= 7.4 containing 0. 5 % DDM). One peak only give
positive result with GPCR assay, fractions representing GPCR were collected , pooled and concentrated by
sucrose. In the second step five active fractions from the previous step were collected and applied once again on
the same column and same conditions. This step gave a single peak that was identical with the peak of GPCR
concentration ,maximum concentration of GPCR that observed in the fractions (34-38) was 18.541 (ng/ml) . The
specific activity for these fractions was 261.14 (ng/mg) protein with yield of 47.717%. The present study a chive
a relatively high purification of GPCR from membrane fraction of a local strain S. cerevisiae with fold
purification 5.094 and a yield of 47.717%. and molecular weight about~55KD.
Proteomics of small proteins from plant tissuesExpedeon
Small genes and the proteins that they encode can play important biological roles including signaling, development, and mediation of plant-microbe interactions in organisms ranging from bacteria to plants to mammals (Frith et al.; Basrai et al.; Galindo et al.; Hemm et al. 2008, 2010; Kastenmeyer et al.). However, genes that encode proteins containing <100 residues are difficult to identify reliably solely by DNA sequence analysis (Dinger et al.)
This document provides an overview of plant biotechnology techniques. It discusses how genes can be manipulated by identifying genes that control traits of interest or modifying existing genes. Genes are then introduced into organisms using transformation methods like Agrobacterium or gene guns. Transformation cassettes containing the gene of interest and selection markers are used. The document explains this process and provides examples like making crops resistant to herbicides or increasing vitamin levels. It also notes there is public controversy around developing and releasing transgenic organisms.
The document discusses techniques for gene manipulation and introduction used in plant biotechnology. It describes how genes can be identified and modified, such as isolating a gene that controls a desired trait or creating a new allele. The modified gene is then introduced into an organism using transformation methods, creating transgenic plants. Specific examples discussed include creating Roundup-resistant crops by introducing a bacterial gene and developing Golden Rice by adding genes to produce vitamin A in rice.
Optimization of Bacillus Subtilis Natto Immobilization Process on Alginate – ...inventionjournals
Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. In this study, Bacillus subtilis natto were immobilized in the alginate – chitosan complex for fermentation of nattokinase enzyme. Six factors affecting the efficiency of immobilization cells were screened by Plackett – Burman design including: concentration of alginate, concentration of chitosan, pH of chitosan, concentration of CaCl2, added cells density, shaking time after supplementing chitosan. Results of optimization have identified two factors affecting the efficiency of cell immobilization. They are concentration of alginate (2.5%) and added cells density (approximately 5.86 million colonies per milliliter). With these two factors optimized and others kept at the normal level, immobilization efficiency reached 90.73%. After Bacillus subtilis natto had been immobilized by optimization of parameters, we conducted application for fermenting nattokinase. For 24 hours of fermentation, nattokinase enzyme activity reached 71.80 ± 0.19 FU/ml. Immobilized Bacillus subtilis natto cells were reused 6 times and on the 6 th time of reuse, nattokinase enzyme activity only decreased 2.7% in compared with the 1st reuse.
Optimization of Bacillus Subtilis Natto Immobilization Process on Alginate – ...inventionjournals
Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. In this study, Bacillus subtilis natto were immobilized in the alginate – chitosan complex for fermentation of nattokinase enzyme. Six factors affecting the efficiency of immobilization cells were screened by Plackett – Burman design including: concentration of alginate, concentration of chitosan, pH of chitosan, concentration of CaCl2, added cells density, shaking time after supplementing chitosan. Results of optimization have identified two factors affecting the efficiency of cell immobilization. They are concentration of alginate (2.5%) and added cells density (approximately 5.86 million colonies per milliliter). With these two factors optimized and others kept at the normal level, immobilization efficiency reached 90.73%. After Bacillus subtilis natto had been immobilized by optimization of parameters, we conducted application for fermenting nattokinase. For 24 hours of fermentation, nattokinase enzyme activity reached 71.80 ± 0.19 FU/ml. Immobilized Bacillus subtilis natto cells were reused 6 times and on the 6 th time of reuse, nattokinase enzyme activity only decreased 2.7% in compared with the 1st reuse.
33.Expression, Production and Purification of Proteinases from Aspergillus spp.Annadurai B
This document summarizes research on the expression, production, and purification of proteinases (proteases) from 18 strains of Aspergillus. The researchers found that Aspergillus flavus and Aspergillus sojae had the highest mycelial dry weight, indicating maximum growth. Aspergillus tamarii and Aspergillus awamori showed the highest protein content. Aspergillus nidulance had the highest proteinase content. The proteinases were purified using ammonium sulfate precipitation and column chromatography. SDS-PAGE analysis revealed a protease band with a molecular weight of 60,000 Da. The study demonstrates that certain Aspergillus strains are good producers of extracellular proteinases, which
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significant role in maintaining the ecological equilibrium of our planet.Land serves as the foundation for all human activities and provides the necessary materials for
these activities. As the most crucial natural resource, its utilization by humans results in different
'Land uses,' which are determined by both human activities and the physical characteristics of the
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The utilization of land is impacted by human needs and environmental factors. In countries
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to significant land degradation, adversely affecting the region's land cover.
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3. PROTEOMICS
• It is the study of “Proteome”.
• The word "proteome" is a blend of "protein" and "genome”.
• Large scale study of Proteins.
• Particularly their structures and functions.
• Study of full set of proteins in a cell type or tissue, and changes during various conditions
Involves
protein-protein interactions
organelle composition
protein activity patterns and
protein profiles.
3
4. Functions of protein
Function Description
Membrane transport
Membrane proteins are used for facilitated diffusion and active transport, and also for
electron transport during cell respiration and photosynthesis.
Hormones Play vital role for growth and development of plant.
Receptors
Binding sites in membranes and cytoplasm for hormones, neurotransmitters, tastes and
smells, and also receptors for light in the eye and in plants.
Packing of DNA
Histones are associated with DNA in eukaryotes and help chromosomes to condense
during mitosis.
4
5. The Virtue of the Proteome
Proteome = protein compliment of the genome
Proteomics = study of the proteome
Protein world = study of less abundant proteins
Transcriptomics = often insufficient to study functional aspects of genomics
5
6. What is the Proteome?
DNA
RNA
Protein
Modified Proteins/
Functional Protein
Transcription
The collection of proteins in a cell
Translation
Post-translational
modification (PTM)
6
7. Proteomics
Proteomics is an attempt to describe biological state and qualitative
and quantitative changes of protein content of cells and extracellular
biological materials under different conditions to further understand
biological processes.
A large-scale characterization and functional analysis of
the proteins expressed by a genome
The term proteomics was first coined by Marc Wilkins in
1994 to make an analogy with genomics.
7
9. Why Proteomics?
• Whole Genome Sequence –complete, but it does not show how proteins function or
biological processes occur.
• Post-translational modification –proteins sometimes chemically modified or regulated
after synthesis.
• Protein – protein interactions.
• Proteins fold into specific 3-D structures which determine function.
