Isolation of sequences flanking the Trichoderma asperellum task1 gene using a single specific primer PCR and their use for gene knockout

Gene recombination has been widely used in bacteria, yeast and other lower organisms for gene function research, but its application in filamentous fungi is uncommon because long homologous sequences are required. In this study, first, the task1 gene encoding a mitogen-activated protein kinase (MAPK) during fungal growth, mycoparasitic interaction, and biocontrol was cloned, and then we devised a novel and efficient PCR-based technique to amplify unknown regions adjacent to known genes using a single, specific primer. The feasibility of this technique was demonstrated by isolating sequences flanking the task1 gene of Trichoderma asperellum. The flanking regions obtained were used to construct a binary vector to knock out T. asperellum task1 gene by a Agrobacterium tumefaciens-mediated transformation method.