2.1 Strain, media and plasmids
Cryptococcus neoformans B-3501A (serotype D) was used as WT
strains. YPDA (a complete medium containing 1% yeast extract, 2%
peptone 2% glucose and 0.003% adenine sulfate dihydrate, with or
without 2% agar) was for the routine growth of C. neoformans.DOPA medium contained L-dopa (0.2 g/L), L-asparagine (1 g/L), glycine (1
g/L), glutamine (1 g/L), MgSO4 (2.5 g/L),
KH2PO4 (4 g/L), vitamin
B1 (10 mg/L), glucose (1 g/L), with or without agar (20
g/L). For strain selection, G418 (100μg/mL) or hygromycin B (100μg/mL)
was added to the YPDA and the DOPA medium. For the preparation of RNA
extraction, the DOPA medium without L-dopa and glucose was named
laccase-inducing medium. All cultures and plates were incubated at 30℃,
and the liquid cultures needed to continue shaking at 150 rpm/min. The
pSilencer 4.1-CMV neo plasmid was bought from Miao Ling Plasmid
Platform, China. Supplementary Fig. 1 shows the map of pSilencer 4.1-CMV
neo plasmid. The PNK003 vector (Genbank: MW938321) was gifted by
professors Xudong Zhu and Ping Zhang. Supplementary Fig. 3 shows the map
of PNK003 vector, and Supplementary sequence 3 shows the sequencing
result. E. coli DH5α grew on LB medium (containing 0.5% yeast
extract, 1% tryptone, 1% NaCl and 2% agar) with ampicillin to amplify
and extract target vectors in this study. All the reagents were brought
from Sinopharm Chemical Reagent Co., Ltd and Solarbio Science &
Technology Co., Ltd.
Enzymes and primers
BspQI, HindIII-HF and BamHI-HF restriction endonucleases were obtained
from New England Biolabs (Beijing, China). T4 DNA ligase and Taq DNA
polymerases were obtained from Takara (Dalian, China). Yeast
Transformation Kit was obtained from Coolaber (Beijing, China). Ezup
column fungal genomic DNA extraction kits, DNA gel purification kits,
primer synthesis and sequencing were from Sangon Biotech (Shanghai,
China). EndoFree Plasmid Maxi Kit and SYBR qPCR Master Mix were
purchased from Vazyme (Nanjing, China). Evo M-MLV RT Mix Kit with gDNA
Clean for qPCR were purchased from Accurate Biotechnology (Hunan,
China). The primers used in this study are listed in Supplementary Table
1.