• Gain insight into alternative splicing
9
10. Why Proteomics?
• Several levels of regulation from gene to function
• Proteins are the ultimate operating molecules producing the physiological effect
10
15. Fig. 2. Plant Protein Extraction and Fractionation
15
16. Fig. 3. Two-dimensional Gel Electrophoresis
First dimension: IEF (based on isoelectric point)
SDS-PAGE
(basedonmolecularweight)
+ -
acidic basic
High
MW
Low
MW
Sample
Hahne et al., 2010 16
17. Components of a Mass Spectrometer
1. Source – produces gas-phase ions from the sample
2. Mass analyzer – resolves ions based on their m/z ratio
3. Detector – detects ions resolved by the mass analyzer
Sample
Input Ionization
Source
Mass
Analyzer
Detector
m/z
Intensity
Size of arrows indicates the size of the ions, not the flight order or order of detection 17
21. CS-1: Effects of NaCl on protein profiles of
tetraploid and hexaploid wheat species and
their diploid wild progenitors
• Wheat cultivar: Triticum monococcum, Aegilops speltoides, Aegilops tauschii, Triticum durum and Triticum aestivum.
• Wheat seedlings were placed into plastic cups (200 ml)
• Including Hewitt solution without salt and
with salt 100 mmol/l NaCl.
• All solutions were renewed every two days.
• Control and NaCl treated seedlings were grown in a controlled growth during 15 days.
• At the end of this period, the first leaves of control and NaCl-treated seedlings were sampled for electrophoresis analysis.
• Increase in the amount of the proteins may lead to an increase in the tolerance mechanisms towards NaCl salinity of wheat species
Yildiz and Terzi (2008)Afyonkarahisar, Turkey
21
22. Fig. 5. 2D SDS PAGE profiles of the soluble leaf proteins extracted from control (C) and salt (S)-treated seedlings of
Triticum monococcum, Aegilops speltoides, Aegilops tauschii, Triticum durum and Triticum aestivum.
Increased, decreased and completely lost proteins in S
compared to C treatment are indicated by arrow in both
control and salt treatments 22
23. Protein
number
Mr pI
Cultivated wheat species Wild wheat species (progenitors)
Triticum aestivum
(AABBDD)
Triticum durum
(AABB)
Triticum monococcum
(AA)
Aegilops speltoides
(BB)
Aegilops tauschii
(DD)
1 20.3 5.1 -* CL*
2 20.7 5.1 - CL
3 20.7 6.8** - +* - +
4 21.0 5.1 + - CL
5 21.0 6.6 - -
6 21.0 6.8 +
7 21.5 6.6 -
8 21.7 6.7 -
9 21.7 6.9 CL
10 22.0 6.3 + +
11 22.0 6.6 -
12 22.5 6.2 +
13 22.5 6.8 + -
14 22.7 7.1 +
15 22.9 7.0 -
16 23.0 5.4 +
17 23.0 5.5 +
18 23.0 6.7 - +
Table 1. Protein polymorphism in the leaf tissues of cultivated hexaploid and tetraploid wheat species and their diploid wild
progenitors in 100 mmol/l NaCl treatment compared to the control treatment
*CL = completely lost protein; − = protein decreased in amount; + = protein increased in amount **bold numbers in Mr and pI column indicate common proteins between at least two species 23
Cont..
24. Protein
number
Mr pI
Cultivated wheat species Wild wheat species (progenitors)
Triticum aestivum
(AABBDD)
Triticum durum
(AABB)
Triticum monococcum
(AA)
Aegilops speltoides
(BB)
Aegilops tauschii
(DD)
19 23.2 6.5 - - -
20 23.2 7.1 + + - -
21 23.3 6.6 -
22 23.5 6.3 -
23 23.5 6.5 + -
24 23.5 6.7 + -
25 23.5 6.8 + + -
26 23.8 6.3 -
27 23.9 6.2 -
28 24.5 6.3 -
29 29.5 6.2 +
30 30.3 6.2 + -
31 30.4 6.3 +
32 30.6 6.3 +
33 33.4 6.7 +
34 34.8 7.5 + -
35 35.3 7.8 + - +
36 35.4 6.6 +
37 35.4 7.5 + -
*CL = completely lost protein; − = protein decreased in amount; + = protein increased in amount **bold numbers in Mr and pI column indicate common proteins between at least two species
24
Cont..
25. CS-2: Proteomics analysis of salt-induced leaf
proteins in two rice germplasms with different
salt sensitivity
• Seeds of the cultivars Dongjin, and a weedy rice, Dalseongaengmi-44,
were grown hydroponically in a controlled environment growth chamber
• Two week old seedlings were treated with nutrient solution (Sohn et al.
2005) containing 0, 45 and 90 mM NaCl for 4 d.
• The rice leaf samples were then collected and immediately frozen in
liquid nitrogen, after which they were stored at -800C until protein
extraction.
Lee et al. (2011)Jinju, Korea 25
26. Fig. 6. 2-DGE analysis (PEG-fractionated supernatant samples) of leaf proteins in salt-stressed rice
cultivars (Dongjin and Dalseongaengmi-44).
A total of 100 mg proteins were separated by 2-DGE, and then visualized using silver stain.
Arrows and indicate proteins that were up-regulated in response to salt stress.
26
control
control
control
control
90 mM NaCl
90 mM NaCl
90 mM NaCl
90 mM NaCl
Protein no.= 2 Class III peroxidase 29 precursor
3 = Beta-1,3-glucanase precursor
4 = Beta-1,3-glucanase precursor
5 = ATP synthase beta subunit
8 = OSJNBa0086A10.7 (putative transcription factor X2)
16 = Rubisco activase small isoform precursor
20 = Drought-induced S-like ribonuclease
27. Fig. 7. Enlarged views of the up-regulated proteins identified from the PEG-
fractionated pellet samples of the rice leaf tissues.
27
Dongjin
DongjinDalseongaengmi-44
Dalseongaengmi-44
28. Table 2. Abundance ratio of the up-regulated proteins in the leaf tissues of a Dongjin and
Dalseongaengmi-44 rice cultivar after 96 h of salt stress treatment
Spot no.
Abundance ratioz
45 mM 90 mM
1 2.57±0.01y 3.27±0.25
3 2.76±0.91 3.77±1.31
4 3.52±0.21 6.22±0.66
6 1.42±0.15 4.44±1.31
7 1.36±0.34 8.29±0.49
8 1.44±0.07 2.98±0.27
10 2.72±0.81 7.41±0.93
11 4.41±2.29 16.80±3.58
12 2.78±0.54 5.05±0.30
14 7.02±2.99 6.38±0.54
15 2.20±0.43 4.48±0.36
16 6.27±0.93 13.11±1.90
17 2.03±0.62 2.66±0.03
21 5.70±1.41 15.63±1.64
22 3.47±0.72 8.42±0.62
23 3.76±1.04 7.87±2.02
28
Spot no.
Abundance ratioz
45 mM 90 mM
2 1.90±0.74y 3.94±0.97
3 1.65±0.33 6.12±1.66
4 6.41±0.33 22.02±3.32
5 1.88±0.48 7.26±1.01
9 3.85±0.43 14.44±2.50
12 1.42±0.86 6.44±2.68
13 1.09±0.51 5.31±4.47
15 1.06±1.02 5.02±5.47
18 4.57±0.94 8.78±0.86
19 3.34±0.48 7.34±0.97
20 5.65±1.01 13.67±0.68
21 16.08±0.78 19.13±0.97
22 2.10±0.49 5.49±1.27
23 7.73±0.92 15.51±1.12
Dalseongaengmi-44Dongjin
z = The abundance ratios of each spot were measured using a densitometer (Bio-rad) and then
compared with those of controls.
y = Data represent the means9SE of three biological replicates.
