Gene localization of the mogrol biosynthesis pathway was observed in strain CEN-PK2-1C. Strain CEN-PK2-1C was transformed by plasmids pY15-SgCDS-GFP, pY15-SgEPH3-GFP, pY15-CYP87D18-GFP-mCherry-SEC12, and pY15-AtCPR1-GFP-mCherry-SEC12, thereby yielding strains LMOR001, LMOR002, LMOR003 and LMOR004. These strains were streaked onto an SD-Leu plate and cultured at 30°C for 2 days. Later, a single colony was picked into a 2 ml SD-Leu medium, cultured at 30°C and 220 rpm for 24 h, and washed twice with 1× phosphate-buffered saline (0.8% NaCl, 0.02% KCl, 0.144% Na2HPO4, and 0.024% KH2PO4, pH 7.4). Subsequently, 2-μL preparations were directly plated on slides. Strain LMOR001 culture was stained with Nile red solution in acetone (1:10, v/v; 1 mg/ml; Solarbio) and incubated for 60 min in the dark at 25°C to further confirm the specific distribution of SgCDS.
Fluorescence imaging was performed on a Nikon C-HGF microscope with a ×100 oil immersion objective. GFP fluorescence (excitation, 490 nm; emission, 530 nm), mCherry fluorescence (excitation, 580 nm; emission, 615 nm), and Nile red fluorescence (excitation, 561 nm; emission, 615 nm) were detected by microscopy. Image analysis was carried out on the Leica TCS SP8 software package and ImageJ (NIH).
Fluorescence imaging was performed on a Nikon C-HGF microscope with a ×100 oil immersion objective. GFP fluorescence (excitation, 490 nm; emission, 530 nm), mCherry fluorescence (excitation, 580 nm; emission, 615 nm), and Nile red fluorescence (excitation, 561 nm; emission, 615 nm) were detected by microscopy. Image analysis was carried out on the Leica TCS SP8 software package and ImageJ (NIH).