Cloning and its activity analysis of santalene synthase gene (SaSSy) promoter from Santalum album

Objective The primary active component of sandalwood essential oil is santalol, yet the transcriptional regulatory mechanism of key biosynthetic enzyme gene santalene synthase (SaSSy) remains unclear. This study aimed to clone and preliminarily characterize the promoter of SaSSy gene in Santalum album, and to explore its transcriptional regulatory features.

Method The 1 437 bp promoter sequence upstream of ATG start codon of SaSSy gene was cloned from Santalum album using Tail-PCR. Cis-regulatory element analysis was performed to identify functional motifs. Transcription factor binding sites were predicted, and KEGG enrichment analysis was conducted to explore associated regulatory pathways. The transcriptional activity of the promoter was verified via transient expression assays in tobacco leaves. Sandalwood calli were treated with 100 µM methyl jasmonate (MeJA), and SaSSy expression was analyzed by RT-qPCR. GUS activity driven by the promoter was measured to assess MeJA responsiveness. Truncated promoter versions were constructed to identify functional regions.

Result A 1 437 bp promoter sequence of SaSSy was successfully cloned, containing light-, hormone-, and stress-responsive cis-elements. Transcription factor binding site prediction and KEGG analysis revealed enrichment in pathways related to plant hormone signaling, MAPK signaling, plant-pathogen interaction, and circadian rhythm. The promoter demonstrated transcriptional activity in tobacco transient assays. MeJA treatment significantly upregulated SaSSy expression and enhanced promoter-driven GUS activity. A truncated 264 bp core promoter region (PSaSSy-D4) retained full transcriptional activity, comparable to full-length promoter.

Conclusion The present study reveals fundamental characteristics of PSaSSy, confirming that its expression is positively regulated by MeJA, and identifies a core promoter region (−264 bp) containing key cis-acting elements. The MeJA-inducible characteristics of PSaSSy provide experimental evidence for enhancing sandalwood essential oil biosynthesis through exogenous signalling molecules, and offer a candidate target for further elucidating the transcriptional regulatory network of santalol synthesis.