30. Table 4. Salt-stress-induced up-regulated proteins identified by PMF with MALDI-TOF MSz
Spot
no.
NCBI
accession
no.
Protein name Organism
Theoretical Observed
MOWSE
score
My SCx
Mr pI Mr pI
1 ABB47307 ATP synthase F1 beta subunit (chloroplast) O. sativa 60.3 5.3 46.0 6.2 2.31+9 13 31
2 CAH69271 Class III peroxidase 29 precursor O. sativa 34.2 5.3 35.3 5.9 8.50+7 14 55
3 ADD10383 Beta-1,3-glucanase precursor O. sativa 34.8 4.7 33.5 5.6 27509 6 18
4 ADD10383 Beta-1,3-glucanase precursor O. sativa 34.8 4.7 33.2 5.7 92493 7 22
5 BAA90397 ATP synthase beta subunit O. sativa 54.0 5.4 33.0 6.0 3.70+10 15 30
6 CAG34174 Rubisco large chain O. sativa 52.8 6.2 32.9 6.6 95575 11 21
7 CAG34174 Rubisco large chain O. sativa 52.8 6.2 32.3 6.5 1.74+8 19 26
8 NP_918125 OSJNBa0086A10.7 (putative transcription factor X2) O. sativa 28.7 6.7 28.0 6.3 1708 4 18
9 AAX95072 Fructose-bisphosphate aldolase class-1 O. sativa 42.0 6.4 26.0 5.8 41360 9 24
10 AAV44199 Dehydroascorbate reductase O. sativa 23.8 5.8 24.0 6.4 12503 5 39
13 NP_917384 Putative ribosomalprotein L12 O. sativa 18.2 5.4 21.0 5.5 2846 4 40
14 CAJ01693 2-Cys peroxiredoxin O. sativa 28.1 5.7 19.2 5.8 23814 5 33
15 BAD36628 Putative chaperon 21 precursor O. sativa 23.1 5.7 18.2 5.8 3632 5 34
16 AAX95286 Rubisco activase small isoform precursor O. sativa 47.9 5.9 41.0 5.8 3.11+13 24 46
17 CAG34174 Rubisco large chain O. sativa 52.8 6.3 33.5 6.1 8.52+8 22 31
18 Q9LSU1 Proteasome subunit alpha type 5 O. sativa 26.0 4.7 29.0 5.5 1.38+7 7 50
30
zPMF = peptide mass fingerprinting;
MALDI-TOF MS = matrix-assisted laser desorption ionization-time of flight mass spectrometry;
Mr = molecular weight;
pI = isoelectric point.
y = Number of matching peptides.
x = Sequence coverage by PMF.
31. Fig. 8. MS analysis of spot 14. Protein spot 14 was excised and then digested with trypsin, after which the resulting
peptides were analyzed using the QSTAR pulsar-i MS system.
31
(A), MS spectra. The ion (1721.9) marked with an asterisk was analyzed by MS/MS.
(B), the MS/MS spectra of ion 1721.9.
The b ions (b1-6), y ions (y1-10) and the corresponding peptide sequences are shown.
The protein was identified as 2-Cys peroxiredoxin (NCBI accession number AJO1693) by a database
search
32. Table 5. Proteins up-regulated in response to salt-stress-identified by sequence tag analysis with ESI-MS/MSZ
Spot
no.
NCBI
accession no.
Protein name Organism
Theoretical Observed Theoreticaly
(%)
Sequencex
Mr pI Mr pI
11 CAG34174 Rubisco large chain O. sativa 52.8 6.2 23.0 6.0 100 GIQVER
12 AAO65861 Putative actin binding protein O. sativa 16.2 4.9 4.9 5.5 100 SLPADGCR
100 AAGEGGEAPR
100 SPAAADVR
14 CAJ01693 2-Cys peroxiredoxin O. sativa 28.1 5.7 5.7 5.5 100 STINNLAIGR
100 PLISDVTK
100 QGIALR
19 BAD33340 Putative triose phosphate isomerase, Chloroplast precursor O. sativa 32.4 7.0 7.0 5.8 100 AQEVHAAVR
91 VSPEVAGSGIR
100 IGELLEER
100 NSVTSK
20 AAL33776 Drought-induced S-like ribonuclease O. sativa 28.4 5.2 5.2 5.8 100 GVTPGVQ
100 GYPSEDFFVK
100 ENTAVVR
100 YNTAFIK
21 CAG34174 Rubisco large chain O. sativa 52.8 6.2 6.2 5.7 100 GIQVER
22 CAG34174 Rubisco large chain O. sativa 52.8 6.2 6.2 6.8 100 DILAAFR
23 CAG34174 Rubisco large chain O. sativa 52.8 6.2 6.2 5.6 100 EYETK
zESI-MS/MS = electrospray ionization-tandem mass spectrometry;
Mr = molecular weight;
pI = isoelectric point.
y = Percentage of identities for sequence tags.
x = The sequence of the matching peptide.
Increase in concentration of NaCl to increase in conc. of protein,
this upregulated proteins are gives a tolerance mechanism
against a NaCl stress and identify upregulated protein
32
33. CS-3: Differential Proteins Expressed in Rice
Leaves in Response to Salinity and
Exogenous Spermidine Treatments
• protein spots were identified in the leaves of salt-tolerant (Pokkali) and salt sensitive (KDML105) rice cultivars
• Seven-day-old seedlings with three leaves were transplanted to plastic containers holding 20 L full-strength Yoshida nutrient solution (Yoshida et al, 1976)
with pH 5.0 (50 seedlings per container).
• The solution was renewed weekly and the pH was daily adjusted to 5.0.
• Two treatment groups of 30-day old seedlings were pretreated by adding 1.0 mmol/L Spd into nutrient solution.
• After 24 h of Spd pretreatment,
control,
Spermidine (Spd), (1.0 mmol/L Spd)
NaCl (150.0 mmol/L NaCl)
NaCl+Spd treatments
• After 7 d of salt treatment, the leaves were collected, immediately frozen in liquid nitrogen and stored at -80 °C.
• The experiment was carried out in a greenhouse under natural photoperiod
Saleethong et al. (2016)Surin, Thailand 33
34. Fig. 10. Two-dimensional electrophoretic patterns of soluble proteins in rice leaf.
Protein no. = 8 Oxygen-evolving complex protein 1,
this protein are responsive to salt stress and cause changes in the activity of photosystem II (PSII) in coping
with salt stress
34
Pokkali KDML105
(A) Pokkali
(B) KDML105
obtained from
control,
Spermidine (Spd),
NaCl and
NaCl+Spd treatments
35. Table 6. Identification of differential proteins and changes in the intensity of each protein spot in the leaves of Pokkali.
Protei
n
No.
Increasing or decreasing component (Fold)
pI
Mw
(kDa)
Accession no.
