Kawai-Toyooka Hiroko, Kuramoto Chihiro, Orui Kyoko, Motoyama Kentaro, Kikuchi Kazuhiro, Kanegae Takeshi, Wada Masamitsu
Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo, 192-0397 Japan.
Plant Cell Physiol. 2004 Nov;45(11):1648-57. doi: 10.1093/pcp/pch186.
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'.
RNA干扰（RNAi）已成为确定基因功能的强大工具，并被广泛应用于各种生物体中。由于需要生成双链RNA（dsRNA）作为RNAi的诱导剂，因此制备RNAi诱导构建体有些繁琐且耗时，特别是对于全基因组分析中使用的数千个基因而言。为了克服这些问题，我们在蕨类植物铁线蕨中开发了一种更便捷的基因沉默方法，使用双链DNA（dsDNA）作为植物功能分析的模型系统。将与内源基因同源的dsDNA片段导入配子体细胞可诱导序列特异性基因沉默。由于它只需要与靶基因同源的dsDNA片段，因此PCR扩增片段就足以触发基因沉默。对于转化植株，已实现了>90%的最大基因沉默效率。此外，同时转移对应于多个基因的dsDNA片段对单个基因仍具有沉默作用。我们将这种方法称为“DNA干扰”。
