| Objective: DNA methylation is involved in epigenetic processes and plays an important role in epigenetic regulation of eukaryotes,including regulation of growth and development,control of gene expression,and maintenance of genome integrity.The use of drugs to alter methylation status(hypomethylation or hypermethylation)is an effificient method to study DNA methylation.The purpose of this study was to investigate the effects of DNA methylation on the morphology and chemotypes of Pogostemon cablin,so as to clarify the role of DNA methylation in the formation of chemotypes.Methods: The DNA methylation inhibitor 5-azaC was used to demethylate Pogostemon cablin,Using traditional methods to observe and measure the agronomic traits of Pogostemon cablin,and the changes during the growth and development of Pogostemon cablin were analyzed by comparing with the control group,at the same time,GC-MS was used to detect the dynamic changes of index components in Pogostemon cablin,Genomic DNA methylation was detected by high performance liquid chromatography(HPLC),Methyl Rad-Seq(Simplified Genome Methylation Sequencing)technology performs methylated sequencing analysis of two chemotypes of Pogostemon cablin and transcriptome sequencing analysis was performed.Results: 1.After 5-azacytidine treatment,the growth and development of two different chemical types of Pogostemon cablin was significantly inhibited,but the effect on patchouliol-type Pogostemon cablin was more significant.the plants became shorter,the leaves became smaller and the crown became smaller,and with the increase of 5-azaC treatment concentration,the leaves gradually turned yellow,curled and wrinkled,and the excessively high concentration caused the death of the leaves.The growth state of Pogostemon cablin was recovered after the inhibition was lifted.2.The results showed that there was no significant change in the content of patchouli alcohol in patchouliol-type Pogostemon cablin after treatment with different concentrations of 5-azaC,the content of pogostone in 50 μmol/L and 100 μmol/L treatment groups was significantly up-regulated.In 100 μmol/L treatment group,the difference between pogostone and patchouli alcohol was the largest,followed by 50 μmol/L treatment group.After disinhibition,the content of pogostone in the treatment group was significantly increased and decreased the content of patchouli alcohol,while the content of pogostone increased and the content of patchouli alcohol decreased,there was no significant difference between the sum of the two components.After treated pogostone-type Pogostemon cablin with different concentrations of 5-azaC,it was found that the content of patchouli alcohol significantly increased,but the content of pogostone would be significantly decreased with the increase of treatment concentration.After 5-azaC treatment,the ratio of the contents of the two chemical components in the patchouliol-type Pogostemon cablin significantly decreased,and the ratio of the contents of the two chemical components in the pogostone-type Pogostemon cablin significantly increased.The change of the ratio of the two chemical components in pogostone-type Pogostemon cablin was greater than that in patchouliol-type Pogostemon cablin.3.The best HPLC analysis conditions: mobile phase: 10 mM sodium hexane sulfonate: triethylamine(99.8:0.2,V/V)pH 6.9,flow rate: 0.8m L/min,detection wavelength: 273 nm,column temperature: 30℃,sample volume: 20 μL.Methodology investigation showed that the peak shape of the sample was sharp,the separation degree was good,and the results of linearity,precision,reproducibility and stability were very good,which was suitable for the detection of DNA methylation in Pogostemon cablin.The results showed that the DNA methylation level of Pogostemon cablin significantly decreased after 5-azaC treatment,and the DNA methylation level gradually decreased with the increase of 5-azaC concentration.After the removal of inhibition,the DNA methylation level of the treatment group increased significantly,but it was still significantly lower than the control group.In addition,the decrease of DNA methylation level of pogostone-type Pogostemon cablin was greater than that of patchouliol-type Pogostemon cablin.4.Methyl Rad-Seq(Simplified Genome Methylation Sequencing)techniques analyze the methylation sequencing of two chemotypes Pogostemon cablin,the results showed that the methylation sites were mainly distributed in the intergene region,intron,upstream and downstream regions,and the number of methylation sites in intergene region was much higher than that in other gene elements,and there was little difference in the number of methylation sites in intron,downstream region and upstream region.In addition,the number of CG methylation sites was significantly higher than that of CWG,and the number of CG and CWG methylation sites in the