| Rice as one of the world's most important food crops has a wide adaptability and a high-yield performance that make it in a great social significance and an important strategic significance.In the historical context of intensified food crisis and growing population,people around the world are expecting more and more of rice production.It is predicted that the existing rice yields must have been doubled to just meet the demands of the growing population by 2050.However,the rice diseases are plundering a considerable part of rice yields every year,such as rice blast,one of the three major rice diseases,resulting in 11-30%of loss in rice yields annually.Although chemical control and prevention has a significant effect on rice diseases,the use of chemical agents are constrained to some extent because of the limitation in the application period,the enhancement of pathogen resistance,the improvement of concerns on serious environmental pollution and on rice quality.On the contrary,the breeding of resistant varieties and a large scale of promotion have been demonstrated by the majority of producers as the most economical,most effective and safest solution in controlling the diseases over the years.This paper details an interdisciplinary research involved in phytophysiology,biochemistry and quantitative proteomics.In this paper,the studies on leaf samples taken from the rice infected by Magnaporthe grisea responding to the stress in different periods of time and on leaf samples taken from different resistant plants infected by Magnaporthe grisea responding to the stress are conducted by means of in vitro labeling technique(iTRAQ)combined with high-throughput mass spectrometry identification platform(LC-MS/MS).This paper intends to find a protein differentially expressed in the rice infected by Magnaporthe grisea by means of bioinformatics combined with experimental verification,in order to find the resistance mechanism at the molecular level,to lay the foundation for breeding new high-quality and disease-resistant rice varieties,and to make a certain contribution for the following research on the molecular mechanism of rice resistance to Magnaporthe grisea.The main contents and results in this paper:1)In the research of the mechanism of the rice infected by Magnaporthe grisea responding to the stress in different periods of time by means of quantitative proteomics,the proteins are extracted from the leaves of Gumei 2 infected by Magnaporthe grisea(Guyl 1)at Oh,24h and 48h for quantification,enzymolysis and label processing.Then the complex peptide segments are loaded in SCX column for primary separation according to the characteristics of peptide segments.Finally,the spectrum acquisition is conducted by means of high-throughput mass spectrometry identification platform(LC-MS/MS,ABSECX TripleTOFTM 4600).280,528 Oryza sativa protein sequences are searched by means of ProteinPilot4.5 software(AB SCIEX,Foster City).The data acquisition of each component is repeated twice.In other words,in all of 20 components,10 components are collected and integrated into a single group by a software.A compare search in Oryza sativa library is conducted for each group and the results are indicated as Repeat 1 and Repeat 2.In Repeat 1,when FDRis 1.0%,atotal of 33,373 Spectral,15,835 Peptides and 1,604 Proteins have been identified.In Repeat 2,when FDR is 1.0%,a total of 33,004 Spectral,15,800 Peptides and 1,581 Proteins have been identified.The protein supported by two or more peptide segments are included in the results of analysis.It is turned out that there are a total of 1,234 Proteins in Repeat 1 and 1,210 Proteins in Repeat 2.The proteins obtained in two experiments are classified into up-regulated group with the ratio value larger than 1.2 and down-regulated group with the ratio value lower than 0.8.Except for the proteins that are identified only once and trend inconsistently,the remaining proteins are divided into five groups:121 proteins with up-regulated protein expression in the rice infected by Magnaporthe grisea for 24h and 48h,26 proteins with up-regulated protein expression in the rice infected by Magnaporthe grisea for 24h or 48h,34 proteins with down-regulated protein expression in the rice infected by Magnaporthe grisea for 24h or 48h,43 proteins with up-and down-regulated protein expression in the rice infected by Magnaporthe grisea for 24h and 48h,and 148 proteins with down-regulated protein expression in the rice infected by Magnaporthe grisea for 24h and 48h.The differential proteins are taken for bioinformatics analysis,GO classification and KEGG-pathway analysis.It is turned out that these differential proteins play a key role in 79 pathways and involve in more photosynthetic pathways of biological carbon sequestration.61 proteins containing signal peptides found in these differential proteins by secretory protein analysis may be secretory proteins.In GO enrichment analysis,they are found to be accumulated in 15 categories including TCA cycle,NADP metabolic processes and isomerase activity.They are also taken for network interaction analysis,two clusters are find,One cluster is about Photosynthesis related protein,and the other cluster is about Ribosome associated protein.In further analysis,differentially expressed proteins are found in the rice infected by Magnaporthe grisea in different periods of time,among which,trypsin inhibitor is significantly