Proteome terminology was proposed by Australian scientists in 1994 and was defined as: all proteins expressed by the Genome. With the completion of sequencing of several models of biological genomes, proteomics has become an important research method in the functional genome era. Compared with some simple prokaryotes, plant proteomics research is relatively backward. After the genome sequences of Arabidopsis thaliana and rice (Oryza sativa) were published, plant proteomics research became active. The first plant proteomics in 1999 reviewed the advances in proteomic research in plants. Most of these studies have not involved the identification of proteins using mass spectrometry. Today, mass spectrometry has become an important tool for proteomics research. With the expansion of the scope of proteomics research, it is no longer confined to the mere isolation of new proteins. Proteomics has now become a discipline that describes and analyzes an organism's proteins, protein interactions, and protein modifications.
The rice genome consists of 430 Mb bases, and is smaller in cereals. Because its gene is easy to manipulate, it is collinear with other monocotyledonous plants, thus becoming a model organism for monocotyledonous plants. In 2002, the rice cultivars Japonica and Japonica genome maps were completed and the fine mapping of the rice genome was completed at the end of the year. However, the translation from mRNA transcription to protein is not a simple correspondence. The protein that actually performs life functions is the protein, and proteomics sets the genome. Sequence information is linked to the types of proteins in specific tissues and organs. At present, research on rice proteomics is gradually starting to boom.
1 Proteomics research methods The classic research method of proteomics is two-dimensional PAGE (2-DE) technology. In 1975 OFarrell used this technique for the first time to isolate E. coli proteins, and later developed a solid-phase pH gradient (IPC) technique that solved the instability of pH gradients in 2-DE, Gorg et al. The selection of focus parameters is detailed. The gel after two-dimensional electrophoresis is stained (such as silver staining, Coomassie brilliant blue staining, etc.), and then analyzed by map software and subsequent mass spectrometry or amino acid sequence analysis, and finally through the database search to identify the purpose of the protein. There are many commercially available and non-commercial image analysis software such as PD Quest, Melanie, Image 2D Elite, Z3, Progenesis, etc.
The principle of mass spectrometry is to ionize a protein sample and then separate it by mass-to-charge ratio and determine its molecular weight. The emergence of two soft ionization techniques, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS), has facilitated the identification of high-throughput proteins.
The MudPIT multidimensional protein identification technique that has recently emerged separates the protein mixture using a two-way separation technique, using strong cation exchange in the first direction, reverse chromatography in the second direction, and then mass spectrometry. Complementary with the ordinary 2-DE method, it represents the most abundant protein separation and identification technology. In addition to the DIGE technique (differential display gel electrophoresis), different samples were labeled with 3 different fluorescent dyes (Cy2, Cy3, Cy5), electrophoresed on the same gel, scanned by lasers of different wavelengths and emission filters , produces 3 different colors of fluorescent signals. The differences in the positions and amounts of protein spots caused by different gel electrophoresis were overcome, the amount of gel used was reduced, and the comparability between gels was increased.
Many new technologies appearing in proteomics research at present have a great tendency to replace 2-DE. However, taking into account the experimental cost, application ability and extensive use, 2-DE is still promising, especially in combination with immunoassays. It remains an important tool for proteomics research. Functional proteomics studies involve the activity of proteins. Therefore, post-translational modifications and protein-protein interactions need to be considered. Protein interactions are the basis of life activities. All life activities are almost achieved through protein-protein interactions. Among the methods currently used to detect protein interactions are the yeast two-hybrid system method, protein chip method, and phage display technology.
