Warthmann Norman, Chen Hao, Ossowski Stephan, Weigel Detlef, Hervé Philippe
Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.
PLoS One. 2008 Mar 19;3(3):e1829. doi: 10.1371/journal.pone.0001829.
Endogenous microRNAs (miRNAs) are potent negative regulators of gene expression in plants and animals. Artificial miRNAs (amiRNAs)-designed to target one or several genes of interest-provide a new and highly specific approach for effective post-transcriptional gene silencing (PTGS) in plants.
We devised an amiRNA-based strategy for both japonica and indica type strains of cultivated rice, Oryza sativa. Using an endogenous rice miRNA precursor and customized 21mers, we designed amiRNA constructs targeting three different genes (Pds, Spl11, and Eui1/CYP714D1). Upon constitutive expression of these amiRNAs in the varieties Nipponbare (japonica) and IR64 (indica), the targeted genes are down-regulated by amiRNA-guided cleavage of the transcripts, resulting in the expected mutant phenotypes. The effects are highly specific to the target gene, the transgenes are stably inherited and they remain effective in the progeny.
CONCLUSION/SIGNIFICANCE: Our results not only show that amiRNAs can efficiently trigger gene silencing in a monocot crop, but also that amiRNAs can effectively modulate agronomically important traits in varieties used in modern breeding programs. We provide all software tools and a protocol for the design of rice amiRNA constructs, which can be easily adapted to other crops. The approach is suited for candidate gene validation, comparative functional genomics between different varieties, and for improvement of agronomic performance and nutritional value.
内源性微小RNA（miRNA）是植物和动物基因表达的有效负调控因子。人工微小RNA（amiRNA）——设计用于靶向一个或多个感兴趣的基因——为植物中有效的转录后基因沉默（PTGS）提供了一种新的、高度特异性的方法。
我们为栽培稻粳稻和籼稻品种设计了一种基于amiRNA的策略。利用水稻内源性miRNA前体和定制的21聚体，我们设计了靶向三个不同基因（Pds、Spl11和Eui1/CYP714D1）的amiRNA构建体。在日本晴（粳稻）和IR64（籼稻）品种中组成型表达这些amiRNA后，靶向基因通过amiRNA引导的转录本切割而下调，从而产生预期的突变表型。这些效应对靶基因具有高度特异性，转基因能够稳定遗传并且在后代中仍然有效。
结论/意义：我们的结果不仅表明amiRNA能够在单子叶作物中有效触发基因沉默，还表明amiRNA能够有效调节现代育种计划中使用的品种的重要农艺性状。我们提供了所有软件工具和水稻amiRNA构建体设计方案，该方案可轻松应用于其他作物。该方法适用于候选基因验证、不同品种间的比较功能基因组学以及农艺性能和营养价值的改良。
