Construction of gene deletion mutant and complementary strains

Deletion mutants of candidate regulatory genes and complementary strain CPoxMBF1 were constructed using P. oxalicum parental strains ∆PoxKu70 and ∆PoxMBF1, respectively, by homologous recombination according to previously described methods [9]. Briefly, ∆PoxKu70 protoplasts were prepared using OM solution comprising 1.2 mM MgSO₄·7H₂O, 10 mM NaH₂PO₄, 4 g/L lysozyme, 6 g/L lysing enzymes from Trichoderma harzianum (Sigma-Aldrich, St. Louis, MO, USA), and 6 g/L snailase (pH 5.8). Knockout cassettes for each candidate gene, comprising ~ 2 kb of DNA sequence upstream and downstream of the target gene and a 1.8 kb DNA fragment encoding the G418 resistance gene, were constructed by recombinant PCR. Subsequently, ~ 2 µg of each knockout cassette was mixed with a defined number of ∆PoxKu70 protoplasts on ice and transferred to plates containing OCM medium comprising 1 g/L casein enzymatic hydrolysate, 1 g/L yeast extract, and 10 g/L agar, and incubated at 50 °C for 30 min. PDA medium containing 250 μg/mL hygromycin B and 500 μg/mL G418 was then poured onto the surface of the OCM medium and incubated for 5 days at 28 °C, and transformants were selected and confirmed.

To further verify that only the PoxMBF1 gene was deleted in the ∆PoxMBF1 mutant compared with the parental strain ∆PoxKu70 and that the phenotypic alteration of ∆PoxMBF1 was due to deletion of PoxMBF1 in ∆PoxKu70, a complementary strain was constructed. A complementary cassette was integrated at the site of aspartic protease gene {"type":"entrez-protein","attrs":{"text":"POX05007","term_id":"1340571911","term_text":"POX05007"}}POX05007 in deletion mutant ∆PoxMBF1, and included ~ 2 kb of DNA sequence upstream and downstream of {"type":"entrez-protein","attrs":{"text":"POX05007","term_id":"1340571911","term_text":"POX05007"}}POX05007, a 1.2 kb DNA fragment encoding the bleomycin resistance gene, and a 4.7 kb complete PoxMBF1 gene containing its promoter, coding region, and terminator. The complementary cassette was introduced into ∆PoxMBF1 protoplasts, and bleomycin was used for selection of complementary transformants.