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广藿香酮是广藿香(Pogostemon cablin)的重要活性成分，广藿香酰基激活酶2(Pogostemon cablin acyl-activating enzyme 2, PcAAE2)作为参与广藿香酮生物合成途径上的关键酶，对广藿香酮的合成具有重要意义。本研究设计特异引物对PcAAE2基因启动子区域进行克隆，获得1 912 bp启动子DNA序列，命名为PcAAE2Pro,并进行顺式作用元件分析。将截短的启动子片段PcAAE2P1(位于PcAAE2Pro的-1 138～-261 bp)连接到诱饵质粒载体中，转化得到诱饵酵母菌株，再对其进行自激活检测，结果表明100 ng/mL的金担子素A(aureobasidin A, AbA)可以抑制诱饵菌株的自激活。利用广藿香cDNA酵母文库，筛选与PcAAE2P1结合的转录因子，并通过酵母回转验证确定PcNAC048、 PcCOL4和PcbZIP13共3个转录因子与PcAAE2P1具有结合互作关系。进一步通过双荧光素酶分析实验揭示了转录因子PcNAC048、 PcCOL4抑制PcAAE2的转录表达，而PcbZIP13促进PcAAE2的转录作用。本研究为深入挖掘PcAAE2基因的转录调控网络及广藿香酮的生物合成途径奠定了基础。
Abstract：Pogostone is an important active ingredient in Pogostemon cablin. Pogostemon cablin acyl-activating enzyme 2(PcAAE2) as a key enzyme involved in pogostone biosynthesis, plays an important role in pogostone synthesis. In this study, specific primers were designed to clone the promoter region of PcAAE2, and a promoter of 1 912 bp was obtained, named PcAAE2Pro, and cis-acting elements were analyzed. The truncated promoter PcAAE2P1(-1 138～-261 bp in PcAAE2Pro) was connected to the bait vector. The bait yeast strain was obtained and self-activation experiment was completed. The bait vector′s self-activation was completely inhibited when treated with 100 ng/mL aureobasidin A(AbA), as indicated by the experimental results. Using the cDNA yeast library of the Pogostemon cablin, the transcription factors interacting with the PcAAE2P1 were screened, and the interaction between PcNAC048, PcCOL4, and PcbZIP13 with the PcAAE2P1 was confirmed through validation experiments. Furthermore, dual luciferase reporter gene assay revealed that PcNAC048 and PcCOL4 could inhibit the transcription of PcAAE2, while PcbZIP13 could promote the transcription of PcAAE2. This research establishes a foundational framework for further investigation into the transcriptional regulatory network of the PcAAE2 gene and the biosynthetic pathways of pogostone.
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基本信息:
DOI：10.13417/j.gab.044.000537
中图分类号:S567.239
引用信息:
[1]张学苑,邱铠滢,柳凤凤等.广藿香酮生物合成关键基因PcAAE2的启动子克隆及与其结合的转录因子筛选研究[J].基因组学与应用生物学,2025,44(06):537-546.DOI:10.13417/j.gab.044.000537.
基金信息:
广东省自然科学基金项目(2023A1515030194); 广东省现代农业产业技术体系创新团队(南药)项目(2024CXTD24); 广东省乡村振兴战略专项省级项目(2023-NBH-00-022,2024-NPY-00-041)共同资助