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 neede
The first two recombinational repair genes of Magnaporthe grisea were cloned. Analysis of the deduced amino acid sequences revealed that Rhm52 and Rhm54 are Saccharomyces cerevisiae RAD52 and RAD54 homologs, respectively. Phenotypic complementation testing of these genes showed their function in recombinational repair. Both genes were in single copies in M. grisea genome. Expression of these genes was induced by methyl methanesulfonate and ultraviolet radiation as known for other homologs of the RAD52 epistasis group. Higher induction of both genes by oxidative stress and heat shock indicated the probability for recombinational repair during the infection cycle of M. grisea.
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