Fattash Isam, Khraiwesh Basel, Arif M Asif, Frank Wolfgang
Plant Biotechnology, Faculty of Biology, University of Freiburg, Freiburg, Germany.
Methods Mol Biol. 2012;847:293-315. doi: 10.1007/978-1-61779-558-9_25.
MicroRNAs (miRNAs) are ∼21-nt-long small RNAs transcribed from endogenous MIR genes which form precursor RNAs with a characteristic hairpin structure. MiRNAs control the expression of cognate target genes by binding to reverse complementary sequences resulting in cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA sequence of endogenous MIR precursor genes, while maintaining the general pattern of matches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene of interest. During the last decade, the moss Physcomitrella patens emerged as a model plant for functional gene analysis based on its unique ability to integrate DNA into the nuclear genome by homologous recombination which allows for the generation targeted gene knockout mutants. In addition to this, we developed a protocol to express amiRNAs in P. patens that has particular advantages over the generation of knockout mutants and might be used to speed up reverse genetics approaches in this model species.
微小RNA（miRNA）是一类长度约为21个核苷酸的小RNA，由内源性MIR基因转录产生，这些基因形成具有特征性发夹结构的前体RNA。miRNA通过与反向互补序列结合来控制同源靶基因的表达，从而导致靶RNA的切割或翻译抑制。人工miRNA（amiRNA）可以通过交换内源性MIR前体基因的miRNA/miRNA序列产生，同时保持回折结构中匹配和错配的总体模式。因此，对于功能基因分析，可以设计amiRNA靶向任何感兴趣的基因。在过去十年中，小立碗藓作为功能基因分析的模式植物出现，这是基于其通过同源重组将DNA整合到核基因组中的独特能力，这种能力使得能够产生靶向基因敲除突变体。除此之外，我们开发了一种在小立碗藓中表达amiRNA的方案，该方案相对于产生敲除突变体具有特殊优势，并且可用于加速该模式物种中的反向遗传学研究方法。
