Eslami Hamid, Khorramizadeh Mohammad Reza, Pourmand Mohammad Reza, Moazeni Maryam, Rezaie Sassan
Dept. of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Invasive Fungi Research Center/ Dept. of Medical Mycology and Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Iran Biomed J. 2014;18(1):55-9. doi: 10.6091/ibj.1217.2013.
Introduction of the RNA interference (RNAi) machinery has guided the researchers to discover the function of essential vital or virulence factor genes in the microorganisms such as fungi. In the filamentous fungus Aspergillus nidulans, the gene sidB plays an essential role in septation, conidiation and vegetative hyphal growth. In the present study, we benefited from the RNAi strategy for down-regulating a vital gene, sidB, in the fungus A. nidulans.
The 21-nucleotide small interfering RNA (siRNA) was designed based on the cDNA sequence of the sidB gene in A. nidulans. Transfection was performed through taking up siRNA from medium by 6 hour-germinated spores. To evaluate the morphologic effects of siRNA on the fungus, germ tube elongation was followed. Moreover, total RNA was extracted and quantitative changes in expression of the sidB gene were analyzed by measuring the cognate sidB mRNA level by use of a quantitative real-time RT-PCR assay.
Compared to untreated-siRNA samples, a significant inhibition in germ tube elongation was observed in the presence of 25 nM of siRNA (42 VS 21 µM). In addition, at the concentration of 25 nM, a considerable decrease in sidB gene expression was revealed.
Usage of RNAi as a kind of post-transcriptional gene silencing methods is a promising approach for designing new antifungal agents and discovering new drug delivery systems.
RNA干扰（RNAi）机制的引入引导研究人员发现了真菌等微生物中必需的重要或毒力因子基因的功能。在丝状真菌构巢曲霉中，sidB基因在隔膜形成、分生孢子形成和营养菌丝生长中起着至关重要的作用。在本研究中，我们利用RNAi策略下调构巢曲霉中一个重要基因sidB。
基于构巢曲霉sidB基因的cDNA序列设计21个核苷酸的小干扰RNA（siRNA）。通过6小时萌发的孢子从培养基中摄取siRNA进行转染。为了评估siRNA对真菌的形态学影响，观察了芽管伸长情况。此外，提取总RNA，并通过定量实时RT-PCR测定同源sidB mRNA水平，分析sidB基因表达的定量变化。
与未处理siRNA的样品相比，在存在25 nM siRNA（42对21 μM）的情况下，观察到芽管伸长受到显著抑制。此外，在25 nM浓度下，sidB基因表达显著下降。
RNAi作为一种转录后基因沉默方法，是设计新型抗真菌药物和发现新的药物递送系统的一种有前景的方法。
