基于代谢组学筛选人参根际土壤中特异分泌物及其功能解析

Root exudates are signal medium for interactions of plant and soil, playing critical roles in the control of rhizosphere ecological processes and plant growth. Panax ginseng C. A. Mey has been used as a kind of traditional medicine and functional food, especially distributed and cultivated in the Changbai Mountain of the Northeast China. It remains to be investigated how the rhizosphere exudates - mediated interactive network runs among Ginseng root - Rhizosphere soil - Microbial populations. To reveal this issue, the different aged ginseng cultivated in Changbai Mountain base was selected to be studied in this project. First of all, through the continuous ultrasound and vacuum extraction technology, the combination of 6 kinds of solvents with different polarity was adopted here. This extracting system was ready to obtain the as many exudates at rhizosphere of ginseng as possible for metabonomics dissection in the next step in virtue of UPLC/Q - TOF - MS and GC/Q - TOF - MS/MS technology. By the metabomics strategies, we attempt to construct differential and global metabolic profiling and screen particular exudates at ginseng rhizosphere with activity. Next the selected metabolites were exposed to histochemical and metagenomics detection to unravel the changes of seed germination and rhizosphere microbial populations of ginseng plants in relation to Panax root exudates. The illustration of potential allelopathic effects of them on Panax plants and their rhizosphere microbial profiles is able to be probe into the mechanisms of ginseng root - rhizosphere interactions mediated by root exudates. The results could also provide instruction for explanation of ginseng continuous cropping obstacle problem.

根系分泌物是植物与土壤进行互作的信号媒介，对于根际微生态及植物生长等均具有重要调控功能。中国东北长白山地区是药用和保健植物人参（Panax ginseng C. A. Mey）主要分布区，具有悠久的人工栽培历史。其根系分泌物介导下的植物-土壤-微生物的相互作用途径与机理目前还不清楚。本项目以长白山地区不同栽培年限的人参为对象，首先通过连续超声及真空抽提技术，使用6种不同极性提取溶剂，全面高效提取根际与根围土壤中根系分泌物，然后借助UPLC/Q-TOF-MS和GC/Q-TOF-MS联用仪对其开展代谢组学检测和分析，构建其代谢组图谱，筛选人参根部土壤存在的特异性分泌物；其次，利用组织化学、Illumina高通量全基因组测序等技术，研究上述分泌物对人参种子萌发进程以及人参根际土壤微生物群落结构的影响，解析特异性分泌物对人参及其土壤潜在的化感功能，同时也为解释人参连作障碍问题提供理论基础。

本文已建立人参和西洋参主根，侧根，叶片，茎和叶柄这五个不同部位的靶向代谢分析，基于UPLC-MS对35种人参活性成分同时进行定性，定量及比较研究。该方法检测时间短，精准度高，具有较高的灵敏度，回收率和重复性，为今后人参药材的分析提供稳定可靠的检测方法。此外，利用非靶向代谢组学GC-MS平台技术应用到人参和西洋参中对其初级代谢产物进行鉴定分析。结合多元统计分析筛选出鉴定物种间以及组织部位间差异代谢物，同时结合皂苷检测结果将数据整合，最终构建出一个可视化两物种代谢物差异代谢网络，为揭示两物种皂苷合成通路提供新的研究思路。研究结果表明，在人参和西洋参主根，侧根，叶片，茎和叶柄中共鉴定出149种初级代谢产物，其中包含糖14个，酸39个，醇类14个，氨基酸29个，未知化合物24个和29个未分类化合物。最终将GC-MS和LC-MS代谢物相结合构建可视化代谢调控网络，用于详细了解初级代谢产物与皂苷合成的相关性及特异性组织分布。此外，针对C代谢和N代谢作为研究基础对代谢物进行分析，结果表明C代谢和N代谢对人参皂苷合成具有重要作用。人参皂苷含量的积累主要取决于主根和侧根的能量代谢，并且独立于地上部光合作用。此外C同化到C积累的高转换率与人参皂苷的积累有着密切的相关性，主要表现在人参主根中和西洋参侧根中。本研究利用高通量测序技术发掘人参功能基因和皂苷合成关键基因，最终共获得130.90Gb Clean Data，各样品Clean Data均达到7.50Gb，组装后共获得61,574条Unigene。对Unigene进行功能注释，共获得28,956条Unigene的注释结果。通过对比主根，侧根，叶片，叶柄和茎的转录组数据，筛选出存在显著差异高表达基因最多的一组，最终筛选出主根和叶片之间共存在4233个差异表达基因，其中2568个基因显著积累在主根中，另1665个基因高表达在叶片中。人参主根和叶片之间的差异基因主要是参与碳代谢的生物合成，氨基酸生物合成和其他生物合成的相互作用，并在其中挥发主要的调节作用。