DNA methylation of the DC8 gene in carrot (Daucus carota L.) and its association with development

The Dc8 gene in carrot (Daucus carota L.) expresses differentially. The methylation pattern in its promoter during somatic embryogenesis was investigated and the association of Dc8 methylation with its expression was studied by bombarding a Dc8-GUS construct with different methylation patterns into carrot calli and somatic embryos followed by GUS transient assay.;Dc8-GUS expression can be enhanced by 5-azacytidine (5-azaC) treatment of carrot calli and somatic embryos prior to bombardment and decreased by in vitro enzymatic methylation of the plasmid. The plasmid bearing Dc8-GUS obtained from 5-azaC treated E. coli also has a slightly higher expression, which is associated with decreased level of methylation. GUS expression, increased by the 5-azaC treatment, is enhanced by ABA treatment of both calli and somatic embryos and was more prominent in the latter. In vitro methylation of Dc8-GUS by methylases generally resulted in a lower frequency of GUS expression. SssI methylase completely inhibited GUS expression.;Methylation patterns of a segment covering +120 to ;Digestion of DNA with MaeII, HpaII and TagI followed by PCR also revealed differences methylation status at different sites during somatic embryogenesis. The MaeII sites, which are closer to the transcriptional start point, showed greater loss of methylcytosine associated with Dc8 expression than the more distal HpaII and TagI sites.;Two improvements were made to the sodium bisulfite technique. The methylation status can be detected by just one, rather than four types of sequencing termination reactions. The strand origin of the cloned PCR insert derived from bisulfite-modified DNA can be determined through the types of base conversions of the sequencing data.