| Abiotic stresses such as drought,cold,high temperature and salinization threaten agricultural productivity all over the world to a great extent.Among these environmental stressors,osmotic stress is an interrelated stress that can be induced by these abiotic stresses and affects important plant metabolic processes such as cell growth,ion transport,oxidative homeostasis,and photosynthesis.Watermelon often faces a series of abiotic stresses such as drought,salinity and cold damage in production,which affects the quality,yield and even the whole watermelon industry.Therefore,there is an urgent need to use modern biological techniques for effective genetic improvement of watermelon varieties to enhance their resistance to stress.With the continuous development of gene editing technology,great progress has been made in the improvement of agronomic traits,but the genetic information of resistant plants is still poorly.Watermelon,as a drought-tolerant species originating from the Sahara desert,is an ideal model plant for the study of water stress.Plants have developed multiple response mechanisms to cope with external stresses during long-term evolution,including pre-transcriptional epigenetic regulation and classical genetic regulation such as post-transcriptional hormones,transcriptional regulators and stress response genes.In this study,the transcriptome and whole genome bisulfite sequencing were performed on watermelon cells before and after osmotic stress treatment using a high-throughput sequencing platform to provide a theoretical reference for understanding the process of watermelon response to osmotic stress and the molecular mechanism of resistance formation research.The main results are as follows:(1)Using RNA-Seq method,the investigation of the transcriptome analysis of diploid watermelon suspension cells after 100 mmol/L mannitol artificially simulated osmotic stress for 0,2 and 4 h were performed.The results indicated that the gene expression patterns of watermelon suspension cells were similar between 2 and 4 h of mannitol treatment.GO analysis of selected 2,960 DEGs between 0 h and 4 h showed that were mainly concentrated in biological processes,catalytic activity and membrane-bounded organelle among the three main categories of molecular functions,biological processes and cell components process;KEGG Pathway analysis results show that these differentially expressed genes are significantly enriched in carbon metabolism,plant hormone signal transduction,biosynthesis of amino acids,phenylpropanoid biosynthesis,amino sugar and nucleotide sugar metabolism,Glycolysis/Gluconeogenesis,protein processing in endoplasmic reticulum,plant-pathogen interaction,endocytosis and MAPK signaling pathway.Among them,genes related to plant hormone synthesis,osmotic regulator related genes and signal transduction pathways and reactive oxygen species scavenging enzyme genes can rapidly respond to osmotic stress.(2)Whole genome bisulfite sequencing was performed on cells treated with 100 mM mannitol for 4 h and control by the high-throughput sequencing platform Illumina Hiseq TM,and the data showed that the average methylation level of the watermelon genome was highest in the CG contexts,followed by CHG,and lowest in the CHH.The overall methylation level of watermelon decreased after osmotic stress treatment,and demethylation occurred in all three contexts of CG and CHG and CHH,and the most genes with dynamic changes of methylation were found at CHH contexts.742 and 1,584 differentially methylated genes were obtained in the genebody and promoter regions of the CHH contexts respectively,and GO analysis showed that these differentially methylated genes are mainly involved in catalytic activity,membrane components,oxidoreductase activity,protein hydrolysis and ATP binding.In KEGG analysis,these differentially methylated genes were significantly enriched in metabolic pathways,starch and sucrose metabolism,peroxidases,plant hormone signal transduction and biosynthesis of secondary metabolites.The transcriptome data and methylation sequencing data were used for association analysis,and 114 common differential genes were obtained.KEGG analysis revealed that these differentially methylated expression-related genes were mainly involved in RNA transport,starch and sucrose metabolism,plant hormone signal transduction and biosynthesis of secondary metabolites.The relationship between methylation levels and gene expression was further analyzed,and it is hypothesized that demethylation of promoter regions of osmolyte-related genes and phytohormone signaling-related genes is one of the mechanisms by which watermelon responds and adapts to osmotic stress.(3)Exogenous application of inositol significantly reduced MDA accumulation in watermelon cells under osmotic stress,effectively alleviating the inhibition of cell growth by osmotic stress.And the inositol content in watermelon cells gradually increased in response to osmotic stress.Fluorescence quantification and bioinformatics analysis of ClMIPS were performed.The results showed that among the two ClMIPS genes,Cla014489 was predominant and Cla014161 expression was suppressed.Both of their promoter regions contained many stress-responsive and plant hormone-related cis-acting elements that could be induced to be expressed by phytohormones such as ABA,Me JA and SA.It was hypothesized that Me JA and ETH might be intermediate signals to induce Cla014489 expression,and SA and ABA might be intermediate signals to induce Cla014161 expression by phytohormones induced expression pattern changes.(4)Cla014489 encodes 510 amino acids,the molecular weight of the protein is about56.3 k D,and the isoelectric point is 5.40,which is a stable protein.The protein has four conserved structural domains and was found to be closely related to melon and cucumber of the same family by phylogenetic tree analysis.SDS PAGE electrophoresis showed that the gene has inositol-1-phosphate synthetase activity.The constructed recombinant plasmid p ET28a-Cla014489 was transferred into BL21 for prokaryotic expression and found that the expression of ClMIPS protein could improve the tolerance of E.coli under osmotic stress.It was hypothesized that this gene might be a resistance gene.The effect of methylation level on the expression properties of inositol and its related genes was analyzed by DNA methylation inhibitor 5-Azacytidine.The results showed that the decrease in DNA methylation level increased the expression of ClMIPS and promoted the accumulation of inositol.It is speculated that the decrease of ClMIPS methylation status is one of the mechanisms of watermelon cells responding to osmotic stress. |
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