Studies On DNA Methylation Analysis And MET1,DDM1 Cloning During Somatic Embryogenesis Of Larix Spp.

Somatic embryogenesis plays an important role in large-scale propagation of plant, has great value in forestry breeding, germplasm storage, genetic transformation, and especially is a model system to investigate the mechanism of cell differentiation and dedifferentiation. Epigenetic regulation may underlie some novel development processes during somatic embryogenesis. In this study, changes of DNA methylation during somatic embryogenesis of Larix cell line Y35 were detected, and the full-length cDNAs for MET1 and DDM1 homologues from Larix, LlMET1 and LlDDM1, were identified. The main results are following:1. Changes of DNA methylation were detected by methylation-sensitive amplified polymorphism (MSAP) at 14 time points, covering 7 developmental stages of Larix somatic embryogenesis. MSAP analysis shows that an increase occured from day 5 to day 14 after abscisic acid (ABA) induction, no change was detected from day 14 to day 35, and a decrease was observed from day 35 to day 42. These results show alterations in DNA methylation levels during Larix somatic embryogenesis, indicating that DNA methylation, which regulates gene expression at the chromatin level, is involved in somatic embryogenesis.2. DNA hypermethylation and DNA demethylation were determined during Larix somatic embryogenesis. DNA hypermethylation of the cytosine in 5'-CCGG-3' sequence, was most from hour 0.5 to hour 6, immediately after ABA induction, and was least from day 5 to day 7. DNA demethylation of the cytosine in 5'-CCGG-3' sequence was most from day 5 to day 7, and was least from day 14 to day 21, indicating that somatic embryogenesis involves different DNA methylation patterns.3. LlMET1 and LlDDM1 were cloned from Larix. Based on MET1 conserved domain, degenerate primers were designed to amplify the homologue from Larix using the cDNA as tempate. A 403 bp fragment was obtained, and then 3' rapid amplification of cDNA ends (RACE) and 5' RACE were performed to get the full-length cDNA of LlMET1. Using PFAM and CDD online software, LlMET1 was predicted to contain two BAH domains, two DNMT1-RFD domains, two DNA methylase domains and Dcm multi-domains. Phylogenetic analysis indicated that LlMET1 was nearest to MET1 from Elaeis guineensis. 3' end of cDNA for putative decrease in DNA methylation (DDM1) gene which has chromatin remodeling activity was obtained form SSH libary constructed by our lab. 5' RACE were performed to get the full-length cDNA of LlDDM1. The predicted protein sequence of LlDDM1 has DEXDc, HELICc superfamily, SNF2_N,SrmB multi-domains. Phylogenetic analysis indicated that LlDDM1 was nearest to DDM1 from Physcomitrella patens.Taken altogether, somatic embryogenesis involve changes of DNA methylation, and further investgation about LlMET1 and LlDDM1 will discover how DNA methylation functions in somatic embryognesis.