Tanshinone in Salvia miltiorrhiza root is a diterpene quinone compound.Identification of the CYP450 gene involved in tanshinone synthesis has become a key step in analyzing the molecular mechanism of tanshinone biosynthesis pathway.Methods:SmCYP81C16 was screened from the genome and transcriptome data of S.miltiorrhiza and the full-length cDNA sequence was obtained and cloned by RT-PCR method.Bioinformatics analysis was used to analyze the characteristics of physicochemical properties of the coded protein, to predict the secondary structure, conserved domains, etc.of the protein, to establish phylogenetic tree; real-time quantitative PCR method was used to detect the relative expression specificity of this gene in the tissues/organs of S.miltiorrhiza.The overexpressing transgenic hairy root was obtained by genetic transformation, and the content of tanshinones was analyzed by chemical detection and metabolomics.Real-time quantitative PCR method was used to detect the comparative expression levels of key enzymes in tanshinone biosynthetic pathway in hairy roots.Results:The full-length of SmCYP81C16 was 1 497 bp, encoding 498 amino acid residues.The relative molecular weight of this protein was 55.8 kDa.SmCYP81C16 was highly expressed in the stem and the periderm of S.miltiorrhiza.Through chemical detection and metabolomics analysis, it was found that compared with the control strain, in SmCYP81C16 overexpressing strains, the content of tanshinone compounds increased; the expression level of key enzyme genes of tanshinone synthesis pathway in overexpressing hairy roots increased significantly.Conclusion:SmCYP81C16 has the function of positively regulating tanshinone biosynthesis.This study lays a foundation for improving tanshinone biosynthesis content by using biotechnology.