Homologous
protein
Score Function
C/NaCl C/S S/S+NaCl Pr S/NaCl
1A ↑(4.03) ↓(4.58) ↑(4.67) ↓(3.96) 7.4 32
NP_00105638
9
Os05g0574400 377 Similar to malate dehydrogenase
2A - ↓(8.48) - ↓(2.96) 6.7 32
NP_00104371
7
Os01g0649100 369 Similar to malate dehydrogenase
3A ↓(2.68) ↓(3.92) - ↓(2.20) 5.4 32 ABA91631
Fructose
bisphosphate
aldolase
451 Fructose-bisphosphatealdolase activity
4A ↓(1.05) ↓(3.54) ↑(1.57) ↓(2.15) 4.9 29
NP_00104313
4
Os01g0501800 1633
Similar to photosystem II oxygenevolving
complex protein 1
5A ↑(2.00) ↓(11.78) - ↓(17.03) 5.3 24 AAB63603
Triosephosphate
isomerase
251 Triose-phosphate isomerase activity
6A ↑(2.28) - - ↓(3.34) 6.4 24 EEC78412
Hypothetical
protein OsI_18213
251 Putative uncharacterized protein
7A - ↓(3.71) - ↓(6.71) 4.8 23 BAD35228
Putative
chaperonin 21
precursor
606 Chaperonin ATPase activity
8A ↑(1.04) ↓(1.60) ↑(2.36) ↑(1.12) 5.9 21
NP_00105886
3
Os07g0141400 976
Similar to photosystem II oxygenevolving
enhancer protein 2
9A - ↓(6.06) ↑(2.80) ↓ (2.07) 6.6 18 AAA33917
Superoxide
dismutase
60
Superoxide dismutase copper
chaperone activity
35
C/NaCl = control gel was compared with the NaCl gel
C/S = the control gel was compared with the Spd gel,;
S/S+NaCl = Spd gelwas compared with Spd+NaCl gel
Pr S/NaCl = the plants were pretreated Spd, with the plants treated with NaCl
pI, Isoelectric point; Mw, Molecular weight.
36. Table 7. Identification of differential proteins and changes in the intensity of each protein spot in the leaves
of KDML105.
Protei
n
No.
Increasing or decreasing component (Fold)
pI
Mw
(kDa)
Accession no. Homologous protein Score Function
C/NaCl C/S S/S+NaCl Pr S/NaCl
1B ↓(2.02) ↓(1.66) ↑(2.39) ↑(2.91) 7.8 34 NP_001056389 Os05g0574400 386 Similar to malate dehydrogenase
2B - ↓(2.33) ↑(1.53) ↓(1.65) 7.0 34 NP_001043717 Os01g0649100 311 Malate dehydrogenase
3B ↓(1.34) ↓(3.43) - ↓(2.73) 6.4 34 ABG66141 Malate dehydrogenase 193 Malate metabolic process
4B ↓(4.11) ↓(2.01) - ↑(1.97) 5.7 35 ABA91631 Fructose-bisphosphate aldolase 806 Fructose-bisphosphatealdolase activity
5B ↑(1.58) ↓(2.65) ↑(2.73) ↓(1.53) 5.6 27 AAB63603 Triosephosphate isomerase 727 Triose-phosphate isomerase activity
6B ↓(3.41) ↓(4.62) ↑(3.81) ↑(2.81) 6.9 27 EEC78412 Hypothetical protein OsI_18213 248 Putative uncharacterized protein
7B ↓(1.18) ↑(1.43) ↓(6.01) ↓(3.58) 4.7 19 NP_001065834 Os11g0165700 89
Jacalin-related lectin domain containing
protein
8B - ↓(1.49) - ↓(1.28) 6.5 20 2002393A
Oxygen-evolving complex
protein 1
97 Oxygen sensor activity
9B ↑(9.85) ↑(7.57) ↓(1.36) ↓(1.77) 6.8 20 AAA33917 Superoxide dismutase 55
Superoxide dismutase copper chaperone
activity
10B ↓(1.59) ↑(1.48) ↓(2.73) - 8.6 20 NP_001067074 Os12g0569500 100
Thaumatin, pathogenesis-related family
protein
11B ↑(11.94) ↑(5.92) - ↓(1.54) 7.2 17 BAC10110 Copper/zinc-superoxide 71 Antioxidant activity
12B ↑(41.74) ↑(31.19) ↓(1.18) ↓(1.58) 5.8 16 BAD09607
dismutasePutative superoxide
dismutase [Cu-Zn] 389 Antioxidant activity
36
C/NaCl = control gel wascompared with the NaCl gel
C/S = the control gel was compared with the Spd gel,;
S/S+NaCl = Spd gelwas compared with Spd+NaCl gel
Pr S/NaCl = the plants were pretreated Spd, with the plants
treated with NaCl
pI, Isoelectric point; Mw, Molecular weight.
The photosynthetic oxygen-evolving enhancer protein 2 was detected only in Pokkali and
was up-regulated by salt-stress and further enhanced by Spd treatment. that Spd acted directly as
antioxidants and give a tolerant mechanism against NaCl stress.
38. CS-4: Proteomic Analysis of Drought Stress-
Responsive Proteins in Rice Endosperm
Affecting Grain Quality
• IR-64 (Indica cultivar)
• Plants were grown to maturity under greenhouse conditions
• were subjected to 4 days of rapid drought stress starting 3 days before heading
(3DBH).
• Properly filled seeds for proteomic analysis were collected at maturity from both
well-watered and drought-stressed rice plants.
• Sample grains were dehusked, embryo was excised from the seed, and the
remaining endosperm was used for protein extraction.
Manila, Philippines Mushtaq et al. (2008) 38
39. Fig. 11. Effect of drought on spikelet fertility per panicle in IR-64
Two-dimensional gel protein patterns of mature seed endosperm of IR-64 plants drought stressed for 4 days at 3 days
before heading.
Approximately 120 μg of protein was loaded on each gel.
Sizes of molecular markers and the pI range of the first dimension (pI 4-7) are indicated. 39
Sky blue color bar represents spikelet fertility in well watered plants and Gray
color Bar represents spikelet fertility in drought-stressed plants.
spikeletfertility
40. Fig. 12. Behavior of (A) Granule-Bound Starch Synthase (GBSS), (B) Nucleoside diphosphate
Kinase and (C) Globulin, on 2-dimensiosnal gels of grain proteome of rice cultivar IR-
64, under well-watered (WW), and drought-stressed (4DS) conditions.
40
(A) Starch is composed of two distinct polymers, amylopectin and
amylose amylose in endosperm of rice grain is synthesized by a
Granule-Bound Starch Synthase (GBSS),
It is a product of waxy gene, has been improving the quality of rice
grains
GBSS remained unaltered in its behavior,
No effect on grain quality.
(B) NDP is involved in nitrogen metabolism.
In seeds, nitrogen mainly originates from leaves and stems that
mobilize more than 65% of their nitrogen content.
NDP is down-regulation has affected seed filling under drought stress
conditions and consequently, might have affected seed yield.
(C) Globulins are an important source of major endosperm storage
protein
Globulin was down-regulated
Therefore, nutritional quality of rice grains under drought stress
conditions may have been affected
41. Table 8. Abundance ratio of endosperm proteins during drought conditions.
Spot no. pI MW AB ration
1 5.89 41 0.63*
2 6.75 58 1.86*
3 6.73 43 0.31*
4 6.46 43 2.31*
5 5.81 28 0.42*
6 6.84 40 NA
7 5.52 19 4.90*
8 6.55 22 0.55*
9 5.10 36 0.27/0
10 6.40 19 0.73*
11 6.51 65 NA
12 6.80 75 1.05
13 4.17 25 1.56*
NA=data not available 41
42. Table 9. Proteins identified by MS.
Spot
no.
