Objective In order to further study the functional characterization of CaSE3 in Centella asiatica (L.) Urb., the squalene epoxidase gene CaSE3 was cloned, and its promoter activity and expression were analyzed.

Method Based on our published C. asiatica genome database, the gene and promoter of CaSE3 were isolated and cloned. Bioinformatics analysis, GUS histochemical staining and real-time fluorescence quantitative PCR (qRT-PCR), and its gene expression patterns were explored under different treatments, including methyl jasmonate (MeJA), melatonin (MT), 4℃ and polyethylene glycol (PEG).

Result The open reading frame (ORF) of the CaSE3 gene in C. asiatica is 1, 587 bp in length and encodes a protein of 528 amino acids. The CaSE3 promoter sequence was up to 1, 310 bp, and the binding element prediction showed that it contained multiple eukaryotic promoter core elements, MYB/MYC transcription factor binding elements, and light and hormonal response cis-acting elements. The results of promoter activity detection showed that the CaSE3 promoter demonstrated efficiency in driving the expression of the reporter gene GUS. qRT-PCR analysis showed that the expression level of the CaSE3 gene exhibited a clear trend of an initial increase followed by a decrease under MeJA treatment, while it was only significantly up-regulated at 9 h under low-temperature treatment.

Conclusion This study successfully cloned CaSE3, a key candidate gene involved in saponin biosynthesis in C. asiatica, as well as its promoter sequence. Promoter analysis revealed that the CaSE3 gene may be regulated by a variety of transcription factors and plant hormone signals, while functional validation confirmed that the CaSE3 promoter exhibits transcriptional activity. This study establishes a crucial theoretical foundation for comprehensive analysis of the molecular regulation mechanism of CaSE3 in the biosynthesis of triterpene saponins and its metabolic engineering application.