Changes Of DNA Methylation During Cold Acclimation And Cloning Of DNA Methyltransferase Gene(CsDRM2) In Tea Plant

Low temperature is one of the most critical environmental factors that limit plant growth and distribution range. As a kind of economic crop, tea plant(Camellia sinensis(L.)O.Kuntze) is originated from tropical or subtropical regions, and it is vulnerable to low temperature damage. Tea plant resistance to low temperature is based on a period of cold acclimation, and the ability of cold resistance is correlation with tea plant genetic. In the process of cold acclimation, some genes changed and then tea plant can response to low temperature. DNA methylation is one of the major route of epigenetic modifications, it can participate in the responses to environment stresses through the modification of genomic DNA. In order to explore the relationship between DNA methylation and low temperature stress responsiveness in tea plant, methylation sensitive amplified polymorphism(MSAP) and high performance liquid chromatography(HPLC) were used to analyze the changes of DNA methylation level and pattern during different stages of cold acclimatization in this study. At the same time,we cloned Cs DRM2——the important gene that regulate DNA methylation in tea plant, and analysised the bioinformatic characteristics of the gene. To explore the relationship between DNA methylation and the cold hardiness of tea plant, we studied the gene expression patterns in the process of cold acclimation.1. The genome DNA methylation level at different cold acclimation stages are analyzed by HPLC technology.The results showed that there is a certain difference of DNA methylation level among the different stages. The percentage composition of 5-methylcytosine are between 49%~51%, and the DNA methylation levels in fully acclimated and de-acclimated samples were increased compared to non-acclimated sample(CK). The result indicate that low temperature can induce DNA methylation changes, and the overall methylation level showing a rising trend.2. Changes of tea plant genome DNA methylation level and pattern at different stages of cold acclimation were analyzed by MSAP technology. The results showed that 50 selected primer sets totally amplified 905, 968 and 970 methylated sites in non-acclimated sample(CK), fully acclimated and de-acclimated samples, respectively. And the methylation levels were 50.6%, 54.1% and 54.2%, respectively. DNA methylation levels in fully acclimated and de-acclimated samples were increased compared to CK. In addition, DNA demethylation and methylation were occurred among the three stages, but the alterations of the DNA methylation state took the methylation as the major thing. The results indicated that DNA demethylation may participate in cold acclimation progress in tea plant.3. The full-length cDNA sequence of DNA methyltransferase gene CsDRM2 was cloned by using RACE and RT-PCR technique, and submitted to Gen Bank with accession number KR057963. The Cs DRM2 c DNA length was 2 328 bp, and contained a 1 815 bp open reading frame(ORF), encoding 604 amino acid residues. The molecular weight and theoretic isoelectric point of Cs DRM2 protein are 67.39 k D and 4.84, respectively. Comparison of sequences similarityshowed that Cs DRM2 had more than 65% similarity with other reported DRMs. And Cs DRM2 had the typical UBA domain and methyltransferase functional domain at N-end and C-end, respectively. Phylogenetic tree analysis showed that Cs DRM2 had the closest genetic relationship with Sesamum indicum. The results of q RT-PCR showed that Cs DRM2 gene differentially expressed among different cold acclimation stages. The results indicated that Cs DRM2 participate in the DNA methylation progress which response to cold stress in tea plant.