Abstract
Gene silencing technology such as RNA interference (RNAi) is commonly used for the reduction of gene expression in plant cells. Exogenous double-stranded RNA (dsRNA) can induce gene silencing in higher plants. Previously we showed that delivery of dsDNA fragments such as PCR products with an endogenous gene sequence into fern Adiantum capillus-veneris gametophytic cells can induce sequence-specific gene silencing named DNAi (Kawai-Toyooka et al. 2004). In this study, we used a NEO1/PHY3 gene that mediates red light-induced chloroplast movement as a model of DNAi. According to microscopic observation and RT-PCR analysis, PHY3 gene expression and phy3 function was suppressed in DNAi-induced Adiantum gametophytes. Interestingly, this suppression effect still remained in the next generation. Hence, we tried to identify how PHY3 suppression happened during the gene silencing process by DNAi over generation. PCR analyses using methylated cytosine-specific nuclease and cytosine methylation-sensitive restriction enzyme showed that the DNAi gene silencing was mediated by DNA methylation. Methylation profiles in PHY3 gene by bisulfite sequencing will be presented.