MP/Ca Protein name
Accession
no.
Experimental
Mw/pI
Subcellular
localization/
Probability
5 8/28 19 kDa globulin precursor CAA45400 28/5.84
Mitochondrial outer membrane /
0.850
6 6/25 Glutelin type I precursor XP_463450 40/6.84 outside / 0.820
10 3/38
Nucleoside diphosphate
kinase
XP_478187 19/6.4 Microbody peroxisome /0.640
11 15/28
Granule-bound Starch
Synthase
AAC61675 65/6.51 chloroplast stroma / 0.851
12 2/47 B1160F02.9 XP_470940.1 75/6.7 Cytoplasm/0.650
a the number of matched peptides/the percentage of sequence coverage.
42
Identified proteins such as Granule-Bound Starch Synthase (GBSS, Wx protein), which is
thought to play a very important role in starch biosynthesis and quality and is a very crucial
factor in determining rice grain quality.
43. CS-5: Comparative Analysis of Sorghum
bicolor Proteome in Response to
Drought Stress and following Recovery
• 11434, drought tolerant, and 11431, drought sensitive
• Seeds were germinated in Petri dishes for 48 hours.
• Three seedlings were planted in 11 × 11 × 11 cm3 plastic pots filled with 700g soil
• Plants were kept in a greenhouse
• the soil was left to dry until the level of 10% field capacity and left further for 7 days without any watering.
• The plants were rewatered till water drain for recovery.
• third leaves were collected,
• frozen under liquid N2, and
• stored at −80∘C.
• 24 hours after recovery, samples of the third leaves were collected from the remaining plants,
• frozen under liquid N2, and
• stored at −80∘C.
Jedmowski et al. (2014)Frankfurt, Germany 43
44. Fig. 13. RWC of # 11434, drought
tolerant, and # 11431,
drought sensitive, genotypes.
Differentially expressed proteins on 10% SDS-PAGE following
separation on 24 cm nonlinear strips pH 3–10, scanning, and
staining with Colloidal Comassie Brilliant Blue.
Black arrows represent proteins expressed in both genotypes.
represent expressed proteins in # 11434, drought tolerant
genotype, and
white arrows with round bottom represent expressed proteins in #
11431, drought sensitive, genotype. 44
45. Table 10. MALDI-TOF-MS results of picked spots from control, drought treatment and recovery of drought tolerant, #
11434, and susceptible, # 11431, genotypes and their expression values in comparison to control
Spot
ID
NCBI Acc. no. Protein name Mr/pI 𝑆∗ %𝐶 𝑀
# 11434 # 11431
D R D R
01: Metabolism
6 gi/18483235 Methionine synthase/Sorghum bicolor 84135/5.93 96 8% 8 6.7 1.6 6.6
7 gi/18483235 Methionine synthase/Sorghum bicolor 84135/5.93 102 12% 12 5.5
16 gi/226502947 S-adenosylmethionine synthase l Z. mays 42986/5.57 193 19% 10 1.7 1.5
29 gi/666089 P-(S)-hydroxymandelonitrile lyase Sorghum bicolor 41796/4.72 154 13% 8 3.6
02: Energy
1 gi/242096062
Hypothetical protein Sorbidraft of S. bicolor homologues to putative C4 phosphoenolpyruvate carboxylase/Saccharum officinarum
CAC08829.1
108988/5.89 306 29% 28 2.1 3.3 -2
2 gi/242096062 Hypothetical protein Sorbidraft of S. bicolor homologues to putative C4 phosphoenolpyruvate carboxylase/S. officinarum CAC08829.1 108988/5.89 504 30% 33 1.7
3 gi/242096062 Hypothetical protein Sorbidraft of S. bicolor homologues to putative C4 phosphoenolpyruvate carboxylase/S. officinarum CAC08829.1 108988/5.89 331 29% 30 2.3
4 gi/242096062 Hypothetical protein Sorbidraft of S. bicolor homologues to putative C4 phosphoenolpyruvate carboxylase/S. officinarum CAC08829.1 108988/5.89 354 26% 30 2.4
5 gi/242080811 Hypothetical protein Sorbidraft of S. bicolor homologues to aconitate hydratase of Oryza sativa Japonica Q6YZX6.1 108402/6.41 101 11% 13 1.8
9 gi/242051769 Hypothetical protein Sorbidraft of S. bicolor homologues to chloroplast NADP-dependent malic enzyme Z. mays NP 001105313.1 69904/6.23 181 28% 21 -2.8 -2.4
10 gi/242051769 Hypothetical protein Sorbidraft of S. bicolor homologues to chloroplast NADP-dependent malic enzyme Z. mays NP 001105313.1 69904/6.23 199 25% 18 -2.8 -2.7
11 gi/242051769 Hypothetical protein Sorbidraft of S. bicolor homologues to chloroplast NADP-dependent malic enzyme Z. mays NP 001105313.1 69904/6.23 193 26% 18 -2.8 -2.3 -2
17 gi/242059597 Hypothetical protein Sorbidraft of S. bicolor homologues to fructose-1,6-bisphosphat aldolase, cytosol Z. mays NP 001105336.1 38990/6.96 278 23% 12 1.6
45
The search carried out against the entire NCBInr database; SID: spot identification number; Mr/pI: calculated molecular weight and isoelectric point of predicted
proteins; 𝑆: Score; ∗protein scores greater than 84 are significant (𝑃 < 0.05); 𝐶%: percentage of coverage;𝑀: number of peptides matched; 𝐷: drought; 𝑅: recovery.
Cont..
46. Spot
ID
NCBI Acc. no. Protein name Mr/pI 𝑆∗ %𝐶 𝑀
# 11434 # 11431
D R D R
24 gi|195634659 Plastid fructose-1,6-bisphosphate aldolase, Z. mays 41924/7.63 395 34% 19 -2.1 -1.9 -1.8
19 gi|108705994 Plastid glyceraldehyde-3-phosphate dehydrogenase/O. sativa Japonica 34024/4.99 121 19% 11 -1.5
20 gi/255540341 Cytosolic glyceraldehyde-3-phosphate dehydrogenase/Ricinus communis 36930/7.10 157 20% 14 1.6 1.7 1.7
30 gi/30385668 Pyruvate phosphate dikinase/Sorghum bicolor 103021/5.68 94 16% 17 1.9 1.6
03: Transcription
21 gi/242085078 Hypothetical protein Sorbidraft of S. bicolor homologues to RNA-binding protein/Arabidopsis thaliana NP 172405.1 42324/8.89 438 40% 25 -2
28 gi|195637410 40S ribosomal protein S3/Z. mays 25605/9.4 117 46% 12 1.8
05: Protein synthesis
15 gi|195620072 Elongation factor alpha/Z. mays 49534/9.15 225 21% 14 8.5 5 7
04: Protein destination/storage
8 gi|162459902 Nucleoredoxin 1/Z. mays 64058/4.8 168 16% 12 4.4 4.9 4.5
12 gi/242094438 Hypothetical protein Sorbidraft of S. bicolor homologues to Heat shock protein 60 61927/5.47 148 31% 16 2.1
13 gi/242094438 Hypothetical protein Sorbidraft of S. bicolor homologues to Heat shock protein 60 61927/5.47 161 29% 14 2.2
14 gi/242094438 Hypothetical protein Sorbidraft of S. bicolor homologues to Heat shock protein 60 61927/5.47 126 19% 12 1.9 2.3
18 gi/242041951 Hypothetical protein Sorbidraft of S. bicolor homologues to pepsin/retropepsin like aspartat protease/Z. mays ACG35399.1 42799/8.96 79 14% 2
31 gi/242056107 Hypothetical protein Sorbidraft of S. bicolor homologues to mitocondrial processing peptidase/Z. mays NP 001150614.1 54054/6.24 198 28% 12 17
32 gi/145666464 Protein disulfide isomerase/Z. mays 56921/5.01 174 19% 18 2.1 2.5
05: Unclear classification
22 gi/242075782
Hypothetical protein Sorbidraft of S. bicolor homologues to ABA stress- and fruit-ripening inducible-like protein Z. mays
CAA72998.1
28466/4.92 258 65% 22 3.6 4.5 7
25 gi/242035869 Osr40Sc1 like protein 39718/6.27 66 9% 3 5.2
26 gi/242035869 Osr40Sc1 like protein 39718/6.27 201 13% 6 3.6 3.3 3
27 gi/242056773 Hypothetical protein Sorbidraft/S. bicolor 31577/6.06 205 23% 9 3.6 2.9 4.6
33 gi/242095250 Hypothetical protein Sorbidraft/S. bicolor 41333/4.91 222 37% 14 1.9 2.3
46
The drought tolerant genotype proteome analysis indicated that the combined activities of several protein groups may enable the plants to
tolerate drought stress and efficiently recover after removing the stress conditions.
