Biotechnology Research Institute, The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing, China.
Plant Biotechnol J. 2013 Oct;11(8):997-1005. doi: 10.1111/pbi.12093. Epub 2013 Aug 3.
Long hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification-mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down-regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double-stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross-silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA-derived siRNAs were characteristic of Argonaute-binding small RNAs. Our results indicate that RNAi mutant library is a high-efficient approach for genomewide gene identification in plants.
长发夹 RNA (hpRNA) 转基因是研究植物基因功能的有力工具，但尚未有针对植物的基于 hpRNA 转基因的全基因组 RNAi 突变体文库的报道。在这里，我们报告了一种使用改良的滚环扩增介导的 hpRNA (RMHR) 方法构建水稻全基因组基因功能鉴定的 hpRNA 文库。该文库转化水稻后，产生了数千个含有靶向各种功能基因的 hpRNA 的转基因系。hpRNA 系中的靶 mRNA 下调，这与 hpRNA 双链臂对应的 siRNA 的积累相关。来自单个成员的 hpRNA 可以同时沉默一个基因家族的多个成员，但这种交叉沉默的程度取决于成员之间的序列同源性以及匹配 siRNA 的丰度。关键基因的沉默往往会导致严重的表型，但这些转基因系通常在田间存活足够长的时间，以便进行表型和分子分析。小 RNA 的深度测序分析表明，hpRNA 衍生的 siRNA 是 Argonaute 结合小 RNA 的特征。我们的结果表明，RNAi 突变体文库是一种高效的植物全基因组基因鉴定方法。
