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Construction of pDESTR, a GATEWAY Vector for Gene Disruption in Filamentous Fungi

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Abstract

We have constructed pDESTR, a destination vector of gateway system especially for gene targeting and disruption in filamentous fungi. The vector was constructed by removing the multicloning site of pGEM-T easy vector, and inserting hygromycin phosphotransferase gene construct from pCB1004, and a gateway vector conversion cassette. In order to construct a DNA for gene disruption, only an inverse-polymerase chain reaction (PCR) amplification of the restricted, target sequence is needed. After the amplification with a 5′CACC-tagged primer and an ordinary primer, the DNA fragment will be inserted into pENTR/D-TOPO vector and then transferred into pDESTR through LR-recombination reaction. The resulting vector has the disruption construct, after being digested with the restriction enzyme used for the inverse-PCR. The effectiveness of this vector was assessed in Neurospora crassa. The use of pDESTR will therefore simplify the construction of a targeting vector, where multiple ligation steps are usually needed

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Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (16780029).

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  1. Laboratory of Applied Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Sapporo, 060-8589, Japan

    Ayumi Abe, Evelyn B. Elegado & Teruo Sone

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  1. Ayumi Abe
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  2. Evelyn B. Elegado
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  3. Teruo Sone
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Correspondence to Teruo Sone.

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Abe, A., Elegado, E.B. & Sone, T. Construction of pDESTR, a GATEWAY Vector for Gene Disruption in Filamentous Fungi. Curr Microbiol 52, 210–215 (2006). https://doi.org/10.1007/s00284-005-0238-0

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  • DOI: https://doi.org/10.1007/s00284-005-0238-0

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