2 Application of Proteomics in Rice
1) Developmental proteomics studies. Before the concept of proteomics was proposed, many researchers have used 2-DE technology to separate and compare the protein components of various tissues and organs in rice. Komatsu et al. used 2-DE to analyze the proteins of rice embryo, endosperm and leaves. About 600, 100, and 150 protein spots were detected after Masing blue staining. Twenty-seven proteins were identified by N-terminal or internal amino acid sequencing, and more than 70% of proteins were closed at the N-terminus. Tsugita et al. isolated 4892 proteins from 10 tissues and organs of rice, and further analyzed 2.8% of the proteins. Only 1.1% (56) of them were sequenced. Due to limited genome and protein databases at that time, even some sequenced proteins did not know their functions. . The important reproductive organs of rice anthers in the early development of microspore susceptible to environmental influences, Imin through 2-DE separation, silver staining to obtain about 4000 anther proteins, representing the entire genome of 10%, of which 53 were identified, most They are housekeeping proteins, molecular chaperones, glycine-rich proteins, and oncoproteins that regulate translation (first reported in anther research). At the same time, some subcellular structures closely related to metabolism also carried out proteomic studies, such as rice mitochondria, identified 149 proteins by mass spectrometry, and identified the function of 85 of them. When preparing subcellular protein samples, if necessary, micron-laser cutting techniques can be used to enrich specific samples before further lysis. Other subcellular structures involved in the study include the plasma membrane, Golgi apparatus membrane, and chloroplast. The current rice proteome database contains 23 reference maps with detailed sampling locations, sample preparation and electrophoresis conditions, identification methods, and clicks on protein spots to link the corresponding information (http:gene64.dna.affrc.go.jp/rpd/ ). It is very important to establish high-quality rice 2-DE reference maps for accurate annotation of rice proteomics and rice genome function.
2) Proteomics studies under environmental stress. Gene expression is affected by various environmental factors, including biological and abiotic factor stress. Treating rice with various stresses can separate new proteins and genes, and at the same time, can deeply understand the adaptation mechanism of rice to these environmental stresses. Rice is quite sensitive to drought stress, and drought seriously affects rice yield. Salekdeh et al. analyzed the proteomes of rice leaves under drought stress and after restoration of irrigation. It was found that the abundance of 42 protein spots significantly changed under drought stress, thus identifying some drought-responsive proteins, which will help improve rice drought-resistant breeding. And improve crop quality and increase production.
Agrawal et al. used 2-DE combined with amino acid sequencing and immuno-hybridization to study the leaf protein expression of rice seedlings under ozone stress. Compared with the control, there were differences in more than 50 points, among which the main protein RuBisCO related to photosynthesis was reduced and induced. Accumulation of proteins associated with defense, including OsPR5, OsPR10, SOD, APX, calcium binding proteins, etc., These proteins can be used as potential molecular markers for the detection of rice and other plant-related damage associated with ozone stress. Similarly, metal stress can also cause damage to photosynthesis, resulting in fragmentation of large and small subunits of RuBisCO and affecting the expression of defense-related proteins such as OsPR5, OsPR10, and SOD.
Kim et al. used rice pathogens to treat rice suspension culture cells and induced the production of the pathogen-associated protein OsPR10, isoflavone reductase-like proteins OsPR10, and β-glucanase and other proteins for the first time. Effects of rice yellow mottle virus (RYMV) on rice yield, Ventelon-Debout et al. Studies on the proteome of two rice varieties IR64 (sensitive to the virus) and Azucena (relatively insensitive) in suspension culture cells There were significant changes in the expression of 40 and 24 protein spots, respectively, and some were only expressed in IR64, such as HSP70, PR-10a, ethylene-induced proteins, etc. Some were only found in the latter, such as Chaperonin CPN60-2, etc. The glycolysis-related enzymes vary in both varieties.
Rakwal et al. treated rice with jasmonic acid (JA). The appearance of leaves and stems was related to tissue necrosis, RuBisCO subunits were greatly reduced, and a new 28 KD alkaline protease inhibitor and an acidic 17 appeared in stems. KD's disease-associated proteins, which play a potential role in plant defense. Shen et al. used gibberellic acid to treat rice leaf sheaths and found that altered calreticulin was involved in gibberellin signaling to regulate leaf sheath elongation. There are other plant hormones and growth substances such as: abscisic acid, salicylic acid, ethylene and other rice processing and proteomics to further study the expression of specific proteins.