Cont..
47. CS-6: Physiological and proteomic analysis of the
response to drought stress in an inbred
Korean maize line
• Inbred maize (Zea mays L.) line KS140 was subjected to drought stress by
withholding water for 10 days at the V5 or V6 leaf stage
• Plants were cultivated in a greenhouse
• Water was withheld for 10 days commencing at the V6 leaf stage.
• protein spots, and these were identified using MALDI-TOF mass spectrometry.
Kim et al. (2015)Suwon, South Korea 47
48. Fig.14. (A) Relative leaf water content, (B) leaf area, dry
matter of aerial tissue(C) and root (D) of well-watered and
drought-stressed plants at 0, 3, and 10 days after
withholding water.
Fig. 15. (A) Stomatal conductance, (B) net CO2
assimilation rate, (C) water use efficiency, and (D) leaf
chlorophyll content of well-watered and drought-stressed
maize plants at 0, 3, and 10 days after withholding water. 48
49. Representative 2-DE gel of Korean maize inbred line
(KS140).
(A) Well-watered, (B) Ten days drought stress.
A total of 500 µg protein was used for each 2-DE
gel.
Arrows are indicated by differentially expressed
protein spots.
Fig. 16. A close-up view of differentially expressed protein spots is
shown.
49
Protein= 4 = Pathogenesis-related protein 1
5 = TPA: pathogeneis protein 10
7 = Abscisic stress-ripening protein 2-like
29=Heat shock protein 1 (LOC100501536)
51. Spot No.
Accession No. Putative Function Score Expect MP
SC
(%)
Mr(kD)
/pI
Biological
process
Organism
18 P25462 Uncharacterized protein LOC100274579 586 8.5e-052 31 70 42.5/5.65 Zea mays
19 gi|242083462 Glutamine synthetase 228 5.4e-016 15 28 46.3/6.42 Metabolism Zea mays
20 B6TG70 Hypothetical protein 66 9.5 6 19 72.02/8.44
Sorghum
bicolor
21 B6TG70 Mitochondrial-processing peptidase beta subunit 332 2.1e-026 33 51 58.5/5.87 Proteolysis Zea mays
22 Q6L3A1 ATP synthase subunit alpha 381 2.7e-031 35 50 55.8/5.87 Photosynthesis
Saccharum
hybrid
23 P93804 Phosphoglucomutase 301 2.7e-023 28 41 63.3/5.46
Carbohydrate
metabolism
Zea mays
24 C0P4M0 Pyridine nucleotide-disulphide oxidoreductase 95 0.01 9 29 46.6/5.60 Plant stress Zea mays
25 B6T416 Ribulose bisphosphate carboxylase/oxygenase activase 107 0.00067 9 24 48.1/6.29 Photosynthesis Zea mays
26 K7VII1 Putative actin family protein isoform 1 324 1.3e-025 21 52 41.9/5.24 Structure Zea mays
27 P15719 Malate dehydrogenase (NADP) 207 6.7e-014 23 40 47.3/6.49
Carbohydrate
metabolism
Zea mays
28 B4G072 UDP-glucosyltransferase BX9 376 8.5e-031 29 53 50.6/5.22 Metabolism Zea mays
29 C4J410 Heat shock protein 1 (LOC100501536) 319 4.3e-025 33 46 71.2/5.08 Plant stress Zea mays
51
SC, sequence coverage.Mr/pI, Theoretical molecular weight/isoelectric point.
Cont…
Drought affected the relative leaf water content, leaf area, aerial and root tissue dry matter, stomatal conductance,
net CO2 assimilation rate, and water use efficiency. Up regulated protein like two pathogen related proteins (PR)
(PR-1 and PR-10), abscisic stress-ripening protein 2-like protein and heat shock protein 1 (HSP1) shows drought
tolerant mechanism and for development of selective breeding markers for drought tolerance in maize.
52. CS-7: Physiological, Biochemical and Proteomic
Responses of Rice (Oryza sativa L.)Varieties
Godaheenati and Pokkali for Drought Stress
at the Seedling Stage
• Two traditional rice (Oryza sativa L.) varieties,
Godaheenati (4049) and
Pokkali
• were selected to screen for drought stress responses at the vegetative stage.
• A single seed was planted in 8" length soil column with 1" diameter
• Dehydration condition was imposed to the 4-week old seedlings by withdrawing water,
• tissues were harvested at every 24 h after treatment for 5 days.
• The collected leaf tissues were subjected to the following analysis.
• Further screening is recommended for Godaheenati as a drought tolerant rice variety to be used in rice breeding programs.
Jayaweera et al. (2016)Gannoruwa, Sri Lanka 52
53. 53
Fig. 17. Effect of drought on the relative water content of
rice varieties Godaheenati (4049) and Pokkali
Fig. 18. A representative fraction of the 12% acrylamide gel
Differentially expressed proteins of two-week old rice
leaves of Godaheenati (4049):
(A) control and
(B) drought affected
photosystem II oxygen evolving complex protein (N-EGVPPXLTFD) and stated of its role in light harvesting, which could
potentially yield crop plants that are more resistant to Drought stress and prevent inhibitory effects on photosynthesis
Protein no. 2 = photosystem II oxygen evolving complex protein
55. CS-8: Metabolomics and proteomics analyses of
grain yield reduction in rice under abrupt
drought-flood alternation
• Wufengyou 286 (Oryza sativa L.) is the dominant double-cropping super hybrid early rice variety
• Rice was planted in plastic buckets of 24.0 cm height and 29.0 cm inner diameter of the upper
portion, and 23.5 cm inner diameter at the bottom
• Each pot contained approximately 10 kg of dry soil
• the rice abrupt drought-flood alteration stage was set at the panicle differentiation stage.
