Mass spectrometry imaging and single-cell transcriptome reveal metabolic profiles of the postauricular glands of the Bufo gargarizans, and hypothesize Bufotoxin biosynthesis pathways

bufotoxin, which contain alkaloids, small peptides and bufadienolides (BDS), are neurotoxic and cardiotoxic, they are promising medical drugs, but the unclear specifics of bufotoxin in the postauricular glands (PG) and the biosynthetic routes of BDS are unknown. We used MALDI/MS to visualize 1872 components of PG, and established the expression profiles of 9316 cells by single-cell sequencing, which were classified into nine clusters by marker genes. We analyzed the expression differences of key genes for alkaloid, terpene backbone and steroid synthesis, identified optimal and silenced genes involved in key enzymes encoding bufotoxin biosynthesis, and hypothesized the full route of BDS synthesis via by analyzing the cholesterol metabolic pathway. BDS was enriched in EP, FB, and GG2 cells, and genes were expressed mainly in EP and FB cells. Combined with the bulk-RNA transcriptome we suggest that BDS synergistically synthesized by the liver and PG. metabolic mapping of toxin components in the PG of the toad, as demonstrated by our data. The Bufo gargarizans' postauricular glands were separating into single-cell suspensions.