Division of Molecular Biology, Department of Medical Mycology and Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Mycopathologia. 2012 Sep;174(3):177-85. doi: 10.1007/s11046-012-9539-6. Epub 2012 Apr 7.
The introduction of RNA silencing machinery in fungi has led to the promising application of RNAi methodology to knock down essential vital factor or virulence factor genes in the microorganisms. Efg1p is required for development of a true hyphal growth form which is known to be essential for interactions with human host cells and for the yeast's pathogenesis. In this paper, we describe the development of a system for presenting and studying the RNAi function on the EFG1 gene in C. albicans. The 19-nucleotide siRNA was designed on the basis of the cDNA sequence of the EFG1 gene in C. albicans and transfection was performed by use of a modified-PEG/LiAc method. To investigate EFG1 gene silencing in siRNA-treated cells, the yeasts were grown in human serum; to induce germ tubes a solid medium was used with the serum. Quantitative changes in expression of the EFG1 gene were analyzed by measuring the cognate EFG1 mRNA level by use of a quantitative real-time RT-PCR assay. Compared with the positive control, true hyphae formation was significantly reduced by siRNA at concentrations of 1 μM, 500 nM, and 100 nM (P < 0.05). In addition, siRNA at a concentration of 1 μM was revealed to inhibit expression of the EFG1 gene effectively (P < 0.05). On the basis of the potential of post-transcriptional gene silencing to control the expression of specific genes, these techniques may be regarded as promising means of drug discovery, with applications in biomedicine and functional genomics analysis.
RNA 沉默机制在真菌中的引入，使得 RNAi 方法在敲除微生物中必需的生命因素或毒力因素基因方面具有广阔的应用前景。Efg1p 对于真正的菌丝生长形式的发育是必需的，而这种生长形式对于与人类宿主细胞的相互作用以及酵母的发病机制是必需的。在本文中，我们描述了一种在 C. albicans 中展示和研究 EFG1 基因 RNAi 功能的系统的开发。19 个核苷酸的 siRNA 是基于 C. albicans EFG1 基因的 cDNA 序列设计的，转染是通过改良的 PEG/LiAc 方法进行的。为了研究 siRNA 处理细胞中 EFG1 基因的沉默，酵母在人血清中生长；为了诱导芽管，使用含有血清的固体培养基。通过使用定量实时 RT-PCR 测定来测量同源 EFG1 mRNA 水平，分析 EFG1 基因表达的定量变化。与阳性对照相比，siRNA 在 1 μM、500 nM 和 100 nM 浓度下显著降低了真菌丝的形成（P<0.05）。此外，siRNA 在 1 μM 浓度下被证明能有效地抑制 EFG1 基因的表达（P<0.05）。基于转录后基因沉默控制特定基因表达的潜力，这些技术可能被视为有前途的药物发现手段，在生物医学和功能基因组学分析中有应用。
