Jiang Li, Yu Yi-Lin, Jiang Min, Cui Xiu-Ming, Liu Di-Qiu, Ge Feng
Key Laboratory of Panax notoginseng Resources Sustainable Development and Utilization, Faculty of Life Science and Technology, Kunming University of Science and Technology Kunming 650500, China.
Zhongguo Zhong Yao Za Zhi. 2021 Jan;46(1):94-102. doi: 10.19540/j.cnki.cjcmm.20201011.101.
This study cloned the transcription factor gene PnbHLH which held an open reading frame of 966 bp encoding 321 amino acids. This study constructed the overexpression vector of transcription factor PnbHLH of Panax notoginseng. The combination of PnbHLH overexpression and RNAi of the key enzyme gene PnCAS involved in the phytosterol biosynthesis was achieved in P. notoginseng cells, thus exploring the biosynthetic regulation of P. notoginseng saponins(PNS) by the synergistic effect of PnbHLH overexpression and PnCAS RNAi. The results showed that the PnbHLH transcription factor interacted with the promoters of key enzyme genes PnDS, PnSS and PnSE in the biosynthetic pathway of PNS, and then regulated the expression levels of key enzyme genes and affected the biosynthesis of saponins indirectly. Further study indicated that the synergistic effect of PnbHLH overexpression and PnCAS RNAi was a more effective approach to regulate the biosynthesis of saponins. Compared with the wild type and PnCAS RNAi cells of P. notoginseng, the contents of total saponins and monomeric saponins(Rd, Rb_1, Re, Rg_1 and R_1) were increased to some extent in the cell lines of PnbHLH overexpression and PnCAS RNAi. This indicated that the two ways of forward regulation and reverse regulation of saponin biosynthesis showed superposition effect. This study explored a more rational and efficient regulation strategy of PNS biosynthesis based on the advantages of multi-point regulation of transcription factors as well as the down-regulation of by-product synthesis of saponins.
本研究克隆了转录因子基因PnbHLH，其开放阅读框为966 bp，编码321个氨基酸。本研究构建了三七转录因子PnbHLH的过表达载体。在三七细胞中实现了PnbHLH过表达与植物甾醇生物合成关键酶基因PnCAS的RNA干扰相结合，从而通过PnbHLH过表达与PnCAS RNA干扰的协同作用探索三七皂苷（PNS）的生物合成调控。结果表明，PnbHLH转录因子与PNS生物合成途径中关键酶基因PnDS、PnSS和PnSE的启动子相互作用，进而调控关键酶基因的表达水平并间接影响皂苷的生物合成。进一步研究表明，PnbHLH过表达与PnCAS RNA干扰的协同作用是调控皂苷生物合成的更有效方法。与三七野生型和PnCAS RNA干扰细胞相比，PnbHLH过表达与PnCAS RNA干扰细胞系中总皂苷和单体皂苷（Rd、Rb_1、Re、Rg_1和R_1）的含量均有一定程度的增加。这表明皂苷生物合成的正向调控和反向调控两种方式呈现叠加效应。本研究基于转录因子多点调控的优势以及皂苷副产物合成下调，探索了一种更合理、高效的PNS生物合成调控策略。
