Identification And Analysis Of The DNA Methylation Modification Enzyme Gene Family In Peppe

Pepper(Capsicum spp.)is widely cultivated in China and has high nutritional value,economic value and medicinal value.As an important means of epigenetic regulation,DNA methylation is of great significance in the regulation of gene expression in plants.The whole process of DNA methylation in plants needs to be completed by the gene regulation of DNA methylation modifying enzyme.In order to explore the function of DNA methylation modifying enzyme gene in pepper,this study identified all DNA methylation modifying enzyme genes from the sequenced pepper genome,and compared the progressive relationship between gene family members in Arabidopsis,pepper,potato and tomato of the same family as pepper.To explore the expression patterns of DNA methyltransferase gene family in different stages of fruit development and abiotic stress of annual pepper(Capsicum annuum spp.)cultivar "zunla-1",and clone two DNA methyltransferase genes CaDRM1-like3(domains rearranged methyltransferase)and CaDNMT2(DNA methyltransferase 2),bioinformatics analysis was made on the physical and chemical properties of its protein,and the viral silencing recombinant vector and overexpression recombinant vector of CaDNMT2 gene were constructed to preliminarily verify the function of CaDNMT2 gene.The main results of this study are as follows:(1)In the genome of “zunla-1”,a gene family composed of fourteen DNA methylation modifying enzyme genes was identified.It includes ten DNA methyltransferase genes: two MET1(Methyltransferase 1)genes,three CMT(Chromatin domain methyltransferases)genes,four DRM(Domain rearranged methyltransferases)genes and one DNMT2(DNA methyltransferase 2)gene.Four DNA demethylase genes: two DME(DNA glycosylases Demeter)genes and two ROS1(Reporter of silencing 1)genes.Through bioinformatics analysis,it is found that the genes with close genetic relationship among family members have basically the same conserved domains.Clustering divides fourteen genes into four subfamilies,and almost all homologous genes exist in the two species of potato and tomato in the same family.(2)Based on the existing transcriptome sequencing results,the expression of gene family members in different tissues and fruit development of pepper was analyzed.The results showed that only CaMET1 expression in family members was close to 0,and the remaining genes had a certain expression.Among them,the expression of CaDRM1-like2 is the highest in the root,stem and leaf tissues of pepper,the expression of CaROS1-like2 is the highest in the bud tissues of pepper,and the expression of CaROS1-like2 is higher than that of other members of the family during the development and maturation of pepper fruit.(3)The expression patterns of DNA methylation modifying enzyme gene in Pepper under abiotic stress of salt and high temperature were studied by real-time fluorescence quantitative PCR.The results showed that during the 12 hour process of the two abiotic stresses,the members of the gene family produced methylation mechanism,and the expression changed dynamically with the stress time.Under high temperature stress: only the changes of CaCMT2,CaCMT3 and CaMET1 were lower than those without treatment,the remaining genes peaked at 3 hour or 12 hour after treatment,and the expression of CaROS1-like2 was much higher than that of other family members in these two periods.Under salt stress: the expression of family members changed greatly at different time periods.Except CaCMT2,CaCMT3,CaDRM1-like2 and CaDME,the other DNA methylation modifying enzyme genes reached the peak at 12 hour.(4)The full-length CDS of CaDRM1-like3 and CaDNMT2 genes were cloned from the genome of “zunla-1”.The results showed that the total length of CaDRM1-like3 was2036 bp,encoding 678 amino acids,and the total length of CaDNMT2 was 887 bp,encoding295 amino acids.Bioinformatics analysis showed that both were unstable hydrophilic proteins.Subcellular localization predicted that CaDRM1-like3 was located in Golgi apparatus and CaDNMT2 was located in nucleus.The prediction of CaDNMT2 was consistent with the results of subcellular localization test.(5)Construction of VIGS vector,and the fertile materials of pepper were infected.The gene expression and phenotypic changes of the experimental group were observed.The results showed that the expression of the target gene decreased in the leaves and flowers of the test group,indicating that the CaDNMT2 gene of the test material was successfully silenced.The pollen viability of the silenced plants was measured by fluorescence microscope.It was found that the pollen viability of the silenced plants was lower than that of the control group,the pollen viability of the blank control and negative control plants were more than 70%,and the pollen viability of the silenced plants was about 30%.It is speculated that CaDNMT2 gene plays a positive regulatory role in pepper pollen development and germination,this leads to a change in the fertility of peppers.