Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Tasly Pharmaceutical Group Co., Ltd, Tianjin 300410, China.
Int J Biol Macromol. 2022 Jul 1;212:303-313. doi: 10.1016/j.ijbiomac.2022.05.134. Epub 2022 May 21.
Panax notoginseng saponins (PNS) are one of the main active ingredients of Panax notoginseng, a representative plant of the genus Panax. However, the detailed regulation mechanism of PNS biosynthesis remains elusive. Therefore, a sequence of upstream promoters of PnSS and PnSE were cloned and analyzed firstly. GUS quantitative results showed that the upstream promoters could specifically and significantly respond to exogenous GA and ABA signals. To further identify the binding proteins that respond to peripheral hormones, PnCOX11 and PnDCD were screened and identified from the P. notoginseng cDNA library. The Y1H experiment verified the interaction between the above two binding proteins and the promoters. Several online software was used to analyze the domains, secondary structures, three-dimensional structures, and phylogenetic trees of the two binding proteins. Subcellular localization analysis exhibited that PnCOX11 was mainly located in the chloroplast, while PnDCD was located in the cytoplasm and nucleus. Prokaryotic expression demonstrated that the recombinant proteins had a high concentration under the induction of IPTG. This study can provide a fundamental date for the subsequent thorough investigation of the transcription regulatory mechanism of PNS biosynthesis.
三七总皂苷（PNS）是三七的主要活性成分之一，三七是人参属的代表性植物。然而，PNS 生物合成的详细调控机制仍不清楚。因此，本研究首先克隆并分析了 PnSS 和 PnSE 的上游启动子序列。GUS 定量结果表明，上游启动子可以特异性且显著响应外源 GA 和 ABA 信号。为了进一步鉴定响应外周激素的结合蛋白，从三七 cDNA 文库中筛选并鉴定了 PnCOX11 和 PnDCD。Y1H 实验验证了这两个结合蛋白与启动子之间的相互作用。使用几种在线软件分析了这两个结合蛋白的结构域、二级结构、三维结构和系统进化树。亚细胞定位分析表明，PnCOX11 主要定位于叶绿体，而 PnDCD 则定位于细胞质和细胞核。原核表达表明，在 IPTG 的诱导下，重组蛋白的浓度很高。本研究可为后续深入研究 PNS 生物合成的转录调控机制提供基础数据。
