Objective The study was aimed to clone the terpenoid synthase gene CcTPS26 from Cinnamomum cassia Presl, characterize its sequence features, conserved domains, phylogeny and spatiotemporal expression, and obtain soluble recombinant protein via prokaryotic expression, so as to lay a solid foundation for elucidating the molecular mechanism of terpenoid biosynthesis and validating enzymatic function in cinnamon.

Method The full-length coding sequence of CcTPS26 was cloned from C. cassia. Bioinformatics tools were employed to analyze the physicochemical properties, conserved domains, subcellular localization, promoter cis-elements, and phylogenetic relationships of the encoded protein. Molecular docking was performed to evaluated the binding affinity of CcTPS26 with FPP and GPP. The prokaryotic expression vector was constructed and transformed into Escherichia coli BL21(DE3), and recombinant protein expression was induced by IPTG. The molecular weight and solubility were determined by SDS-PAGE, and the protein was purified by Ni²⁺ affinity chromatography. Transcript levels of CcTPS26 in different tissues and phloem at different developmental stages were quantified by qRT-PCR.

Result The full length open reading frame (ORF) of CcTPS26 was 1 686 bp, encoding 561 amino acids with a theoretical molecular weight of 64.94 kDa and an isoelectric point (pI) of 5.60. CcTPS26 was predicted as a hydrophilic, acidic and unstable protein without signal peptide or transmembrane domains, and was primarily localized in the cytoplasm. It contained the Terpene cyclase plant C1 domain and the Class Ⅰ IPS conserved motifs DDXXD and NSE/DTE. Phylogenetic analysis revealed that CcTPS26 shared the highest identity (93.76%) with the TPS from C. burmanni. Abundant stress- and hormone-responsive cis-elements were detected in the promoter region, suggesting transcriptional regulation by stress and hormonal signals. Molecular docking confirmed that CcTPS26 could stably bind GPP and FPP through hydrogen bonds and salt bridges. The soluble recombinant protein of approximately 82.1 kDa was successfully expressed and purified, with the highest purity achieved at 275 mmol/L imidazole elution. CcTPS26 exhibited distinct tissue- and development-specific expression: transcripts were significantly more abundant in fruits, leaves, and the phloem than in flowers and xylem, and peaked in the phloem of 5-year-old trees.

Conclusion The terpene synthase gene CcTPS26 was first cloned and characterized from C. cassia. The encoded protein can specifically recognize the key precursors for terpenoid biosynthesis. Its sequence characteristics, spatiotemporal expression patterns, and optimal conditions for recombinant protein purification were determined. CcTPS26 is predominantly expressed in terpenoid-accumulating tissues and presumably plays an essential role in the biosynthesis of terpenoids. This work provides a key candidate gene for terpenoid metabolism regulation and molecular breeding in C. cassia.