目的 克隆剑叶龙血树Dracaena cochinchinensis中3个查耳酮合酶（chalcone synthase，CHS）基因及其启动子，并解析其序列特征与胁迫表达模式，为黄酮类生物合成调控研究提供基因资源。方法 基于转录组数据克隆DcoCHS1、DcoCHS2和DcoCHS3 3个基因的编码区并开展生物信息学分析；采用实时荧光定量逆转录聚合酶链式反应（Real-time quantitative reverse transcription polymerase chain reaction，RT-qPCR）检测机械胁迫下木质部中的表达动态；构建原核表达载体，利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis，SDS-PAGE）验证重组蛋白表达；通过染色体步移技术获取启动子并预测顺式元件。结果 3个基因编码区长度分别为1 179、1 188和1 173 bp，均含CHS保守结构域及8个保守基序，预测蛋白定位于细胞质。系统进化显示DcoCHS2与DcoCHS3 亲缘较近，DcoCHS1独立成支。机械胁迫处理后3个基因均随处理时间呈上调表达模式，重组蛋白均可在大肠杆菌中成功表达。获得启动子序列长度为1 096～1 239 bp，顺式元件组成存在显著差异：DcoCHS1启动子富含脱落酸响应元件，DcoCHS3启动子含水杨酸和茉莉酸甲酯响应元件，DcoCHS2启动子则以通用型基础元件为主。结论 3个DcoCHS基因在进化关系、启动子调控特征及胁迫响应模式上呈现明显功能分化，提示其在龙血竭黄酮合成过程中发挥不同作用，可为后续解析龙血竭合成调控网络和代谢工程改良提供基因资源。

Objective To clone three chalcone synthase (CHS) genes and their promoters from Dracaena cochinchinensis, characterize their sequence features and stress-responsive expression patterns, and provide genetic resources for studying the regulation of flavonoid biosynthesis. Methods The coding sequences of DcoCHS1, DcoCHS2, and DcoCHS3 were cloned based on transcriptomic data and subjected to bioinformatics analysis. Real-time quantitative reverse transcription PCR (RT-qPCR) was used to examine their expression dynamics in xylem under mechanical stress. Prokaryotic expression vectors were constructed, and recombinant protein expression was verified via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Promoters were isolated using genome walking and analyzed for cis-acting regulatory elements. Results The coding regions of the three genes were 1 179, 1 188, and 1 173 bp in length, respectively. All deduced proteins contained the conserved CHS domain and eight conserved motifs, and were predicted to localize in the cytoplasm. Phylogenetic analysis revealed that DcoCHS2 and DcoCHS3 clustered together, whereas DcoCHS1 formed a separate clade. All three genes were upregulated over time in response to mechanical stress, and the recombinant proteins were successfully expressed in Escherichia coli. The promoter lengths ranged from 1 096 to 1 239 bp, with distinct cis-element compositions: the DcoCHS1 promoter was enriched in abscisic acid-responsive elements, the DcoCHS3 promoter contained salicylic acid- and methyl jasmonate-responsive elements, and the DcoCHS2 promoter was predominantly composed of general basal elements. Conclusion The three DcoCHS genes exhibited marked functional divergence in phylogenetic relationships, promoter regulatory logic, and stress-responsive patterns, suggesting that they play differentiated roles in flavonoid biosynthesis in dragon's blood. These findings provide candidate genetic resources for further dissection of the regulatory network and for metabolic engineering applications.

R282.12

云南省“兴滇英才支持计划”青年人才专项；云南省职工创新项目补助资金项目：龙血竭可持续环剥诱导技术研究；云南省高层次科技人才及创新团队选拔专项：云南省傣药南药资源保护与利用创新团队（202405AS350020）