Gentic Analysis Of Chrysanthemum CmMET1 Gene Interfered

The CmMET1 gene has been transferred into callus of Chrysanthemum cultivar “ZJL” by RNAi vector using Agrobacterium-mediated transformation,and the expression of CmMET1 was reduced in differentiation seeding.To understand the effect the effect of RNAi interference on Chrysanthemum cultivars,we transplanted the transgenic plants to the experimental field and observed and recorded the plant height and blade shape and flowering-time,and other phenotypic changes.We employed the techniques of real-time fluorescence quantitaive PCR to detect the expression of related genes of DNA methylation and demethylation and flowering genes FT.The techniques of MSAP and HPLC were conducted to detect the DNA methylation level of Chrysanthemum.To study the transmission of RNAi silencing signal between different cultivars of Chrysanthemum.Chrysanthemum cultivars GQH and HSJQ were grafted onto transgenic plants ZJL(treatment group)and non-transgenic ZJL(control group)as scions.The results were follows:(1)The effect of RNAi interference CmMET1 on the expression of CmMET1 of rootstocks and scions cultivars: Compared with controled group,the CmMET1 gene expression quantity of transgenic plants ZJL with RNAi-CmMET1 vector was 46.51%,the CmMET1 gene expression quantity of Chrysanthemum scions cultivars GQH and HSJQ was 58.67% and 52.5% respectively.The expression of CmMET1 gene of three Chrysanthemum cultivars was lower than the control group,this result demonstrated that the RNAi silencing signal could move to the scions through grafting position,but the interference effect is different in different Chrysanthemum cultivars,which may be due to the difference of target sequence of Chrysanthemum cultivars.(2)Effect of RNAi interference CmMET1 on phenotype of Chrysanthemum rootstocks and scions: The transgenic plants had shorter internodal length,lower plant height,aging tendency,less and shoal blade incision and top leaves with yellow spots,wich could be self-recovery when flowering stage.The time to capitulum bud appearance and early blooming were significantly shorter 10 d,8 d,12 d and 9 d than those of the control for Chrysanthemum cultivars ZJL and HSJQ respectively.Capitulum development of Chrysanthemum cultivar GQH was significantly accelerated 14 d compared with that of the control.(3)Effect of RNAi interference CmMET1 on the expression of other methyltransferases and demethylation enzyme of Chrysanthemum rootstocks and scions: The expression of methyltransferase CmDRM2 was up-regulated by 31%,29% and 21% respectively when the expression of CmMET1 was decreased,the expression of CmDME was down-regulated by 26.42%,14.99% and 31.26% compared with the those of the control respectively.(4)Effect of RNAi interference CmMET1 on DNA Methylation leveal of Chrysanthemum rootstocks and scions: The whole genome DNA methylation rate of rootstocks and scions were decreased by 14.88%,11.2% and 13.84% respectively compared with the control(HPLC).The DNA methylation rate of CG site was lower 12.6%,8.9%,13% than those of the control respectively.in addition to GQH,CG site methylation model had changed(MSAP).The DNA methylation rate of transposon was also decreased(iPBS-MSAP).From the degree of decline could be seen: the decrease in CmMET1 gene activity mainly affected the methylation rate of CG sites.(5)Effect of DNA hypomethylation on the expression of FT-likes of Chrysanthemum rootstocks and scions: There were three FT-likes genes families in Chrysanthemum,the expression FTL was downregulated and FTL2 and FTL3 were up-regulated.