The entire cDNA sequence of the candidate gene c26719 was cloned and completely sequenced. The open reading frame of c26719 (GenBank: KX904822) is 1455 bp in length and encodes a protein of 484 amino acids with a molecular weight (MW) of 54,894.74 Da and a theoretical isoelectric point of 8.36. InterproScan analysis revealed that the protein encoded by c26719 belongs to the CYP450 superfamily, Eclass, group I with oxidoreductase activity. Analysis using the ScanProsite tool showed that the protein contains a cytochrome P450 cysteine heme-iron ligand signature (FSGGPRLCPG) at the position 422–431 aa (Fig. S1). The neighbor-joining tree of C-22 hydroxylase showed that the amino acid sequence of c26719 is most closely related to an orthologue from V. californicum (Fig. 3), a plant that also belongs to the Liliaceae and whose major secondary metabolites are steroid alkaloids. The BLAST results showed that the amino acid sequence of c26719 shared 82% identity with VcCYP90B27v1 (AJT59558.1), which prompted us to designate it as PpCYP90B27. Phylogenetic comparisons indicate that PpCYP90B27 is located in the cluster of the CYP90B subfamily, comprising the CYP90Bs that are responsible for the brassinosteroid biosynthesis pathway (Fig. 3), but has no more than 55% homology to other members, with especially different amino acids in the cytochrome P450 cysteine heme-iron ligand signature composition (Fig. S1). This suggests that PpCYP90B27 is involved in steroid C-22 hydroxylation, but the exact metabolic pathway it is specifically involved was uncertain.