• Drought treatment continued for further 2 d until the soil was white and cracking, and the plants
were wilting and withered (imitating severe drought).
• For submergence treatment, plants in soil-containing pots were completely submerged in a high
water-filled square box (1.35 m height) in a greenhouse
Xiong et al. (2018)Jiangxi, China 55
56. Fig. 19. Analysis of yield and physiological indexes: (a) yield per plant, (b) soluble protein content, (c)
SOD activity, (d) CAT activity, (e) POD activity, (f) MDA content.
56
CK0: Control
CK1: drought
CK2: floods;
T1: abrupt drought-flood alteration
In plants under flooding stress, the electron transport chains of mitochondria and chloroplasts is blocked, and the intracellular energy charge is reduced.
All these factors could promote the production of reactive oxygen species (ROS)
POD, CAT, SOD, and glutathione-S-transferase usually act as ROS scavengers to reduce oxidative damage caused by oxidative stress in plants
57. Fig. 20. Hierarchical cluster analysis of changed metabolite pools.
57
Hierarchical trees were drawn based on detected changes of metabolites in spikes of rice under different water treatments:
(a) T1 vs CK0 comparison treatment,
(b) T1 vs CK1 comparison treatment, and
(c) T1 vs CK2 comparison treatment.
Columns represent the repetition between different treatments, while rows represent different metabolites.
Red and green colors indicate increased and decreased metabolite concentrations, respectively.
CK0: Control
CK1: drought
CK2: floods
T1 : abrupt drought-flood alteration.
58. Fig. 21. Venn diagram the differentially expressed
proteins (DEPs) between alteration.
58
Fig. 22. Summary of up- and down-regulation of
differentially expressed proteins (DEPs)
Results for abrupt drought-flood alternation at the young spike differentiation stage responsive proteins.
CK0: Control
CK1: drought
CK2: floods
T1 : abrupt drought-flood alteration. Activity of SOD activity, CAT activity, POD activity and MDA content
and also up regulated protein protect the rice plant to against drought-
flood effect
T1 vs CK0 T1 vs CK1
T1 vs CK2
60. CS-9: Comparative Proteomic Analysis Provides New
Insights into Chilling Stress Responses in Rice
• To gain a better understanding of chilling stress responses in rice (Oryza
sativa L. cv. Nipponbare), we carried out a comparative proteomic analysis.
• Three-week-old rice seedlings were treated at 6 °C for 6 or 24 h and then
recovered for 24 h.
• Chilling treatment resulted in stress phenotypes of rolling leaves, increased
relative electrolyte leakage, and decreased net photosynthetic rate.
Yan et al. (2005)Shanghai, China 60
61. Fig. 23. The physiological responses induced by chilling stress
in rice.
Three-week-old seedlings were treated at 6 °C for 0, 6, and 24 h and then were allowed to recover for 24 h (R24 h).
The relative electrolyte leakage, the Pn = photosynthetic rate, the Gs = stomatat conductance, and the
intercellular CO2 concentration (Ci) are shown in B, C, D, and E, respectively. 61
62. 62
(A) 2-DE gel of the control sample.
(B) 2-DE gel of sample treated at 6 °C for 6 h.
Temporal changes of differentially expressed proteins after
chilling treatment and recovery.
Fig. 24. Representative 2-DE gels of rice leaf proteins.
63. Fig. 25. Venn diagram analysis of the differentially expressed proteins at each
chilling time point.
63
The number of differentially expressed spots up- or down-regulated at a particular
time point(s) are shown in the different segments.
A, the down-regulated proteins.
B, the uregulated proteins.
R24 h, recovery for 24 h.
64. 64
The protein excised from gels was digested with trypsin, and the resulting peptides were analyzed using the 4700 roteomics
Analyzer.
A, the MS spectra.
The ion 2047.08 marked with an asterisk was analyzed by MS/MS.
B, MS/MS spectra of ion 2047.08.
The y ions (y3– y14) and the corresponding peptide sequence are shown.
The protein was identified as ascorbate peroxidase (NCBI accession number BAB17666) after database searching.
Fig. 26. Identification of spot 71 by MS
65. 65
a Sequence coverage of
matched peptides.
b The sequence of
matched peptides
The identification of novel
cold-responsive proteins
provides not only new
insights into chilling stress
responses but also a good
starting point for further
dissection of their functions
using genetic and other
approaches
66. CS-10: Proteomic analysis of cold acclimation in
winter wheat under field conditions
• wheat (Triticum aestivum L. cv Pishgam) under field conditions
• fully expanded upper leaves of wheat plants in each sampling date were harvested and
then stored at -80 °C.
• proteome analysis was carried out for four sampling dates including
• T1 (4 Nov: before the beginning of cold acclimation),
• T2 (23 Nov: initiation of cold acclimation; LT tolerance=~-6 °C),
• T3 (26 Dec: vernalization fulfillment; LT tolerance=~ -15 °C) and
• T4 (21 Feb: early reproductive growth stage LT tolerance=~-10 °C).
• Changes induced in leaf proteins were studied by two dimensional gel electrophoresis
and quantitatively analysed using image analysis software.
Janmohammadi et al. (2014)Iran 66
67. 2-DE gel analysis of proteins extracted from
leaves of Pishgam winter wheat harvested at
different developmental stages.
Panel shows the reference map derived from
computerized image analysis performed by
using Progenesis Same Spots software.
Numbers indicate the variable protein spots.
A large number of all the selected proteins
were part of the photosynthetic apparatus,
confirming the key role of the chloroplast
machinery during LT acclimation.
Accordingly, proteome analysis of organelles
such as the chloroplast and plasma membrane
may be applied to widen our information about
LT tolerance.
67
Protein no.=
Fig. 27.
68. Table 12. Differentially expressed proteins during different developmental stages in winter wheat identified by MALDI-
TOF MS
Spot No.a Protein name Accession no. Organism Database Fold of variationb Proteome comparisons
Increased
771 70 kDa heat shock protein gi|290131414 Triticum aestivum NCBI 2.13 T2/T1
1357 Putative fructose-bisphosphate aldolase 35_1820 Oryza sativa HarvestHv 2.08 T2/T1
2432 MADS-box protein 35_27943 Oryza sativa HarvestHv 2.05 T2/T1
1042 RuBisCO large subunit gi|2493650 Triticum aestivum NCBI 1.95 T2/T1
641 Os03g0108400 gi|255674149 Oryza sativa NCBI 1.88 T2/T1
1292 actin gi|281485191 Persea americana NCBI 1.86 T2/T1
1133 NADH dehydrogenase subunit 1 gi|18378414 Cucurbita argyrosperma NCBI 1.76 T3/T2
1169 Thioredoxin-like protein 35_50073 Oryza sativa HarvestHv 1.68 T3/T2
2199 Oxygen-evolving enhancer protein 2 gi|131394 Triticum aestivum NCBI 1.44 T3/T2
2234 Manganese superoxide dismutase gi|125663927 Triticum aestivum NCBI 1.40 T3/T2
2169 NADH dehydrogenase subunit 1 gi|18378406 Cucurbita ecuadorensis NCBI 1.36 T3/T2
2351 metal ion transmembrane transporter gi|240256271 Arabidopsis thaliana NCBI 1.34 T3/T2
2190 Oxygen-evolving enhancer protein 2, 35_1423 Oryza sativa HarvestHv 1.96 T4/T3
1510 ribosomal protein S1 gi|159161283 Cuscuta exaltata NCBI 1.92 T4/T3
1108 UDP-glucose pyrophosphorylase gi|88866516 Oryza sativa NCBI 1.84 T4/T3
1359 Fructose-bisphosphate aldolase Ta_TC235339 Arabidopsis thaliana TIGERPoa 1.79 T4/T3
2648 ribulose 1,5-bisphosphate carboxylase, large subunit Ta_TC263613 Arabidopsis thaliana TIGERPoa 1.78 T4/T3
1007 RuBisCO large subunit-binding protein subunit alpha gi|134102 Triticum aestivum NCBI 1.70 T4/T3
a = Spot number represents the number on the master gel
b = Fold of protein variation is calculated by standardizing the mean of the normalized spot volumes of samples at different harvesting times (T1, T2, T3
and T4) with the mean of the normalized spot.
