Objective: To obtain the key enzyme genes involved in cardiac glycosides biosynthesis pathway

acetyl-CoA C-acetyltransferase (AACT) gene

from leaves of Lepidium apetalum seedlings

and conduct sequence analysis and prokaryotic expression analysis. Method: Based on the transcriptome data of L. apetalum

specific primers were designed to obtain cDNA sequence of LaAACT gene

construct prokaryotic expression vector pET-32a-LaAACT and induce the expression of recombinant LaAACT protein. Result: LaAACT gene had ORF full length of 1 212 bp

which encoded a protein of 403 amino acid residues. Sequence analysis indicated that LaAACT protein located in cytoplasm had no transmembrane domain and signal peptide

and the conserved domains of thiolase super family were detected. Phylogenetic analysis indicated that LaAACT protein had the closest relationship with AACT protein from cruciferous plant. The recombinant LaAACT protein was successfully expressed in E. coli BL21 (DE3) cells and finally

the recombinant LaAACT protein was purified through Ni2+ affinity chromatography. Conclusion: LaAACT gene was isolated from leaves of L. apetalum seedlings for the first time

and the stable prokaryotic expression system of pET-32a-LaAACT was constructed. This study could provide fundamental information for antibody preparation of LaAACT protein and provide basis for functional researches of LaAACT gene in cardiac glycosides biosynthesis pathway.