Transcriptomic Analyses of Akebiae Fructus and Identification of Genes Related to Triterpenoid Saponin Biosynthesis

Akebiae Fructus (AF) is the dry and near-ripe fruit of Akebia trifoliata (Thunb.) Koidz. Triterpenoid saponins are important characteristic components of AF, but their molecular biosynthetic pathway has not yet been identified. In this study, DNBSEQ was used to sequence the transcriptome of the five stages of AF from May to September. A total of 96.87 Gb of data were assembled, including 115,430 unigenes with an average sequence length of 1445 bp. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses for metabolic pathway enrichment identified 434 unigenes involved in two metabolic pathways related to triterpenoid saponin biosynthesis, of which 255 encoded 19 key enzymes in the triterpenoid saponin biosynthetic pathway. Sequence analyses and homology modeling of the key enzyme, beta-amyrin synthase (beta-AS), which is implicated in triterpenoid saponin biosynthesis, showed that it has a conserved substrate-binding domain. Differentially expressed genes (DEGs) were identified by comparing gene expression levels between September and other months, and multiple genes encoding key enzymes, such as beta-AS, and the transcription factors (TFs) that are involved in triterpenoid saponin biosynthesis were further analyzed. The chemical constituents of triterpenoid saponins were identified via ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry (UHPLC-QE-MS), and triterpenoid saponin content in AF at different developmental periods was determined by high performance liquid chromatography (HPLC). Finally, the expression levels of some unigenes encoding key enzymes were fully verified using quantitative real-time polymerase chain reaction (qRT-PCR). Herein, we elucidate the biosynthetic pathway of triterpenoid saponins in AF and its key enzymes, laying a foundation for future investigations of AF biosynthesis regulation.