68
69. Spot No.a Protein name Accession no. Organism Database Fold of variationb Proteome comparisons
Decreased
2199 Oxygen-evolving enhancer protein 2, chloroplastic gi|131394 Triticum aestivum NCBI 0.68 T2/T1
2351 metal ion transmembrane transporter gi|240256271 Arabidopsis thaliana NCBI 0.54 T2/T1
2169 NADH dehydrogenase subunit 1 gi|18378406 Cucurbita ecuadorensis NCBI 0.52 T2/T1
1821 oxygen-evolving complex protein 1 gi|739292 Triticum aestivum NCBI 0.50 T2/T1
2265 undecaprenyl diphosphate synthase, putative gi|255582903 Ricinus communis NCBI 0.50 T2/T1
2191 Oxygen-evolving enhancer protein 2 gi|131394 Triticum aestivum NCBI 0.48 T2/T1
2207 dehydroascorbate reductase gi|259017810 Triticum aestivum NCBI 0.48 T2/T1
1062 Os05g0291700 protein gi|3176645 Oryza sativa HarvestHv 0.58 T3/T2
1237 Transcription factor, putative gi|255574095 Ricinus communis NCBI 0.48 T3/T2
1042 RuBisCO large subunit-binding protein subunit beta, chloroplastic gi|2493650 Triticum aestivum NCBI 0.48 T3/T2
1244 Translational elongation factor Tu 35_976 Oryza sativa HarvestHv 0.46 T3/T2
1069 ATP synthase CF1 beta subunit gi|14017579 Triticum aestivum NCBI 0.46 T3/T2
1366 ribulose 1,5-bisphosphate carboxylase activase isoform 1 gi|167096 Hordeum vulgare NCBI 0.44 T3/T2
1357 Putative fructose-bisphosphate aldolase 35_1820 Oryza sativa HarvestHv 0.43 T3/T2
948 protein P0668H12.12 35_24827 Oryza sativa HarvestHv 0.49 T4/T3
1502 Putative aldehyde oxidase-like protein 35_3416 Oryza sativa HarvestHv 0.44 T4/T3
689 Cytochrome P450-like 35_12050 Oryza sativa NCBI 0.40 T4/T3
525 Electron transporter, putative gi|255576550 Ricinus communis NCBI 0.38 T4/T3
2690 cell-autonomous heat shock cognate protein 70 gi|26985223 Cucurbita ecuadorensis NCBI 0.38 T4/T3
1335 Photosystem II 44 kDa reaction center protein (P6 protein) Og_TC291300 Oryza sativa TIGERPoa 0.36 T4/T3
69
The abundance of some metabolic regulator, ion transporter, redox and photosynthetic proteins increased by achieving maximum LT tolerance in treatment
T3- (26 Dec: vernalization fulfillment; LT tolerance=~ -15 °C). By initiation of the reproductive phase treatment T4- (21 Feb: early reproductive growth stage
LT tolerance=~-10 °C) the abundance of some proteins that mainly participate in photosynthesis and carbon metabolism significantly increased
70. Nandha et al. (2018)Junagadh, India 70
Identify the characteristic change in two wheat genotypes viz, GW 451 (heat tolerant) and WH 147 (heat
susceptible) from flag leaf.
Seeds of both genotypes were grown up to boot leaf stage (around 52 days after sowing).
Flag leaves from both genotypes were collected as control samples and other set of plants were treated at 40ºC for
one hour and treated leaves were collected as treated samples.
proteins reported in heat tolerant genotype was higher than heat susceptible genotype.
71. Fig. 28. Spots detected on gel photograph of A) control leaf sample of heat susceptible genotype (WH 147);
B) treated leaf sample of heat susceptible genotype (WH 147); C) control leaf sample of heat
tolerant genotype (GW 451); D) treated leaf sample of heat tolerant genotype (GW 451)
A
C
B
D
71
control
control
WH 147 WH 147
GW 451
GW 451
Heat treated
Heat treated
74. Table 15. Numbers of spots identified in 2DE in control and treated leaf sample in both
genotypes
Control Leaves Treated Leaves Total No. of Spots
GW 451 WH 147 GW 451 WH 147 GW 451 WH 147
pI (2-4) 41 17 21 28 62 45
MW (KDa) 14-97 14-97 14-102 14-99
pI (4-6) 35 41 30 45 65 86
MW (KDa) 18-97 12-97 14-107 14-97
pI (6-8) 41 27 27 40 68 67
MW (KDa) 17-97 12-100 18-97 13-97
pI (8-10) 53 33 38 51 91 84
MW (KDa) 12-97 14-97 14-97 14-97
Total Spots 170 118 116 164 286 282
Total 288 280 568
74
Various proteins were reported with highly versatile amount of molecular weight
indicated the tolerance level against heat stress.
75. Plant proteomic analysis allows for the large-scale study of molecular changes occurring at the
protein level.
Proteomics has already been used to evaluate abiotic stress-responsive proteins in important crop
species such as rice, wheat, maize and sorghum.
Proteome studies the complete set of proteins encoded by the genome and thus complement the
transcriptome studies.
The first group includes proteins that function in abiotic stress tolerance such as chaperones, late
embryogenesis abundant (LEA) proteins, osmotin, mRNA-binding proteins, key enzymes for
osmolyte biosynthesis, water channel proteins, metabolites transporters, detoxification enzymes,
and various proteases.
These proteins can then serve as molecular markers in marker-assisted selection and breeding
programs or in transgenic approaches to improving plant drought tolerance.
75
Slide 8: Applications of Proteomics
Voice: Both methods of research may have many applications. For example, drug discovery, mice knockout that glow in the dark and even disease mechanisms
ATP may play a role in the acclimation of rice seedlings to anaerobic conditions created by oxidative stress. Therefore, up-regulation of this protein in salt-stress. ensitive Dalseongaengmi-44 may help to produce additional energy, which is required to decrease damage caused by salt stress.
Class III peroxidase 29 precursor = The imposition of abiotic stresses can give rise to excess concentrations of ROS in plant. These ROS are potentially harmful since they
cause membrane damage and Dalseongaengmi-44 likely plays a role in ROS scavenging and improving the structural strength of cells.
the OEC proteins are responsive to salt stress and cause changes in the activity of photosystem II (PSII) in coping with salt stress
necessary to combine proteomics, metabolomics, and physiology to clarify the abrupt drought-flood alternation response mechanism of rice yield reduction during the panicle differentiation stage.