Construction Of RNAi Vector Of Three Chrysanthemum DNA Methylation Related Gene Ang Genetic Transformation

In order to reveal the relationship between DNA methylation related genes and the apparent traits of Chrysanthemum,we used enzyme digestion-linked method to construct the RNA interference vector of CmDRM2,CmCMT3 and CmDME gene conservative region in Chrysanthemum,and try to build Chrysanthemum leaf disc regeneration system,and expected to obtain transgenic Chrysanthemum plants.At the same time we are going to reveal the expression pattern of CmDRM2,CmCMT3,CmMET1,and CmDME genes of different parts of Chrysanthemum in flowering period,and transgenic Chrysanthemum plants,and understand the relative expression of DNA methylation related genes in Chrysanthemum,using Real-time fluorescence quantitative PCR(RT-PCR)technology.The main results are as follows:(1)The conserved sequences of CmDRM2,CmCMT3,and CmDME genes in ChrysanthemumThe study process that we searched for the conserved sequences of CmDRM2,CmCMT3,and CmDME genes in Chrysanthemum,each family of genes was used to refer to the corresponding genes of 9 species,and 3 DRM2 alleles,CMT3 class alleles,and 4 DME alleles were obtained.This further proved that the genes we acquired were the target genes.By clustering analysis 9 DNA methylation gene obtained in Chrysanthemum and related gene in other species,we know that 3 DRM2 alleles,2 CMT3 alleles,and 4 DME alleles of Chrysanthemum respectively together with other species of DRM2,CMT3,and DME gene.This further proved that the genes we acquired were the target genes.DNA MAN software was used to compare the 3 family genes homology,the conserved regions of these 3 family genes were obtained,which were 1868bp,722bp,and 1096/181 bp,respectively.The primers were designed according to the conserved region of them,with varieties of Chrysanthemum "Nan Nong Li Ya","Nan Nong Hong Xiu","Zi Die Fan Fei","Zi Jing Ling" and "Guo Qing Hong",and "Sam" cDNA as template,and PCR amplification,sequencing.Again using DNA MAN software analysis:The homology of CmDRM2,CmCMT3,and CmDME gene conserved sequences in 6 Chrysanthemum cultivars were all at the same time,which was 100%.(2)The expression patterns of CmDRM,CmCMT3,CmDME,and CmMET1 genes in Chrysanthemum.By using the technology of RT-PCR,to analyze expression of CmDRM2,CmCMT3,CmDME and CmMET1 gene in petal,stems,leaves,roots and shoots of "Nan Nong Li Ya".The expression of CmDRM2 gene in the tissues of Chrysanthemum from high to low was the stem,petal,root,leaf and foot bud;The expression of CmDME gene in the tissues of Chrysanthemum from high to low was the stem,petal,foot bud,root,and leaf;The expression of CmCMT3 gene in petal and stem segment was similar,the expression in root was the second,but expression in leaf and foot bud were the lowest;The expression of CmMET1 gene in the stems and leaves of Chrysanthemum was higher,but the expression in the petal,root and foot buds was lower.Among them,the expression of CmDRM2 and CmDME genes in stem segments were much higher than that in other parts.The expression of CmDRM2 gene was higher than that of CmDME gene in 5 tissues of Chrysanthemum,and the expression of CmDME gene was higher than that of CmCMT3 and CmMET1 gene.(3)Construction of RNA interference vectorThe RNA interference vector of Chrysanthemum CmDRM2,CmCMT3,and CmDME genes were constructed byenzyme digestion-linked method,respectively,and were tested by Bacterial fluid PCR and double enzyme digestion,respectively.(4)The establishment of regeneration system of Chrysanthemum leaf disc geneticThe regeneration medium by orthogonal experimental design of Chrysanthemum leaf discs were prepared,9 kinds of culture medium,each treatment has 3 replications,repeat 3 times,and statistical analysis using SPASS software:The optimal differentiation medium of Chrysanthemum "Zi Die Fan Fei"leaf disc was MS+3%sucrose+0.7%agar+1.0 mg/L 6-BA+0.75 mg/L NAA,differentiation rate was 71.1%;The optimal differentiation medium of Chrysanthemum "Nan Nong Liya" and "Nan Nong Hong Xiu" leaf disc was MS+3%sucrose+0.7%agar+1.0 mg/L 6-BA+0.5 mg/L NAA.The differentiation rates were 100%and 80%,respectively.(5)Genetic transformation of leaf disc of RNAi vector of Chrysanthemum CmDRM2 and CmCMT3 geneRNAi interference vector of CmDRM2 and CmCMT3 gene of Chrysanthemum were transformed by Agrobacterium mediated,regenerated plants were obtained.Plant regeneration we got through the kanamycin marker screening and quantitative identification of RT-PCR transgenic plants.The results were as follows:]0 CmDRM2 transgenic plants were obtained,and 3 CmCMT3 transgenic plants were obtained.(6)Relative expression of transgenic Chrysanthemum plantsCompared with the control group,the relative expression of CmDRM2 in the CmDRM2 transgenic plants was reduced by 61.14%;Compared with the control group,the relative expression of CmCMT3 in transgenic CmCMT3 plants was reduced by 46.19%;In CmDRM2 transgenic plants,CmCMT3 and CmMET1 DNA methyltransferase enzyme gene expression was 42.88%and 11.14%,respectively.Compared with the control group,the relative expression of CmDME gene was down 30.76%;In CmCMT3 transgenic plants,CmDRM2 and CmMET1 DNA methyltransferase enzyme gene expression compared with the control group,the average of 19.75%and 5.85%,respectively.Compared with the control group,the relative expression of CmDME gene was down 41.88%.The expression of other DNA methyltransferase genes in the transgenic plants of Chrysanthemum CmDRM2 or CmCMT3 transgenic plants was up-regulated,while the expression of CmDME gene was down regulated.