3) Proteomics studies of mutants. Mutants are important materials for genetic research, and genetics can be found by just seeking genetics. The comparative study of the mutants and the wild plant proteome can visualize differentially expressed proteins. Further study of these proteins can help reveal the physiological and biochemical mechanisms of the mutants.
Komatsu et al. performed a 2-DE separation of proteins from rice green and albino seedlings, sequenced N-terminal and internal amino acids and found that the proteins involved in photosynthesis only appeared in green shoots, and the precursors in albino seedlings could not participate in photosynthesis; ascorbic acid. Peroxidase occurs only in albino seedlings and plays a role in cell protection. Chlorophyll mutants are beneficial to the study of photosynthesis in higher plants, chloroplast inheritance and development. Wang Yuzhong et al.'s comparison of the 2-DE maps before and after chlorosis of the temperature-sensitive chlorophyll deficient mutant rice showed that P1 was specifically deleted in the chlorotic part of the plant, and after regreening, P1 was normally expressed, suggesting that the protein is closely related to chlorophyll metabolism. Temperature-sensitive genic male sterile rice is an important breeding material for two-line genic male sterile rice, and is fertile at low temperatures. Xie Jinyun et al. used 2-DE separation, sterility fingerprinting, and database searching for infertility and fertile anthers samples. From infertility to fertility maps, proteins significantly up-regulated include chitinase, acid phosphatase, and glutenin. Precursors, etc., significantly down-regulated the protein glutamate transcarbamylase. Lesion Mimic Mutants usually produce systemic allergic necrotic plaques in the absence of pathogens and show increased resistance to various pathogens. Tsunezuka et al. used 2-DE for the three plaque formation stages of a cdr2 mutant. Compared with the wild type, 37 proteins were differentially expressed, of which 28 were up-regulated and 9 were expressed in the mutant. Down-regulation, identified by mass spectrometry, differentially expressed protein spots are associated with defense. In addition, 27 proteins are metabolizing enzymes, suggesting that the programmed cell death of this mutant is associated with active metabolism.
Modern life science research is closely related to the development of bioinformatics, and bioinformatics is not only collecting data, but more importantly, processing, analyzing, interpreting, and developing and applying these data. At present, various kinds of nucleic acid sequence databases, genome databases, protein sequences, and structural databases are increasingly perfected, laying the foundation for the rapid and accurate study and identification of proteins. In addition to the need for sophisticated mass spectrometry instruments and associated hardware support for the high-throughput identification of proteins, the final identification results are directly related to the major databases. The corresponding software provided on the Internet supports online identification, and the identification of the protein's attribution is performed by sorting the homology matching scores. Of course, the success rate of this search is directly related to the accumulation of various information databases of protein species sources. The main websites related to rice proteomics research are: SWISS-2DPAGE database Chinese mirror site (http://cn.expasy.org/ch2d/,), providing two-dimensional electrophoresis reference maps and online search software, etc. Rice Proteome Database Rice Proteome Database (http://gene64.dna.affrc.go.jp/rpd/,); Anther Proteome Profiling Website (http://semele/anu.edu.au/2d/2d.html,).
The key issue in proteomics research is the reproducibility and resolution of isolated mixed proteins, given the limitations of traditional 2-DE (eg, the lack of separation of basic proteins, membrane proteins, and hydrophobins), new technologies The emergence and application are inevitable. 2-DE still occupies an important position in the current science of protein separation, and complementing other methods with rapid, high-throughput features will be a trend for the study of proteomes now and for a long time to come.
A database of rice proteomes has been established, including two-dimensional electropherograms of various tissues, sub-cells, and different developmental stages. The proteomics study of rice under various environmental stresses and various rice mutants is also an important research content, and the reference map established in this case will also be of great significance because it directly improves the quality of rice and The correlation of production. At present, rice proteomics mainly focuses on the changes of genome expression under the influence of environmental factors. The complete rice proteomics also requires in-depth study of protein post-translational modifications (PTMs) and complex network interactions between proteins. This is proteomics. A difficult task for research.
