不同产区太子参次生代谢物差异机制的iTRAQ定量蛋白质组学研究

Pseudostellariae Radix, a kind of staple traditional Chinese medicine, has the functions of strengthening spleen, replenishing Qi, moistening lung and producing fluids. It has large clinical consumption for its positive effects. However, due to the difference of ecological environment, the variation of its effective components accumulation is big, and its quality is uneven. The key of manufacturing procedure of Pseudostellariae Radix is how to ensure its high and uniform quality. During the quality forming process of Pseudostellariae Radix, the environment ecology factors impact the synthesis and accumulation of its secondary metabolites through a certain way. But mechanism of the differences of the secondary metabolites of Pseudostellariae Radix growing in different ecological environments is unclear, which affects its quality improvement. This project will adopt the technology of plant metabolomics to filter and identify the biomarkers of metabolic differences of Pseudostellariae Radix from different production regions, and apply iTRAQ quantitative proteomics to screen and analyze the differential expression proteins of Pseudostellariae Radix from different regions. The mechanism of the differences of second metabolites in Pseudostellariae Radix will be discussed, through the correlation analysis of the biomarkers of metabolic differences and the differential expression proteins from Pseudostellariae Radix, and looking for the goal differential expression proteins, and combining with its biological function. Our research will provide the basic information for exploring the quality forming process, which will be demonstration and guidance to study effective constituents of Chinese materia medica and its quality formation mechanism.

太子参为大宗中药材,具益气健脾、润肺生津之功能,疗效确切,临床用量大。然而,由于生态环境的不同,造成太子参有效成分的积累变异较大,质量参差不齐。如何在药材生产过程中，保证太子参的优质和质量均一已成为太子参生产过程中的关键问题。在太子参品质形成过程中，环境生态因子可通过一定途径影响太子参次生代谢物的合成与积累，然而不同生态环境太子参次生代谢物的积累差异机制如何尚未阐明，从而影响了太子参药材品质的提升工程。本项目采用植物代谢组学技术筛选和鉴定不同产区太子参次生代谢物差异的生物标记物，应用iTRAQ定量蛋白质组学方法筛选和分析不同产区太子参的差异表达蛋白质，通过对太子参代谢差异标记物与差异蛋白质的关联分析，寻找其目标差异蛋白质，结合其生物学功能，探讨不同产区太子参次生代谢物差异机制。从而为探索太子参药材品质形成过程提供基础资料，对研究中药材有效成分成因及品质形成机制具有示范性和指导。

太子参为大宗常用中药材，前期研究发现，种质资源、生态环境会造成太子参有效成分的积累变异较大，药材质量参差不齐。如何保证太子参的优质和质量均一已成为太子参生产过程中的关键问题。鉴于此，本项目综合评价了不同种质、产地、采收加工太子参药材的质量，系统分析了不同生态型、不同产区太子参化学成分的差异及其机制。基于UFLC- QTRAP-MS/MS结合统计分析考察种质、生境、采收加工对太子参中多元指标成分的影响；基于De novo组装的RNA-Seq高通量测序技术对不同生态型、产地太子参药材进行转录组学分析，根据差异基因表达量结合参与的代谢通路筛选目标差异表达基因；基于iTRAQ技术对不同生态型、产地太子参进行定量蛋白质组学研究，根据差异蛋白表达量结合生物信息学分析筛选目标差异蛋白质；基于1H-NMR、UPLC-Triple TOF-MS/MS结合多元统计分析技术对不同生态型、产地太子参中化学成分的差异性进行分析，找出差异显著的化学成分及其变化规律；用双变量相关分析中Spearman相关系数对野生与栽培太子参、传统产区与种植基地太子参中的RNA-Seq转录组、iTRAQ定量蛋白质组、1H-NMR或UPLC-Triple TOF-MS/MS代谢组进行关联分析，找出相关性显著的目标差异基因、目标差异蛋白质和代谢差异标志物。结果表明：种质、生境及采收加工对太子参药材品质形成均有影响； 野生与栽培太子参中，筛选出12,054个差异表达基因（DEGs），上调DEGs 3017个，下调DEGs 9037个，找到6个目标差异基因；筛选出332个差异蛋白质，上调差异蛋白127个，下调差异蛋白205个，找到71个目标差异蛋白；筛选出37差异化学成分。传统产区与种植基地太子参中，筛选出11,535个DEGs，上调DEGs 5988个，下调DEGs 5547个，找到45个目标差异基因；筛选出140个差异蛋白质，上调差异蛋白54个，下调差异蛋白86个，找到44个目标差异蛋白；筛选出22差异化学成分。分析得到有显著关联的差异化学成分与目标差异蛋白质、目标差异蛋白质与目标差异基因以及差异化学成分与目标差异基因。本项目研究为探索太子参有效成分成因及其药材品质的形成机制及提升工程提供基础资料。