Establishment of an efficient RNA silencing system in Trichoderma koningii using DsRed as a reporter
Jazyk angličtina Země Spojené státy americké Médium print-electronic
Typ dokumentu časopisecké články, práce podpořená grantem
- MeSH
- fluorescence MeSH
- genový knockdown metody MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- luminescentní proteiny analýza genetika MeSH
- malá interferující RNA genetika MeSH
- mikrobiální genetika metody MeSH
- rekombinace genetická MeSH
- reportérové geny MeSH
- RNA interference * MeSH
- stanovení celkové genové exprese MeSH
- transformace genetická MeSH
- Trichoderma genetika MeSH
- western blotting MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fluorescent protein 583 MeSH Prohlížeč
- luminescentní proteiny MeSH
- malá interferující RNA MeSH
We aimed to establish an efficient RNA interference (RNAi) system in the industrially important filamentous fungus Trichoderma koningii using the DsRed protein as a reporter of the silencing process. To accomplish this, a DsRed expression cassette was transformed into T. koningii, and a recombinant strain that stably expressed DsRed was obtained. Next, a vector-directing expression of a DsRed hairpin RNA was constructed and transformed into the T. koningii recipient strain. Approximately 79 % of transformants displayed a decrease in DsRed fluorescence, and expression of DsRed in some transformants appeared to be fully suppressed. Characterization of randomly selected transformants by genomic DNA PCR analysis, real-time PCR quantification, and western blot confirmed downregulation of gene expression at different levels. The RNA silencing approach described here for T. koningii is effective, and the DsRed reporter gene provides a convenient tool for identification of silenced fungal transformants by their DsRed fluorescence compared to the control strain. The results of this study demonstrate the power of RNAi in T. koningii, which supports the use of this technology for strain development programs and functional genomics studies in industrial fungal strains.
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