娄彻氏链霉菌 D74菌丝多糖促进丹参毛状根生长和丹参酮积累

Mycelial polysaccharide from Streptomyces rochei D74 promotes growth and tanshinone accumulation of Salvia miltiorrhiza hairy roots

摘要:

目的 以娄彻氏链霉菌( Streptomyces rochei) D74为材料，系统分离纯化其菌丝体多糖( Streptomyces rochei polysaccharide, SRP)，解析其精细结构，并评价纯化多糖组分对丹参毛状根生长及丹参酮生物合成的影响及其作用机制。 方法 采用热水浸提法提取粗多糖，经乙醇沉淀、Sevag法脱蛋白后依次通过DEAE-52阴离子交换柱与Sephadex G-100凝胶柱进行纯化。运用傅里叶变换红外光谱 (Fourier transform infrared spectroscopy, FT-IR)、高效液相色谱-质谱联用(high performance liquid chromatography-mass spectrometry, HPLC-MS)及核磁共振(nuclear magnetic resonance, NMR)等技术对主要活性组分SRP-W-2的理化性质及结构进行系统表征。进一步通过测定丹参毛状根生物量、丹参酮含量及关键合成基因表达水平，评估SRP-W-2对毛状根生长与丹参酮合成的调控作用。 结果 SRP-W-2的得率为2.41%，主要由葡萄糖与半乳糖组成(物质的量比为12.53:1)。结构解析表明，SRP-W-2的主链由→4)-α- d-Glc p-(1→和→4)-α- d-Gal p-(1→单元构成，分支点位于→4,6)-α- d-Glc p-(1→和→4,6)-α- d-Gal p-(1→，分支结构为α- d-Glc p-(1→4)-α- d-Glc p-(1→。生物活性实验显示，SRP-W-2可显著促进丹参毛状根生物量与丹参酮类成分的积累。在50 mg/L SRP-W-2处理15 d后，毛状根干重提升37.52%；隐丹参酮 (cryptotanshinone, CT)、二氢丹参酮I (dihydrotanshinone I, DT-I)、丹参酮I (tanshinone I, T-I)和丹参酮IIA (tanshinone IIA, T-IIA)含量分别提高至对照组的19.0倍、6.4倍、2.8倍和4.8倍。基因表达分析进一步表明，SRP-W-2可上调丹参酮合成途径关键基因 HMGR、 DXS、 DXR和 GGPPS的表达。 结论 娄彻氏链霉菌D74来源的多糖组分SRP-W-2可同步促进丹参毛状根的生长与丹参酮的生物合成，具备作为高效诱导剂的潜力，为丹参资源的开发提供了新策略。

Abstract:

Objective To systematically isolate and purify the polysaccharide from the mycelium of Streptomyces rochei D74 (SRP), elucidate its fine structure, and evaluate the effect of the purified polysaccharide fraction on the growth of Salvia miltiorrhiza hairy roots and the biosynthesis of tanshinones, along with the underlying mechanism. Methods The crude polysaccharide was extracted using hot water, which was followed by ethanol precipitation and deproteinization via the Sevag method. Further purification was performed using DEAE-52 anion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The physicochemical properties and structural features of the main active fraction, SRP-W-2, were systematically characterized by Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography-mass spectrometry (HPLC-MS), and nuclear magnetic resonance (NMR). The effects of SRP-W-2 on hairy root growth and the biosynthesis of tanshinones were assessed by measuring biomass, tanshinone content, and the expression levels of key biosynthetic genes. Results SRP-W-2 was obtained with a yield of 2.41%. It was primarily composed of glucose and galactose at a molar ratio of 12.53:1. Structural analysis revealed that the backbone of SRP-W-2 consisted of →4)-α- d-Glc p-(1→ and →4)-α- d-Gal p-(1→ residues, with branching points at →4,6)-α- d-Glc p-(1→ and →4,6)-α- d-Gal p-(1→. The side chain was identified as α- d-Glc p-(1→4)-α- d-Glc p-(1→. Bioactivity assays demonstrated that SRP-W-2 significantly enhanced both the biomass of S. miltiorrhiza hairy roots and the accumulation of tanshinones. After 15 d of treatment with 50 mg/L SRP-W-2, the dry weight of the hairy roots increased by 37.52%. Meanwhile, the content of cryptotanshinone (CT), dihydrotanshinone I (DT-I), tanshinone I (T-I), and tanshinone IIA (T-IIA) was increased by 19.0-fold, 6.4-fold, 2.8-fold, and 4.8-fold, respectively. Gene expression analysis further indicated that SRP-W-2 up-regulated key genes involved in the tanshinone biosynthetic pathway, including HMGR, DXS, DXR, and GGPPS. Conclusion The polysaccharide fraction SRP-W-2 from S. rochei D74 simultaneously promoted the growth of S. miltiorrhiza hairy roots and the biosynthesis of tanshinones, demonstrating its potential as an effective elicitor. This study provided a new strategy for the utilization and development of S. miltiorrhiza resources.