The growing variety of rice-related sequence information databases, such as gene sequences and expressed sequence tags (ESTs), are of great interest for the in-depth study of their physiological activities and genetic mechanisms. With the continuous enrichment of various databases, the development of bioinformatics itself, and the improvement of mass flux and other high-throughput support technologies, large-scale rice functional genome research will be conducted. In short, the integration of the rice protein database and its genome database will help us to understand rice as an important crop as a whole, and the proteomics research of rice will also lay a solid foundation for the functional genome research of other cereal crops.
The rice genome consists of 430 Mb bases, and is smaller in cereals. Because its gene is easy to manipulate, it is collinear with other monocotyledonous plants, thus becoming a model organism for monocotyledonous plants. In 2002, the rice cultivars Japonica and Japonica genome maps were completed and the fine mapping of the rice genome was completed at the end of the year. However, the translation from mRNA transcription to protein is not a simple correspondence. The protein that actually performs life functions is the protein, and proteomics sets the genome. Sequence information is linked to the types of proteins in specific tissues and organs. At present, research on rice proteomics is gradually starting to boom.
1 Proteomics research methods The classic research method of proteomics is two-dimensional PAGE (2-DE) technology. In 1975 OFarrell used this technique for the first time to isolate E. coli proteins, and later developed a solid-phase pH gradient (IPC) technique that solved the instability of pH gradients in 2-DE, Gorg et al. The selection of focus parameters is detailed. The gel after two-dimensional electrophoresis is stained (such as silver staining, Coomassie brilliant blue staining, etc.), and then analyzed by map software and subsequent mass spectrometry or amino acid sequence analysis, and finally through the database search to identify the purpose of the protein. There are many commercially available and non-commercial image analysis software such as PD Quest, Melanie, Image 2D Elite, Z3, Progenesis, etc.
The principle of mass spectrometry is to ionize a protein sample and then separate it by mass-to-charge ratio and determine its molecular weight. The emergence of two soft ionization techniques, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS), has facilitated the identification of high-throughput proteins.
The MudPIT multidimensional protein identification technique that has recently emerged separates the protein mixture using a two-way separation technique, using strong cation exchange in the first direction, reverse chromatography in the second direction, and then mass spectrometry. Complementary with the ordinary 2-DE method, it represents the most abundant protein separation and identification technology. In addition to the DIGE technique (differential display gel electrophoresis), different samples were labeled with 3 different fluorescent dyes (Cy2, Cy3, Cy5), electrophoresed on the same gel, scanned by lasers of different wavelengths and emission filters , produces 3 different colors of fluorescent signals. The differences in the positions and amounts of protein spots caused by different gel electrophoresis were overcome, the amount of gel used was reduced, and the comparability between gels was increased.
Many new technologies appearing in proteomics research at present have a great tendency to replace 2-DE. However, taking into account the experimental cost, application ability and extensive use, 2-DE is still promising, especially in combination with immunoassays. It remains an important tool for proteomics research. Functional proteomics studies involve the activity of proteins. Therefore, post-translational modifications and protein-protein interactions need to be considered. Protein interactions are the basis of life activities. All life activities are almost achieved through protein-protein interactions. Among the methods currently used to detect protein interactions are the yeast two-hybrid system method, protein chip method, and phage display technology.
