• • Full-length transcriptome sequencing yielded 372,483 high-quality transcripts, with 128,646 annotated, enabling comprehensive gene discovery in a non-model medicinal plant where reference genomes are absent; this reduces reliance on fragmented short-read assemblies and supports accurate isoform-level quantification for sterol pathway engineering.
• • Tissue-specific sterol accumulation was significant (P < 0.05), with stems and leaves exhibiting higher sterol content than roots; this spatial partitioning implies that harvest strategies should target aerial tissues for sterol extraction, potentially improving yield per hectare by avoiding root biomass losses.
• • WGCNA identified 43 coexpression modules, and the MEblue module contained six key sterol biosynthetic genes (FPPS, SQS, CAS, GPPS); these rate-limiting enzymes of the MVA pathway are directly linked to cycloartenol precursor supply, offering validated targets for metabolic engineering to enhance sterol titers.
• • RT-qPCR validation of the six key genes showed expression trends consistent with transcriptome TPM values, confirming data reliability; this cross-platform concordance (P < 0.05) establishes a robust pipeline for screening candidate genes in medicinal plants, reducing false discovery rates in downstream functional studies.