Screening And Bioinformatics Analysis Of Differentially Expressed Proteins And MiRNAs In Rice Root Tips In Response To Methylmercury Stress

Objective: Rice is a hyperaccumulator of methylmercury.Rice comsumption has become the main route of methylmercury exposure for people dwelling in mercury mining areas,posing a potential health exposure risk.Elucidating mechanism of absorption and transfer of methylmercury can provide basic theoretical guidance for regulating absorption of methylmercury,and thus effectively controlling its contamination in rice.In this study,the distribution characteristics of methylmercury and physiological and biochemical indicators induced by methylmercury stress on rice were analyzed.Screening differentially expressed proteins and miRNAs in rice root tips that respond to methylmercury stress were conducted by using techniques of i TRAQ and high-throughput sequencing.The molecular mechanism of absorption and transport of methylmercury in rice was discussed,aiming for future study on the controlling of rice methylmercury accumulation and reducing the health risk of methylmercury exposure.Methods: A pot experiment was conducted and rice species of Liang you 302(Indica)was applied in this study.Four levels of methylmercury concentration were set: 0ng/g,50ng/g,150ng/g,300ng/g,with three pots per group and three plants per pot.Concentrations of methylmercury in rice tissues were measured by cold atomic fluorescence spectrometry with water-phase ethylation-gas chromatography(GC-CVAFS).The proline(Pro),total protein(TP),peroxidase(POD),catalase(CAT),total superoxide dismutase(T-SOD),malondialdehyde(MDA),and chlorophyll of stems and leaves were measured by commercial kits.The technique of i TRAQ was used to screen the differentially expressed proteins in rice root tips in response to methylmercury stress.Simultaneously,miRNA sequencing of rice root tips was performed using byhigh-throughput sequencing technology to screen differentially expressed miRNAs,and the bioinformatics as well as target genes of miRNA were analyzed.Results: 1.Distribution characteristics of methylmercury in different tissues of rice under methylmercury stressed: The methylmercury concentrations in 3 stressed group of root,stem and leaves was higher than that in control group(p<0.05).The concentrations of methylmercury increased with the increase concentration of treatment.The distribution of methylmercury in rice tissues of the control group showed root> stem> leaf,and in the dose-stressed group showed stem> root> leaf.2.The physiological and biochemical indicators of rice under methylmercury stress:(1)The proline in 3 methylmercury stressed groups in root were not significantly different than the control group,while the proline of stems and leaves in 3methylmercury stressed groups were higher than the control group(p<0.05).(2)The total protein of roots and stems in the high-dose stressed group was less than the control group(p<0.05),while the total protein of leaves in low-dose and medium-dose stressed groups were less than the control group(p<0.05).(3)POD activity of roots in 3 methylmercury stressed groups was higher than the control group(p<0.05),but POD activity of stem in 3 methylmercury stressed groups were no significant difference,and POD of leaves in the low-dose stressed group was less than the control group(p<0.05).(4)CAT of rice in 3 methylmercury stressed groups were not significantly different with the control group.(5)SOD of roots in 3methylmercury stressed groups were higher than the control group(p<0.05),SOD of stems in medium and high-dose stressed groups were less than the control group(p<0.05),and SOD of leaves in medium and high dose stressed groups were less than the control group(p<0.05).(6)MDA of root in 3 methylmercury stressed groups were higher than the control group(p<0.05),MDA of stem in the medium and high-dose stressed groups were less than the control group(p<0.05),and MDA of leaf in low-dose stressed group was higher than the control group(p<0.05).(7)There were no significant difference of chlorophyllin of stem and leaves between 3methylmercury stressed groups and the control group.3.i TRAQ screening differentially expressed proteins:(1)According to fold change(?1.20 or ?0.83)andP<0.05,a total of 88 differentially expressed proteins were identified,of which 32 proteins were up-regulated and 56 proteins were down-regulated.Approximately 15 differentially expressed proteins associated with characteristic of metal ion binding,including germination-like proteins and lipoxygenase.(2)Differential proteins are involved in 15 biological processes,11 cell components,10 molecular functions,and involve 28 metabolic pathways.4.High-throughput sequencing to screen differentially expressed miRNAs:(1)Compared with the control group,there were 39 differential miRNAs in the low-methylmercury stressed group(consisting of 17 significantly up-regulated and 22 significantly down-regulated),52 differential miRNAs in the middle-methylmercury stressed group(consisting of 27 significantly up-regulated and 25 significantly down-regulated),and 18 differential miRNAs in the high-methylmercury stressed group(consisting of 9 significantly up-regulated and 9significantly down-regulated).(2)Target genes of differential miRNAs that have annotative were predicted,consisting of 285 in the low-dose group,537 in the medium-dose group,and 426 in the high-dose group.(3)Target genes of differential miRNAs in low-methylmercury stressed group involved in 295 biological processes,157 molecular functions,and 46 cellular components,in middle-methlymercury stressed group involved in 361 biological processes,73 molecular functions,and 185 cellular components,and in high-methylmercury stressed group involved in 317 biological processes,170 molecular functions,and 43 cellular components.(4)Differential miRNA target genes participate in related pathways,including 23 in the low-methylmercury group,45 in the middle-methylmercury group,and 24 in the high-methylmercury group,which were mainly involved in biosynthesis,metabolic processes,and signal transduction.Conclusions: 1.Methylmercury is toxic to rice,causing changes in physiological and biochemical indicators.Most enzyme activity accumulates in the roots.Methylmercury concentrations in the rice increase with the increase of treatment concentration.Rice can accumulate methylmercury.2.Differentially expressed proteins mainly respond to methylmercury stress in rice by participating in endoplasmic reticulum protein processing,sucrose and starch metabolism,oxidative phosphorylation and glyoxylic acid and dicarboxylic acid estermetabolism pathways.3.Differentially expressed miRNA target genes are mainly involved in biological processes such as biosynthesis,metabolic processes and signal transduction.4.Differentially expressed proteins and miRNAs play important roles of the molecular regulatory network of rice in response to methylmercury toxicity.