目的 为解析黄果枸杞Lycium barbarum var. L. auranticarpum类胡萝卜素转录调控机制，克隆了枸杞LbaPDS基因及其启动子，并进行了表达分析。方法 以宁夏产黄果枸杞为研究材料，克隆LbaPDS基因及其启动子，采用RT-qPCR技术分析LbaPDS基因在根、茎、叶、花、和果实不同发育期，以及高温、低温、干旱、盐等非生物胁迫下的表达模式，通过构建LbaPDS-GFP载体，确定LbaPDS蛋白的亚细胞定位，利用PlantCare数据库预测启动子顺式作用元件，构建该启动子与GUS融合表达载体，并异源转化拟南芥，通过GUS组织化学染色初步确定转基因拟南芥中LbaPDS启动子的表达强度及表达特异性。结果 枸杞LbaPDS基因全长1 749 bp，编码582个氨基酸。LbaPDS基因在果实成熟期高表达，在根和茎中低表达。在高温、低温、干旱以及盐等非生物胁迫下，LbaPDS基因的表达发生显著性变化。LbaPDS蛋白定位在叶绿体中。LbaPDS启动子序列中存在厌氧诱导、激素响应、生长发育响应等多种顺式作用元件。GUS组织化学染色结果表明，LbaPDS启动子在果荚中高表达，在根中低表达。结论 成功克隆了LbaPDS基因及其启动子；LbaPDS基因在果实成熟期高表达，高温、低温、干旱、盐等非生物胁迫显著影响LbaPDS基因的表达，其启动子主要在拟南芥果荚高表达；LbaPDS蛋白定位在叶绿体。为深入探究LbaPDS基因及其启动子的功能及枸杞类胡萝卜素转录调控机制研究提供了研究基础及参考依据。

Objective To analyze the mechanism of transcriptional regulation of carotenoid in Huangguogouqi (Lycium barbarum var. auranticarpum), the LbaPDS gene and its promoter were cloned and functionally analyzed. Methods L. barbarum L. var. auranticarpum from Ningxia was used as the study material. The LbaPDS gene and promoter were cloned, and their expression patterns of LbaPDS gene in roots, stems, leaves, flowers, and fruits, as well as under abiotic stresses such as high temperature, low temperature, drought, and salinity were analyzed by qRT-PCR. The subcellular localization of the LbaPDS protein was determined by constructing LbaPDS-GFP fusion expression vector. The PlantCare database was used to predict the cis-acting elements of the promoter. A fusion expression vector of this promoter and GUS was constructed, and Arabidopsis thaliana was heterologously transformed. The expression intensity and specificity of LbaPDS in transgenic A. thaliana were preliminarily determined by GUS histochemical staining. Results The LbaPDS gene obtained in this study was 1 749 bp in length and encoded 582 amino acids. The LbaPDS gene was highly expressed during the maturation period, and minimally expressed in roots and stems. The expression of LbaPDS gene underwent significant changes under abiotic stresses such as high and low temperatures, drought, and salt stress. The LbaPDS protein was localized in chloroplasts. PlantCare analysis revealed multiple cis-acting elements in the LbaPDS promoter sequence, including anaerobic-inducible, light-responsive, hormone-responsive, and growth and development responsive elements. To further analyze the function of the LbaPDS promoter, a GUS fusion expression vector was constructed. The expression sites were mainly high concentrated in fruit pods by GUS histochemical staining. Expression of this gene was low detected in roots. Conclusion The LbaPDS gene and its promoter have been successfully cloned. The LbaPDS gene has been observed to exhibit elevated levels of expression during the maturation stage of fruit development. Temperature, drought, salt, and other abiotic stresses have been demonstrated to significantly affect the expression of the LbaPDS gene, whose promoter is predominantly expressed in pods. The LbaPDS protein was localized in chloroplasts.This study establishes a foundational framework and serves as a reference point for subsequent investigations into the functional roles of the LbaPDS gene and its promoter, as well as the transcriptional regulatory mechanisms of carotenoids in L. barbarum.

R286.12

宁夏回族自治区重点研发计划项目(2022BBF02008,2025BBF02004);宁夏自然科学基金优秀青年基金项目(2023AAC05049);国家自然科学基金项目(32460098)