2.6　Gene-knockout and retro-complementation

The target genes were deleted by the split-marker homologous recombination knockout method(Liang et al.，2014).The method uses gene-knockout cassettes containing overlapping truncations of selectable marker to integrate homologues in transformation(Liang et al.，2014).In this study，the marker gene was the hygromycin-resistance gene，allowing selection of the deleted mutation on selective media.For gene retro-complementation，the plasmid p KOV21 was used as the vector and G418 was used as the selected marker.Fragments with HMT(histone methyltransferase)or HDAC(histone deacetylase)genes，including the native promoter and terminator，underwent PCR and were cloned into the vector p KOV21 with the In-fusion HD Cloning Kit(Takara).Retro-complementation vectors，containing the retro-complementation fragments，were confirmed by sequencing.Protoplasts were produced by 20g/L Driselase(Sigma-Aldrich，St.Louis，MO，USA)and lywallzyme(Guangdong Institute of Microbiology，Guangzhou，China)digestion for 4～5h at 33℃.Gene-knockout cassettes and retro-complementation vectors were transformed by the polyethylene glycol-mediated protoplast transformation method(Liang et al.，2014).After incubation at 28℃for 3～5d，transformed colonies were transferred to PDA plates with 200μg/mL hygromycin B or 500μg/mL G418 sulfate for the second round of selection.The deletion mutants were verified by genomic DNA extraction and PCR identification with D-F/D-R，1F/2R，and 5F/4R，and the retro-complementation strains were verified by PCR with primers of D-F/D-R.(https://www.daowen.com)