DNA interference: a simple and efficient gene-silencing system for high-throughput functional analysis in the fern adiantum |
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Authors: | Kawai-Toyooka Hiroko Kuramoto Chihiro Orui Kyoko Motoyama Kentaro Kikuchi Kazuhiro Kanegae Takeshi Wada Masamitsu |
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Institution: | Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo, 192-0397 Japan. |
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Abstract: | RNA interference (RNAi) has become a powerful tool for determining gene function and is used in a wide variety of organisms. Since it is necessary to generate double-stranded RNA (dsRNA) as an inducer for RNAi, preparation of RNAi-inducing constructs is somewhat cumbersome and time consuming, especially for the thousands of genes used in a genome-wide analysis. To overcome these problems, we have developed a more convenient gene-silencing method in the fern Adiantum using double-stranded DNA (dsDNA) as a model system for functional analysis in plants. Delivery of dsDNA fragments homologous to an endogenous gene into gametophytic cells can induce sequence-specific gene silencing. As it only requires dsDNA fragments homologous to a target gene, PCR-amplified fragments are enough to trigger gene silencing. Maximum gene silencing efficiencies of >90% have been achieved for transformed plants. In addition, simultaneous transfer of dsDNA fragments corresponding to multiple genes still has a silencing effect for individual genes. We term this approach 'DNA interference'. |
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