决明StTCP11调控蒽醌类化合物生物合成的分子机制

Molecular mechanism of StTCP11-mediated anthraquinone biosynthesis in Senna tora L.

摘要:

决明(Senna tora L.)种子是蒽醌类化合物生物合成的核心器官，其药用价值高度依赖于蒽醌类化合物的含量。TCP转录因子在植物次级代谢产物生物合成中发挥关键调控作用，但TCP转录因子是否调控蒽醌类化合物的积累还未见报道。因此，本研究通过实时荧光定量PCR (quantitative real-time polymerase chain reaction, qRT-PCR)分析StTCP11在种子不同生长发育阶段的表达模式，构建过表达载体以获得StTCP11过表达转基因决明株系及空载体对照决明株系，探究其对种子中蒽醌类化合物积累的影响；结合酵母单杂交(yeast one-hybrid, Y1H)与双荧光素酶报告基因(dual-luciferase reporter assay, Dual-LUC)实验，解析StTCP11与蒽醌类化合物生物合成关键酶的互作关系。结果表明，StTCP11在种子发育后期表达量较高，StTCP11过表达株系种子中橙黄决明素、大黄酚、大黄素及大黄素甲醚含量较对照显著提高；Y1H与Dual-LUC实验证实StTCP11可直接结合StDXS启动子并激活其转录，进而正向调控蒽醌类化合物的积累。本研究揭示了StTCP11通过靶向调控StDXS转录促进决明种子蒽醌合成的分子机制，为解析决明种子品质调控网络及优质新品种选育提供了关键理论依据。

Abstract:

The seeds of Senna tora L. are the core organs for the biosynthesis of anthraquinones, and their medicinal value is highly dependent on the content of anthraquinones. However, TCP transcription factors play a key regulatory role in the biosynthesis of plant secondary metabolites. Nevertheless, whether TCP transcription factors regulate the accumulation of anthraquinone compounds has not been reported. Therefore, in this study, qRT-PCR analysis was performed to determine the expression pattern of StTCP11 at different growth and development stages of S. tora seeds. An overexpression vector was constructed to obtain two types of S. tora lines: StTCP11-overexpressing lines and empty vector (control) lines, which were used to explore the effect of StTCP11 on the accumulation of anthraquinones in seeds. The interactions between StTCP11 and key enzymes in anthraquinone biosynthesis were analyzed by yeast one-hybrid (Y1H) and dual-luciferase (Dual-LUC) reporter gene assays. The results showed that the expression level of StTCP11 was higher at the late stage of seed development. Compared with the control, the StTCP11-overexpressing lines showed significantly increased content of aurantio-obtusin, chrysophanol, emodin, and physcion in the seeds. Y1H and Dual-LUC assays confirmed that StTCP11 could directly bind to the promoter of StDXS and activate its transcription, thereby positively regulating the accumulation of anthraquinones. This study revealed the molecular mechanism of StTCP11 in promoting anthraquinone synthesis in S. tora seeds through targeted regulation of StDXS transcription, which provided a key theoretical basis for resolving the quality regulation network of S. tora seeds and breeding new high-quality varieties.