Identification of key genes governing diester alkaloid biosynthesis in different tissues of Aconitum kusnezoffii

Introduction: Aconitum kusnezoffii is a valuable medicinal plant in Mongolian medicine, and diester alkaloids including aconitine (AC), hypaconitine (HA), and mesaconitine (MA) are its main active and toxic components. However, the tissue-specific distribution of these alkaloids and the key genes involved in their biosynthesis remain unclear. This study aimed to clarify the content differences of diester alkaloids in different tissues and identify related biosynthetic genes. Methods: The contents of AC, HA, and MA in cortex, phloem, and xylem of Aconitum kusnezoffii were determined by high-performance liquid chromatography (HPLC). Transcriptome sequencing was performed on the three tissues, and functional annotation was carried out using NR, Swiss-Prot, and Pfam databases. Candidate genes involved in alkaloid biosynthesis were screened with E-Value < 1e-10 and Identity ≥ 70%. Multiple sequence alignment and phylogenetic tree construction were performed using MEGA software to analyze the evolutionary relationships of OMT enzymes and BAHD acyltransferases. Results: HPLC results showed that the xylem had the highest content of diester alkaloids. Several candidate enzyme genes related to alkaloid biosynthesis were identified. The expression patterns of OMT and BAHD genes in different tissues were consistent with the trend of alkaloid accumulation. Phylogenetic analysis indicated that these enzymes were evolutionarily conserved. Discussion: This study clarified the tissue-specific accumulation pattern of diester alkaloids in Aconitum kusnezoffii and identified key biosynthetic genes. The findings reveal the evolutionary characteristics of related enzymes and provide a theoretical basis for further exploring the biosynthetic mechanism of diester alkaloids.