广东农业科学2026，Vol.53Issue(5)：49-62,14.DOI:10.16768/j.issn.1004-874X.2026.05.004
肉桂萜类合酶CcTPS26基因克隆与表达模式分析
Cloning and Expression Pattern Analysis of the Terpene Synthase Gene CcTPS26 in Cinnamomum cassia Presl
摘要
Abstract
[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.关键词
肉桂/萜类合酶基因/克隆/生物信息学分析/表达特征Key words
Cinnamomum cassia Presl/terpene synthase gene/cloning/bioinformatics analysis/expression characteristics分类
农业科技引用本文复制引用
陈柳君,祝滢,邱锦霞,明如宏,覃政豪,李良波,黄鼎,姚绍嫦..肉桂萜类合酶CcTPS26基因克隆与表达模式分析[J].广东农业科学,2026,53(5):49-62,14.基金项目
"协同"行动计划(广西区域创新能力提升计划)项目(桂科XT2600870037) （广西区域创新能力提升计划）
中央引导地方科技发展资金资助项目(桂科XT2600870037) （桂科XT2600870037）
广西研究生教育创新计划项目(YCSY2025010) （YCSY2025010）
广西大学生创新训练计划项目(C202408) （C202408）
