Ye Li1, Hui Feng1, Lihua Jin1, Xiulan Xin1, Qipeng Yuan2. 1. Department of Biotechnology, Beijing Polytechnic, No. 9, Liang Shuihe First Street, Yi Zhuang Economic & Technological Development Zone, Beijing, 100176, China. 2. State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, No. 15 East Road of North Third Ring, Chao Yang District, Beijing, 100029, China. yuanqp@mail.buct.edu.cn.
Abstract
PURPOSE: There are several studies on the use of RNA interference (RNAi) for gene function analysis in fungi. However, most studies on filamentous fungi are based on in vitro-transcribed or -synthesized small interfering RNA (siRNA), and only a few have reported the use of vector-based RNAi. Here we want to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungi. METHODS: Molecular techniques were employed to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungus Blakeslea trispora. RESULTS: We characterized the mouse U6 promoter and utilized it for the expression of shRNA in B. trispora. Using real-time polymerase chain reaction and western blotting analyses, we confirmed the decrease in the mRNA and protein expression of carRA, respectively, in cells transformed with the mouse U6 promoter-driven shRNA expression vector. This indicated that the shRNA was transcribed from the mouse U6 promoter and correctly processed into siRNA and that the mouse U6 promoter exhibited transcription ability in the filamentous fungi. CONCLUSIONS: The results suggest that the mouse U6 promoter that drives the expression of shRNA vectors may serve as a novel tool for RNAi induction in filamentous fungi.
PURPOSE: There are several studies on the use of RNA interference (RNAi) for gene function analysis in fungi. However, most studies on filamentous fungi are based on in vitro-transcribed or -synthesized small interfering RNA (siRNA), and only a few have reported the use of vector-based RNAi. Here we want to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungi. METHODS: Molecular techniques were employed to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungus Blakeslea trispora. RESULTS: We characterized the mouse U6 promoter and utilized it for the expression of shRNA in B. trispora. Using real-time polymerase chain reaction and western blotting analyses, we confirmed the decrease in the mRNA and protein expression of carRA, respectively, in cells transformed with the mouse U6 promoter-driven shRNA expression vector. This indicated that the shRNA was transcribed from the mouse U6 promoter and correctly processed into siRNA and that the mouse U6 promoter exhibited transcription ability in the filamentous fungi. CONCLUSIONS: The results suggest that the mouse U6 promoter that drives the expression of shRNA vectors may serve as a novel tool for RNAi induction in filamentous fungi.
Authors: Silvia Calo; Francisco E Nicolás; Ana Vila; Santiago Torres-Martínez; Rosa M Ruiz-Vázquez Journal: Mol Microbiol Date: 2011-12-19 Impact factor: 3.501
Authors: Raphaël Duvoisin; Mary A Ayuk; Gabriel Rinaldi; Sutas Suttiprapa; Victoria H Mann; Clarence M Lee; Nicola Harris; Paul J Brindley Journal: Transgenic Res Date: 2011-09-28 Impact factor: 2.788
Authors: Juan P de Haro; Silvia Calo; María Cervantes; Francisco E Nicolás; Santiago Torres-Martínez; Rosa M Ruiz-Vázquez Journal: Eukaryot Cell Date: 2009-08-07