Dragon’s blood is a traditional medicine in which flavonoids are the main bioactive compounds; however, the underlying formation mechanism of dragon’s blood remains largely poorly understood. Chalcone isomerase (CHI) is the key enzyme in the flavonoid biosynthesis pathway. However, CHI family genes are not well understood in Dracaena cambodiana Pierre ex Gagnep, an important source plant of dragon’s blood. In this study, 11 CHI family genes were identified from D. cambodiana, and they were classified into three types. Evolutionary and transcriptional profiling analysis revealed that DcCHI1 and DcCHI4 might be involved in flavonoid production. Both DcCHI1 and DcCHI4 displayed low expression levels in stem under normal growth conditions and were induced by methyl jasmonate (MeJA), 6-benzyl aminopurine (6-BA, synthetic cytokinin), ultraviolet-B (UV-B), and wounding. The recombinant proteins DcCHI1 and DcCHI4 were expressed in Escherichia coli and purified by His-Bind resin chromatography. Enzyme activity assay indicated that DcCHI1 catalyzed the formation of naringenin from naringenin chalcone, while DcCHI4 lacked this catalytic activity. Overexpression of DcCHI1 or DcCHI4 enhanced the flavonoid production in D. cambodiana and tobacco. These findings implied that DcCHI1 and DcCHI4 play important roles in flavonoid production. Thus, our study will not only contribute to better understand the function and expression regulation of CHI family genes involved in flavonoid production in D. cambodiana but also lay the foundation for developing the effective inducer of dragon’s blood.

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