transcription start site(TSS)and transcription stop site(TTS)regions was significantly lower than that in other regions.However,the number of CG methylation sites in the gene region was significantly higher than that in other regions,and CWG methylation sites were mainly distributed in the upstream and gene region.GO enrichment analysis was carried out on the differentially methylated genes in different comparison groups of two chemotypes of Pogostemon cablin,Comparison of pogostone-type and patchouliol-type Pogostemon cablin(LT-CK and HN-CK)groups,differential methylated genes were found to be mainly concentrated in pathways related to the regulation of growth and development,normal body activities,DNA replication,transcription and translation.Comparison of treatment group and control group of patchouliol-type Pogostemon cablin(HN-50 and HN-CK),it was found that the differential methylation genes were mainly concentrated in the pathways related to the regulation of life activities,gene expression regulation and terpene biosynthesis.Comparison of treatment group and control group of pogostone-type Pogostemon cablin(LT-50 and LT-CK),it was found that the differentially methylated genes were mainly concentrated in the pathways related to photosynthesis,transcription and translation,and the regulation of life activities.KEGG enrichment analysis of differentially methylated genes between two chemical types of Pogostemon cablin(LT-CK and HN-CK)groups,it was found that the differential methylation genes were mainly concentrated in the biosynthesis of pantothenic acid and coenzyme A,amino acid metabolism,diterpenoid biosynthesis,vitamin B6 metabolism,folic acid biosynthesis,splicosome,glucose metabolism and other pathways,which affected the growth and development,photosynthesis,translation process and the synthesis of terpenes in Pogostemon cablin.Comparison between treatment group and control group of patchouliol-type Pogostemon cablin(HN-50 and HN-CK),it was found that differential methylation genes were mainly concentrated in pathways related to metabolic processes such as glucose metabolism,amino acid metabolism,nitrogen metabolism and tricarboxylic acid cycle,which affected the growth and development and the terpenes biosynthesis of patchouliol-type Pogostemon cablin.Comparison between treatment group and control group of pogostone-type Pogostemon cablin(LT-50 and LT-CK),it was found that the differential methylation genes were mainly concentrated in the pathways related to photosynthesis-antenna protein,citric acid cycle,glycolysis/glucose metabolism,amino acid metabolism,pantothenic acid and coenzyme A biosynthesis,splice and other pathways,which affected the photosynthesis,growth and development and the terpenes biosynthesis of pogostone-type Pogostemon cablin.5.In this analysis,the transcriptome sequencing of 12 samples with reference was completed,After analysis and differential screening of the expression levels of four different protein-coding genes,the number of different genes was obtained as: 15660,15254,33500 and 35263,respectively.GO enrichment analysis showed that genes related to DNA integration,DNA recombination,negative regulation of cytokinin activation signal and regulation of tryptophan metabolism were all upregulated,while genes related to regulation of carbohydrate utilization and phosphate absorption were significantly down-regulated after 5-azaC treatment in Pogostemon cablin.And the genes associated with nucleic acid binding,RNA-directed DNA polymerase activity,peptide chain endonuclease activity and endonuclease activity were significantly up-regulated.Genes related to photosynthesis,chloroplast,chloroplast thylakoid membrane and regulation of phytol biosynthesis were significantly down-regulated.KEGG enrichment analysis showed that the genes related to plant hormone signal transduction and folate biosynthesis in Pogostemon cablin were down-regulated,which would lead to the failure of auxin,cytokinin and other hormones related to plant growth and development,and indirectly inhibit the growth and development of Pogostemon cablin.The carbon fixation and photosynthesis-related genes in organisms were significantly down-regulated,and the metabolism and degradation of amino acids prevented the utilization of amino acids in Pogostemon cablin,which indirectly affected the growth,development and photosynthesis of Pogostemon cablin.Conclusion: Based on the above results,this study suggests that DNA methylation plays an important role in regulating the growth and development and formation of chemotypes of Pogostemon cablin.Exogenous application of 5-AZAC could lead to changes in DNA methylation level and differential expression of various genes in Pogostemon cablin,thus affecting the growth and development of patchouli and the formation of chemical types of Pogostemon cablin. |
PDF Full Text Request