Regulation Of DNA Methylation On Synthesis And Accumulation Of Ascorbic Acid In Tomato Fruit

Tomato(Solanum lycopersicum)is an important model plant in horticultural research.It is rich in ascorbic acid and other nutrients.DNA methylation is an apparent modification in animals and plants,and plays an important role in plant growth and development.The regulation of DNA methylation on the nutritional quality of tomato fruit AsA remains to be explored.This project started with the key genes identified in AsA pathway in the laboratory,and explored the effects of methylation regulation on AsA content in tomato through bioinformatics and molecular biology methods.The main results of this topic are as follows:1.The GMP,GME and other genes that had been identified in the laboratory were compared by bioinformatics methods to find homologous genes,and other genes related to AsA pathways were searched by consulting the literature.A total of 61 AsA biosnytheisis-related genes were obtained and the expression of AsA-related genes were analyzed based on RNA-seq data of different developmental stages of tomato.The results showed that AC had more than 18 genes significantly expressed at 17 days after flowering,and AC had more than 14 genes significantly expressed at 42 days after flowering.Further study of the methylation level of AsA-related genes during maturation showed that most of the DNA methylation was CG type,the gene coding region and promoter region were more susceptible to methylation regulation relative to the downstream of the gene during fruit development.2.Based on the data of transcriptome,the changes of AsA related genes in CRISPR lines of SlDML2 and SlMET1 were analyzed.The results showed that there were 5 genes regulated by methylation in tomato CRISPR lines of SlDML2 and SlMET1 respectively.They were GR-1,GalUR-5,GME-1,AO-1 and APX-7.The methylation level of AsA-related genes in the above lines were further analyzed,and the GalUR-5 gene in AsA synthesis was significantly regulated by methylation.3.AC seeds and peduncles were treated with water and methyltransferase inhibitors(5-azaC)respectively,the seed germination,fruit development and maturity were investigated.The results showed that the seed germination rate after 5-azaC treatment reached 95%,while the control only had a germination rate of 72%;the treated fruits matured about 5 days earlier.The content of ascorbic acid was determined.The results showed that the content of AsA in leaves treated with 5-azaC increased by 37% compared with the control;the content of AsA in fruits treated with 5-azaC increased by 44% compared with the control during the breaker stage.4.The expression of GalUR-5 in leaves and fruits was up-regulated after 5-azaC treatment.The methylation level of GalUR-5 was determined by enzyme digestion PCR.The results showed that the 5-azaC-treated fruits had lower levels of methylation at 25 days post-flowering and 38 days post-flowering than the controls.5.When the activity of GalUR enzyme was measured,it was found that the activity of enzymes increased in leaves treated with 5-azaC,and it was more significant at fruit ripening stage,indicating that the decrease of DNA methylation level promoted the enzyme activity of GalUR enzyme,thereby promoting the accumulation of AsA.The conserved domain,physicochemical properties and tissue expression profiles of GalUR-5 were analyzed by bioinformatics.The results showed that the conserved domain was Aldo_ket_red,which was identical to the GalUR conserved domain in strawberry,and the GalUR amino acid idendity with strawberry was 25%.