2 Application of Proteomics in Rice
1) Developmental proteomics studies. Before the concept of proteomics was proposed, many researchers have used 2-DE technology to separate and compare the protein components of various tissues and organs in rice. Komatsu et al. used 2-DE to analyze the proteins of rice embryo, endosperm and leaves. About 600, 100, and 150 protein spots were detected after Masing blue staining. Twenty-seven proteins were identified by N-terminal or internal amino acid sequencing, and more than 70% of proteins were closed at the N-terminus. Tsugita et al. isolated 4892 proteins from 10 tissues and organs of rice, and further analyzed 2.8% of the proteins. Only 1.1% (56) of them were sequenced. Due to limited genome and protein databases at that time, even some sequenced proteins did not know their functions. . The important reproductive organs of rice anthers in the early development of microspore susceptible to environmental influences, Imin through 2-DE separation, silver staining to obtain about 4000 anther proteins, representing the entire genome of 10%, of which 53 were identified, most They are housekeeping proteins, molecular chaperones, glycine-rich proteins, and oncoproteins that regulate translation (first reported in anther research). At the same time, some subcellular structures closely related to metabolism also carried out proteomic studies, such as rice mitochondria, identified 149 proteins by mass spectrometry, and identified the function of 85 of them. When preparing subcellular protein samples, if necessary, micron-laser cutting techniques can be used to enrich specific samples before further lysis. Other subcellular structures involved in the study include the plasma membrane, Golgi apparatus membrane, and chloroplast. The current rice proteome database contains 23 reference maps with detailed sampling locations, sample preparation and electrophoresis conditions, identification methods, and clicks on protein spots to link the corresponding information (http:gene64.dna.affrc.go.jp/rpd/ ). It is very important to establish high-quality rice 2-DE reference maps for accurate annotation of rice proteomics and rice genome function.
2) Proteomics studies under environmental stress. Gene expression is affected by various environmental factors, including biological and abiotic factor stress. Treating rice with various stresses can separate new proteins and genes, and at the same time, can deeply understand the adaptation mechanism of rice to these environmental stresses. Rice is quite sensitive to drought stress, and drought seriously affects rice yield. Salekdeh et al. analyzed the proteomes of rice leaves under drought stress and after restoration of irrigation. It was found that the abundance of 42 protein spots significantly changed under drought stress, thus identifying some drought-responsive proteins, which will help improve rice drought-resistant breeding. And improve crop quality and increase production.
Agrawal et al. used 2-DE combined with amino acid sequencing and immuno-hybridization to study the leaf protein expression of rice seedlings under ozone stress. Compared with the control, there were differences in more than 50 points, among which the main protein RuBisCO related to photosynthesis was reduced and induced. Accumulation of proteins associated with defense, including OsPR5, OsPR10, SOD, APX, calcium binding proteins, etc., These proteins can be used as potential molecular markers for the detection of rice and other plant-related damage associated with ozone stress. Similarly, metal stress can also cause damage to photosynthesis, resulting in fragmentation of large and small subunits of RuBisCO and affecting the expression of defense-related proteins such as OsPR5, OsPR10, and SOD.
Kim et al. used rice pathogens to treat rice suspension culture cells and induced the production of the pathogen-associated protein OsPR10, isoflavone reductase-like proteins OsPR10, and β-glucanase and other proteins for the first time. Effects of rice yellow mottle virus (RYMV) on rice yield, Ventelon-Debout et al. Studies on the proteome of two rice varieties IR64 (sensitive to the virus) and Azucena (relatively insensitive) in suspension culture cells There were significant changes in the expression of 40 and 24 protein spots, respectively, and some were only expressed in IR64, such as HSP70, PR-10a, ethylene-induced proteins, etc. Some were only found in the latter, such as Chaperonin CPN60-2, etc. The glycolysis-related enzymes vary in both varieties.
Rakwal et al. treated rice with jasmonic acid (JA). The appearance of leaves and stems was related to tissue necrosis, RuBisCO subunits were greatly reduced, and a new 28 KD alkaline protease inhibitor and an acidic 17 appeared in stems. KD's disease-associated proteins, which play a potential role in plant defense. Shen et al. used gibberellic acid to treat rice leaf sheaths and found that altered calreticulin was involved in gibberellin signaling to regulate leaf sheath elongation. There are other plant hormones and growth substances such as: abscisic acid, salicylic acid, ethylene and other rice processing and proteomics to further study the expression of specific proteins.
3) Proteomics studies of mutants. Mutants are important materials for genetic research, and genetics can be found by just seeking genetics. The comparative study of the mutants and the wild plant proteome can visualize differentially expressed proteins. Further study of these proteins can help reveal the physiological and biochemical mechanisms of the mutants.
