Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China.
Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, 310018, China.
J Plant Physiol. 2022 Aug;275:153756. doi: 10.1016/j.jplph.2022.153756. Epub 2022 Jun 24.
The main active ingredients of the traditional Chinese medicinal plant, Panax notoginseng, are the Panax notoginseng saponins (PNS). They can be synthesized via the mevalonate pathway; PnSS and PnSE1 are the key rate-limiting enzymes in this pathway. In this study, an interaction between PnMYB2 and the key enzymes was identified and characterized from the P. notoginseng cDNA library using the Y1H technique. Subsequently, X-α-gal color reaction confirmed the interaction between PnMYB2 and the upstream sequences of PnSS and PnSE1 promoters. Full-length cDNA sequence of PnMYB2 was isolated and characterized. PnMYB2 has an open reading frame of 864 bp, encoding 287 amino acids. 3D structural analysis of PnMYB2 indicated that its structure was similar to that of the template. Phylogenetic analysis revealed that PnMYB2 and PgMYB2 are highly homologous and belong to the R2R3 MYB transcription factor (TF). Subcellular localization analysis showed that PnMYB2 was localized in the nucleus. The recombinant protein PnMYB2 was successfully obtained through prokaryotic expression and was confirmed to be an inclusion body protein. Furthermore, electrophoretic mobility shift assay (EMSA) experiments demonstrated that PnMYB2 specifically binds to MYB core and AC-rich elements. This study provides a theoretical basis for transcriptional regulation of saponin biosynthesis in P. notoginseng.
中药三七的主要活性成分是三七总皂苷（PNS）。它们可以通过甲羟戊酸途径合成；PnSS 和 PnSE1 是该途径中的关键限速酶。本研究采用 Y1H 技术从三七 cDNA 文库中鉴定和表征了与关键酶相互作用的 PnMYB2。随后，X-α-半乳糖颜色反应证实了 PnMYB2 与 PnSS 和 PnSE1 启动子上游序列的相互作用。分离并表征了全长 cDNA 序列的 PnMYB2。PnMYB2 有一个 864 bp 的开放阅读框，编码 287 个氨基酸。PnMYB2 的 3D 结构分析表明其结构与模板相似。系统发育分析表明，PnMYB2 和 PgMYB2 高度同源，属于 R2R3 MYB 转录因子（TF）。亚细胞定位分析表明 PnMYB2 定位于细胞核。通过原核表达成功获得了重组蛋白 PnMYB2，并证实其为包涵体蛋白。此外，电泳迁移率变动分析（EMSA）实验表明 PnMYB2 特异性结合 MYB 核心和 AC 丰富元件。本研究为三七皂苷生物合成的转录调控提供了理论依据。