increased.An increase in protease inhibitor(PI)is verified at mRNA level by quantitative PCR.Protease inhibitors are a class of substances that can inhibit the activity of proteolytic enzyme.PI can be combined with the active site and allosteric site of protease to inhibit the catalytic activity of the enzyme or to prevent the proenzyme from translating into an active enzyme.Therefore,it can prevent the degradation of in vivo unnecessary proteins,regulate protein metabolism and regulate the physiological activities of various proteases.PI in many plants can also inhibit the action of in vivo proteases in some pathogenic microorganisms and insects,which is a natural defense system of plants.Interestingly,putative dioscorin protein and dioscorin are detected as storage proteins of yam while screening out differential proteins.This protein has been demonstrated with an activity of trypsin inhibitor.In the results we have identified,the expression level of putative dioscorin protein is significantly increased when the rice is infected by Magnaporthe grisea,which is consistent with the tendency of trypsin inhibitor.A sharp decline in the expression levels of photosynthesis-related large Rubisco subunit,Rubisco subunit binding-protein alpha subunit has been found when the rice is infected by Magnaporthe grisea.The expression levels of energy metabolism-related ATP synthase CF1 beta subunit and ATP synthase CF1 alpha chain are up-regulated when the rice is infected by Magnaporthe grisea.2)In the research of the mechanism of different resistant plants infected by Magnaporthe grisea responding to the stress by means of quantitative proteomics,the proteins are extracted from the leaves of Gumei 2 and Heigu infected by Magnaporthe grisea(Guy11)at 24h for quantification,enzymolysis and label processing.Then the complex peptide segments are loaded in SCX column for primary separation according to the characteristics of peptide segments.Finally,the spectrum acquisition is conducted by means of high-throughput mass spectrometry identification platform(LC-MS/MS,ABSECX TripleTOFTM 4600).Oryza sativa protein sequences are searched by means of ProteinPilot4.5 software(AB SCIEX,Foster City).When FDR is 1.0%,the protein supported by two or more peptide segments are included in the results of analysis.There are a total of1,196 Proteins in Repeat 1 and 1,180 Proteins in Repeat 2.Those with the ratio value larger than 1.2 are up-regulated,and those with the ratio value lower than 0.8 are down-regulated.Except for the proteins that are identified only once and trend inconsistently,there are 220 proteins up-regulated and 240 proteins down-regulated in resistant plants.The differential proteins are taken for bioinformatics analysis,GO classification and KEGG-pathway analysis.It is turned out that these differential proteins play a key role in 81 pathways and involve in more gluconeogenic/glycolytic pathways.68 proteins containing signal peptides found in these differential proteins by secretory protein analysis may be secretory proteins.In GO enrichment analysis,they are found to be accumulated in 11 categories including photosynthesis,carboxylic acid metabolic processes and chloroplast composition.They are taken for network interaction analysis.Also two clusters are find,One cluster is about energy metabolism related protein,and the other cluster is about Ribosome associated protein.In the research content 1),the expression levels of trypsin inhibitor are significantly increased when Gumei 2 resistance to Magnaporthe grisea is infected by Magnaporthe grisea,which has been validated in MS results in quantitative PCR experiments.In the research of the mechanism of different resistant plants infected by Magnaporthe grisea responding to the stress,the expression levels of trypsin inhibitor in the rice resistance to Magnaporthe grisea(Gumei 2)infected by Magnaporthe grisea are not significantly increased than non-resistant plant(Heigu),which is consistent with what happens when Peng is infected by phytophthora infestans in tomato leaves.In a comprehensive analysis combining two parts of the results,a lot of hydrolyzed proteins may be needed to provide energy when the rice is infected by Magnaporthe grisea.The trypsin inhibitor is highly expressed in the rice in order to antagonize the invasion of Magnaporthe grisea,thus inhibiting the activity of trypsin.The expression level of dioscorin,a storage protein of yam,with the activity of Trypsin inhibitor,is also sharply increased,thus inhibiting the activity of trypsin and ultimately achieving the antagonism of infection by Magnaporthe grisea.The expression of photosynthesis metabolism-related proteins including Rubisco activase large isoform precursor and Rubisco subunit binding-protein alpha subunit in the rice resistance to Magnaporthe grisea are significantly down-regulated than the rice infected by Magnaporthe grisea.As a result,it is speculated that the rice infected by Magnaporthe grisea can antagonize the invasion of Magnaporthe grisea by destroying o photosynthesis and reducing energy production by means of down-regulating the expression of these two key proteins.For different resistant rice infected by Magnaporthe grisea,ATP synthase CF1 beta subunit as an energy metabolism-related protein is limitedly up-regulated,while ATP synthase CF1 alpha chain is basically unchanged. |
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