Komatsu et al. performed a 2-DE separation of proteins from rice green and albino seedlings, sequenced N-terminal and internal amino acids and found that the proteins involved in photosynthesis only appeared in green shoots, and the precursors in albino seedlings could not participate in photosynthesis; ascorbic acid. Peroxidase occurs only in albino seedlings and plays a role in cell protection. Chlorophyll mutants are beneficial to the study of photosynthesis in higher plants, chloroplast inheritance and development. Wang Yuzhong et al.'s comparison of the 2-DE maps before and after chlorosis of the temperature-sensitive chlorophyll deficient mutant rice showed that P1 was specifically deleted in the chlorotic part of the plant, and after regreening, P1 was normally expressed, suggesting that the protein is closely related to chlorophyll metabolism. Temperature-sensitive genic male sterile rice is an important breeding material for two-line genic male sterile rice, and is fertile at low temperatures. Xie Jinyun et al. used 2-DE separation, sterility fingerprinting, and database searching for infertility and fertile anthers samples. From infertility to fertility maps, proteins significantly up-regulated include chitinase, acid phosphatase, and glutenin. Precursors, etc., significantly down-regulated the protein glutamate transcarbamylase. Lesion Mimic Mutants usually produce systemic allergic necrotic plaques in the absence of pathogens and show increased resistance to various pathogens. Tsunezuka et al. used 2-DE for the three plaque formation stages of a cdr2 mutant. Compared with the wild type, 37 proteins were differentially expressed, of which 28 were up-regulated and 9 were expressed in the mutant. Down-regulation, identified by mass spectrometry, differentially expressed protein spots are associated with defense. In addition, 27 proteins are metabolizing enzymes, suggesting that the programmed cell death of this mutant is associated with active metabolism.
Modern life science research is closely related to the development of bioinformatics, and bioinformatics is not only collecting data, but more importantly, processing, analyzing, interpreting, and developing and applying these data. At present, various kinds of nucleic acid sequence databases, genome databases, protein sequences, and structural databases are increasingly perfected, laying the foundation for the rapid and accurate study and identification of proteins. In addition to the need for sophisticated mass spectrometry instruments and associated hardware support for the high-throughput identification of proteins, the final identification results are directly related to the major databases. The corresponding software provided on the Internet supports online identification, and the identification of the protein's attribution is performed by sorting the homology matching scores. Of course, the success rate of this search is directly related to the accumulation of various information databases of protein species sources. The main websites related to rice proteomics research are: SWISS-2DPAGE database Chinese mirror site (http://cn.expasy.org/ch2d/,), providing two-dimensional electrophoresis reference maps and online search software, etc. Rice Proteome Database Rice Proteome Database (http://gene64.dna.affrc.go.jp/rpd/,); Anther Proteome Profiling Website (http://semele/anu.edu.au/2d/2d.html,).
The key issue in proteomics research is the reproducibility and resolution of isolated mixed proteins, given the limitations of traditional 2-DE (eg, the lack of separation of basic proteins, membrane proteins, and hydrophobins), new technologies The emergence and application are inevitable. 2-DE still occupies an important position in the current science of protein separation, and complementing other methods with rapid, high-throughput features will be a trend for the study of proteomes now and for a long time to come.
A database of rice proteomes has been established, including two-dimensional electropherograms of various tissues, sub-cells, and different developmental stages. The proteomics study of rice under various environmental stresses and various rice mutants is also an important research content, and the reference map established in this case will also be of great significance because it directly improves the quality of rice and The correlation of production. At present, rice proteomics mainly focuses on the changes of genome expression under the influence of environmental factors. The complete rice proteomics also requires in-depth study of protein post-translational modifications (PTMs) and complex network interactions between proteins. This is proteomics. A difficult task for research.
The growing variety of rice-related sequence information databases, such as gene sequences and expressed sequence tags (ESTs), are of great interest for the in-depth study of their physiological activities and genetic mechanisms. With the continuous enrichment of various databases, the development of bioinformatics itself, and the improvement of mass flux and other high-throughput support technologies, large-scale rice functional genome research will be conducted. In short, the integration of the rice protein database and its genome database will help us to understand rice as an important crop as a whole, and the proteomics research of rice will also lay a solid foundation for the functional genome research of other cereal crops.
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