Screening Differential Proteins Of Rice Root Tip Tissues With Mercury Stress By TMT Technique And Their Bioinformatics Analysis

Objective:Edible rice is one of the most important ways of mercury exposure in typical mercury mining areas in China.In order to reveal the molecular mechanism of rice?Oryza sativa L.?adapting to heavy metal mercury stress from the protein level,the physiological and biochemical responses and total mercury distribution caused by mercury stress were discussed.Characteristics,simulating the pollution of paddy field environment in mercury mining area,to achieve the purpose of discovering the significant difference of mercury-related proteins after stress root tip,in order to provide clues for regulating the molecular biological mechanism of mercury absorption in rice,thereby reducing the risk of mercury exposure in the population.Methods:A pot experiment was conducted in this study.The jointing stage rice?Jinyou 431?was used as the material.The concentration of mercury chloride was set at 0 mg·kg-1 and 50 mg·kg^-1,150 mg·kg^-1,300 mg·kg^-1.Then,the isotope relative labeling and the absolute quantitative?TMT?technique were used to screen the differentially expressed proteins in the root tip of the rice in response to heavy metal mercury stress?p<0.05?.The bioinformatics analysis was performed on all the differential proteins.Proteomics parallel reaction monitoring?PRM?technique was used to identify target proteins with significant responses to mercury stress.Results::1.Effect of mercury stress on physiological and biochemical responses of rice?1?The content of proline in rice leaves and stems increased slightly with the increase of Hg²⁺concentration,but the content of proline in roots increased by478.74%,548.68%and 184.79%compared with the control;?2?rice The activity of SOD in roots and leaves was higher than that in the control group,and the concentration of Hg²⁺was negatively correlated with the activity of SOD.?3?The data showed that the total protein content of each group in different parts of rice was less,and there was no obvious trend change.?4?The MDA content of rice roots and leaves was lower than that of the control in the low dose group under Hg²⁺stress,and the MDA in the high concentration group.The content was 2.29 times of the control;?5?the POD activity in rice stems and leaves showed a low concentration-induced inhibition of Hg²⁺stress;?6?GSH in rice rhizomes stimulated in low concentration group,medium and high concentration The group may be involved in a large number of free radical scavenging or PCs and decreased.2.Distribution characteristics of total mercury content in different tissue parts of rice The trend of total mercury content in rice increased with the increase of HgCl2 stress concentration.The total mercury content in roots>main stem>root tip>leaf>stem>ear in different organs,compared with the control group and each test group,the highest content of mercury in the root of high dose group was 703.03±44.35?g/kg.3.TMT technology screens for significant differential proteins in response to mercury chloride stressUnder the condition of change factor?1.3,P<0.05,a total of 5,253 proteins were identified in this study,of which 4108 proteins contained quantitative information.After the experiment was removed,the statistics contained 364 differential proteins,of which 258 were up-regulated,106 The expression is down-regulated.Among them,12 proteins were found to be closely related to mercury stress,and all of them were significantly expressed in each stress group.Gene ontology?GO?results in the expression of significant differential proteins that are primarily characterized by catalytic activity,binding transport activity,and antioxidant activity.The visual display of the Kyoto Encyclopedia of Genes and Genomes?KEGG?pathway shows that the expression of significant differential proteins is mainly concentrated in metabolicpathways,secondarymetabolitebiosynthesis,phenylpropanoid biosynthesis,etc.Process,false discovery rate?FDR<0.05?.4.Large-scale verification of PRM technologyAccording to the analysis of the discussion and the results of mass spectrometry,combined with the characteristics of PRM test and the principle of protein screening,20 differentially expressed proteins were selected for protein verification by parallel reaction monitoring experiments.The overall trend of TMT and PRM results was consistent?upregulation protein was greater than 1.3,the down-regulated protein is less than 0.77?,and the differentially-changed protein screened may be a marker protein of rice root tip tissue in response to mercury metal stress.Conclusion:1.Mercury stress has an effect on the physiological activities of different tissues in rice.The activity of low concentration stimulating enzyme is enhanced,while the biochemical index is inhibited at different concentrations.The total mercury content increases with the increase of HgCl₂ stress concentration.2,364 significant differential proteins,23 related pathways,and 12 stress group co-variation proteins may directly or indirectly participate in the molecular mechanism of damage caused by mercury stress.3,Large-scale validation of 20 differential protein expression levels consistent with proteomic results,the identified differentially altered protein may be a marker protein for rice root tip tissue response to mercury metal stress.