内生菌生物转化提高穿山龙中薯蓣皂苷元含量的作用机制研究

Diosgenin is not only the effective constituent in Dioscorea nipponica, but also the important material for synthesizing more than three hundreds of steroid hormone and contraceptives in China and the world. The market demand of diosgenin is so large that it results in a shortage of the resources. In our previous research, more than one hundreds of endophytic fungi were isolated from the plant of Dioscorea niopponica. In these fungi, the functional strains that are capable of increasing diosgenin content to 70%-90% in the process of biotransformation were screened, and a patent application for the early study has been filed. A further studies on the effective mechanism will be conducted in this proposal. The precursors, related protein and regulating genes in the process of biotransformation will be investigated with the techniques of fingerprint, compound separation and purification, gene differential expression and proteomics, etc. The mechanism of increasing diosgenin content by endophytic fungus biotransformation will be revealed in three levels: chemistry, biochemistry and molecular biology. The research results will not only supply the theroetial fundation for the patent application, but also the technology can be extend to use in the other crude drugs that contain the same precursors as Dioscorea nipponica to develop new resources of diosgenin production on the basis of clarifying the mutual effect between the endophytic fungi and Dioscorea nipponica. Moreover, the study can provide the important information for the investigation of diosponica biosynthetic pathway and regulating genes.

薯蓣皂苷元不仅是穿山龙等药材中的有效成分，而且是目前国内外合成300多种甾体激素和避孕药物的重要原料，市场需求大，资源紧缺。课题组在前期工作中，自穿龙薯蓣植物中分离出100余种内生真菌，并从中筛选出经过发酵可使穿山龙药材中薯蓣皂苷元含量提高达70%-90%的功能菌种，已申报发明专利。本申请拟在此基础上对其作用机制进行深入研究，采用指纹图谱与模式识别、化合物分离纯化、蛋白质组学和基因差异表达等技术，寻找内生菌生物转化薯蓣皂苷元的前体物质、相关功能蛋白和调控基因，从化学、生物化学和分子生物学三个层面揭示内生菌提高药材中薯蓣皂苷元含量的作用机制。其研究结果不仅为该专利的实际应用提供理论依据，而且在阐明内生菌与药材交互作用的基础上，可将这一技术推广应用于含有相同底物成分的其它原料药材，开辟薯蓣皂苷元生产的新资源。同时，本研究还可为薯蓣皂苷类成分生物合成途径和控制基因等方面的探索提供必要的理论信息。

薯蓣皂苷元不仅是穿山龙等药材中的有效成分，而且是目前国内外合成300 多种甾体激素和避孕药物的重要原料，市场需求大，资源紧缺。本研究从穿龙薯蓣植物中分离出100 余种内生真菌，并从中筛选出经过发酵可使穿山龙药材薯蓣皂苷元含量显著提高的功能菌种，建立了内生菌固体发酵和液体发酵提高穿山龙中薯蓣皂苷元含量的方法。在此研究基础上，确定了内生菌生物转化提高穿山龙中薯蓣皂苷元含量的正丁醇极性部位，发现并验证了内生菌生物转化穿山龙薯蓣皂苷元的呋甾皂苷类前体物质，分离并鉴定了内生菌生物转化穿山龙薯蓣皂苷元的前体化合物，从化学层面初步揭示了内生菌提高穿山龙药材薯蓣皂苷元含量的作用机制。其研究结果为阐明内生菌生物转化提高穿山龙中薯蓣皂苷元含量的作用机制及该技术和方法的实际应用具有理论和实际意义。