Construction of pDESTR, a GATEWAY vector for gene disruption in filamentous fungi.
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.
Laboratory of Applied Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Sapporo, 060-8589, Japan.
Ayumi
A
Abe
Evelyn B
EB
Elegado
Teruo
T
Sone
D001203:Ascomycota / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004271:DNA, Fungal / Q000032:analysis; D005656:Fungal Proteins / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000235:genetics*; D009492:Neurospora crassa / Q000235:genetics / Q000648:ultrastructure; D016133:Polymerase Chain Reaction
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; C407465:FR protein, Neurospora crassa; D005656:Fungal Proteins