Identification and functional verification of key genes involved in alkaloid biosynthesis in Pinellia ternata

Introduction Pinelliae Rhizoma (PR), the dried tuber of Pinellia ternata (Thunb.) Breit, has been used medicinally in China for centuries. PR contains several active ingredients, including alkaloids, which serve as important indicators of PR quality. However, the complete alkaloid biosynthesis pathway in P. ternata has not yet been elucidated. Methods In this study, LC-MS analysis was used to identify two cultivated varieties of P. ternata with distinct alkaloid profiles. Transcriptome sequencing was performed to obtain unigenes and annotate secondary metabolic pathways. Key enzyme and transcription factor genes involved in alkaloid synthesis were further screened, and functional verification was conducted via gene overexpression in P. ternata callus. Results Transcriptomic profiling yielded 63,106 unigenes, many of which were mapped to diverse secondary metabolic pathways. Based on reference sequences, we predicted the putative alkaloid biosynthetic pathways and identified numerous genes encoding key enzymes and transcription factors. Notably, several genes related to monoterpenoid indole alkaloid and benzylisoquinoline alkaloid pathways, such as MAO , NCS I , NCS II , and TyrAT , exhibited significant differential expression between the two varieties. Furthermore, overexpression of MAO , NCS II , and TyrAT in P. ternata callus significantly altered precursor accumulation and alkaloid metabolism. Discussion Our findings establish a molecular framework for understanding the regulation of alkaloid biosynthesis in P. ternata , providing essential insights for the quality control of PR. The differentially expressed genes (e.g., MAO , NCS II , TyrAT ) and the results of overexpression experiments provide direct evidence for their roles in alkaloid metabolism. This study not only fills the gap in the current understanding of the complete alkaloid biosynthetic pathway in P. ternata but also offers essential theoretical insights and technical support for the quality control of PR in medicinal applications.