Aconitum carmichaelii is a high-value medicinal herb widely used across China,Japan,and other Asian countries.Aconitine-type diterpene alkaloids (DAs) are the characteristic compounds in Aconitum.Although six transcriptomes,based on short-read next generation sequencing technol-ogy,have been reported from the Aconitum species,the terpene synthase (TPS) corresponding to DAs biosynthesis remains unidentified.We apply a combination of Pacbio isoform sequencing and RNA sequencing to provide a comprehensive view of the A.carmichaelii transcriptome.Nineteen TPSs and five alternative splicing isoforms belonging to TPS-b,TPS-c,and TPS-e/f subfamilies were identified.In vitro enzyme reaction analysis functional identified two sesqui-TPSs and twelve diTPSs.Seven of the TPS-c subfamily genes reacted with GGPP to produce the intermediate ent-copalyl diphosphate.Five AcKSLs separately reacted with ent-CPP to produce ent-kaurene,ent-atiserene,and ent-13-epi-sandaracopimaradie:a new diterpene found in Aconitum.AcTPSs gene expression in conjunction DAs content analysis in different tissues validated that ent-CPP is the sole precursor to all DAs biosynthesis,with AcKSL1,AcKSL2s and AcKSL3-1 responsible for C20 atisine and na-pelline type DAs biosynthesis,respectively.These data clarified the molecular basis for the C20-DAs biosynthetic pathway in A.carmichaelii and pave the way for further exploration of C19-DAs biosyn-thesis in the Aconitum species.