Bamhi

AOL_s00188g306 of A. oligospora was knocked out using protoplast transformation based on homologous recombination (Si et al., 2022 (link)). The hygromycin-resistant gene (hygR) was selected as the screening marker gene, and knockout plasmids with the hygR screening marker gene were constructed using the pEASY-Uni-seamless Cloning and Assembly kit (Transgen Biotech, Beijing, China).
The genomic DNA of A. oligospora was extracted using a Fungal Genome Extraction Kit (Solarbio, Beijing, China). The upstream and downstream homologous arm sequences of g306 were amplified using primers g306-3F/g306-3R and g306-5F/g306-5R (Supplementary Table S1) with genomic DNA as a template. The hygR fragment was amplified from the pUC57-HygR plasmid using the primers Hph-F/Hph-F (Supplementary Table S1). The pUC19 plasmid was double digested using restriction enzymes XbaI and BamHI (Transgen Biotech, Beijing, China), and the three fragments and linearized pUC19 plasmid were linked using a pEASY-Uni-seamless Cloning and Assembly kit (Transgen Biotech). The ligation product was transformed into E. coli DH5α, and the recombinant plasmid (pUC19-g306KO-hygR) was screened on LB plates containing ampicillin and verified via sequencing.