Ziv Carmit, Yarden Oded
Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.
Methods Mol Biol. 2010;638:77-100. doi: 10.1007/978-1-60761-611-5_6.
The availability of a large number of gene-disrupted mutants (either from natural mutants' collections or from knockout projects) is a great advantage for functional analysis studies. However, disfunction of many fungal genes, involved in key developmental processes, leads to dramatic and pleotropic changes in cell morphology, conferring a major difficulty in studying null mutants. Therefore, obtaining variable levels of reduction in gene expression, especially of essential genes or genes whose impaired expression confers a pleiotropic phenotype, is extremely beneficial for studying their function. Here, we describe the use of RNAi as a gene silencing mechanism, in a manner that might facilitate the functional analysis of such essential genes. Two alternative strategies for the construction of an RNAi-induced inverted-repeat construct are demonstrated and a third alternative is suggested. In addition, DNA-mediated transformation of conidia by electroporation, RNA extraction from fungal mycelium and northern blot analysis are described in detail.The experimental results presented, demonstrate that RNAi can be employed as a gene silencing tool in Neurospora crassa, both for nonessential (al-2) and essential (cot-1) genes, resulting in a range of stable, partially silenced mutants, exhibiting different phenotypes.
大量基因敲除突变体（来自天然突变体库或基因敲除项目）的可得性对于功能分析研究而言是一个巨大优势。然而，许多参与关键发育过程的真菌基因功能失调会导致细胞形态发生显著且多效性的变化，这给研究基因敲除突变体带来了重大困难。因此，实现基因表达水平的不同程度降低，尤其是对于必需基因或其表达受损会导致多效性表型的基因，对于研究它们的功能极为有益。在此，我们描述了以一种可能有助于此类必需基因功能分析的方式使用RNA干扰作为基因沉默机制。展示了构建RNA干扰诱导的反向重复构建体的两种替代策略，并提出了第三种替代方案。此外，还详细描述了通过电穿孔对分生孢子进行DNA介导的转化、从真菌菌丝体中提取RNA以及Northern印迹分析。所呈现的实验结果表明，RNA干扰可作为粗糙脉孢菌中的一种基因沉默工具，用于非必需基因（al-2）和必需基因（cot-1），从而产生一系列稳定的、部分沉默的突变体，表现出不同的表型。
