Abstract: The main components of the medicinal plant Ilex pubescens Hook. et Arn. are the pentacyclic triterpenoids with various chemical structures. The modification of the triterpenoid skeleton is closely related to a class of key enzymes downstream of the biosynthetic pathway, cytochrome P450 monooxygenase(CYP450). In this study, unigenes with a length of more than 1100 bp were retrieved from the previously obtained Ilex pubescens transcriptome data, and a cytochrome P450 gene(Unigene 0036170)proposed to have the oxidative function of pentacyclic triterpene C-28 was screened through phylogenetic tree analysis. The gene was named as IpAO2. The open reading frame of IpAO2 is 1443 bp, encoding 480 amino acids. RNA was extracted from the tender leaves of Ilex pubescens, and cDNA was obtained by reverse transcription using the RNA as the template. The specific primers AO2-F and AO2-R were designed according to the DNA sequence of IpAO2, and the IpAO2 gene was amplified by PCR using high fidelity enzyme. The IpAO2 gene was ligated into the pEASY cloning vector to obtain pEASY-IpAO2 plasmid. The ligation product was transformed into E. coli Trans1-T1 competent cells and positive clones were screened. The pESC-TRP plasmid was digested with restriction endonucleases to obtain a linearized vector. Primers V-AO2-F and V-AO2-R were designed and the DNA fragment was amplified by PCR using the pEASY-IpAO2 plasmid as template. The DNA fragment was ligated to the vector pESC-TRP to obtain pESC-TRP-IpAO2 recombinant plasmid. The pESC-TRP-IpAO2 plasmid was transformed into a strain of S. cerevisiae capable of efficiently synthesizing amyrin, and the expression of IpAO2 protein was induced with galactose. After extracting the total protein of recombinant S. cerevisiae, Western blot analysis was carried out through the 6×His antigen tag linked to the C-terminus of the target protein. The results showed that the recombinant protein was synthesized in accordance with the expected size. After 7 days of induction and cultivation, the cells of recombinant yeast were collected. The metabolites of cells were extracted and detected by GC-MS after silanization. Through GC-MS analysis of recombinant yeast metabolites, it was found that the protein encoded by the IpAO2 gene can oxidize α-amyrin and β-amyrin to ursolic acid and oleanolic acid, respectively, indicating that IpAO2 is a pentacyclic triterpene C-28 oxidase gene. Meanwhile, the transmembrane domain and protein tertiary structure of the IpAO2 were predicted using the Phyre2 online tool, the results showing that IpAO2 is a transmembrane protein. This study is of great significance to elucidate the biosynthetic mechanism of triterpenoids of Ilex pubescens, and lays a foundation for the further utilization of the metabolic engineering to produce triterpenoids of Ilex pubescens in S. cerevisiae.