人参药效成分形成与积累的生理生态机制研究

The research of formation theory and control technology on the efficacious components in Chinese medicinal materials was an important science problem in the Chinese medicine science and its interrelated cross subjects. It was an important base controlling medicinal materials quality to realize syllabify the laws of formation and accumulation of efficacious components and their ecological, physiological and molecule biological mechanism, at present, also was a bottleneck controlling medicinal materials quality. Therefore, to develop the basic theory research of the laws of formation, accumulation and change on the medicinal materials quality from the visual angle of many subjects, and to drive the technique innovation in the medicinal materials quality control through understanding laws and clarifying mechanism, and to achieve the aim of "safety, efficiency, stabilization and controllability" in the medicinal materials quality were an important study mission in the longer time. In the project, we would study the relativity of ginsenoside content with key enzyme gene expression difference and with ecological factors through determining total and individual ginsenoside content and key enzyme gene expression in the different organs and parts of Panax ginseng under the different environments and observing microcosmic climate, light and soil factors at the sample plot as well as their macrocosmic observation data at the local observatory. The dominant ecological factors influencing ginsenoside formation and accumulation as well as physiological regulation mechanism would be understood through the dynamic analysis of ginsenoside content and key enzyme gene expression under the different environments as well as the artificial regulation experiments of dominant ecological factors.

中药质量形成理论与控制技术研究已成为中药学及其交叉学科领域关注的重大科学问题之一。清晰地认识药效成分形成积累规律及其调控机制，是实现药材质量控制的重要基础，也是目前药材质量控制技术研究的瓶颈。为此，从多学科视角，开展药材质量形成、积累、变化规律研究，通过认识规律阐明机制推动药材质量控制技术的创新，实现药材质量"安全、有效、稳定、可控"的目标，是未来较长时期的重要研究任务。本项目拟通过对不同环境条件下，栽培1-5年生人参主要单体皂苷与总皂苷含量不同季节和年际动态测定，以及人参皂苷生物合成途径关键酶基因表达差异分析，并对采样地进行气候、光照和土壤等生态因子同步微观定位观测，结合采样区气象台网宏观观测数据，研究人参皂苷含量与关键酶基因表达量及生态因子的相关性。通过不同生态环境下人参皂苷含量与关键酶基因表达量的动态变化分析，以及生态因子人工调控研究，阐明影响人参皂苷合成的主导因子及其生理调控机制。

以栽培人参为研究对象，采样区覆盖人参道地产区15个县市，116个样地，跨3.4个纬度，5.5个经度，高差1354m。获得人参总皂苷及8种单体皂苷含量、9个关键酶基因表达量，以及土壤pH、电导率、盐浓度及主要土壤元素等数据13000余个。基于GIS地理信息技术，获得116个样地9个气象因子数据。在暖温、温和、温凉和温寒4个气候区进行了定位取样。采用小气候定位观测系统进行了24小时生长季连续观测。从宏观空间尺度和微观时间尺度上，开展了人参皂苷含量、人参皂苷合成关键酶基因表达与气候、土壤、海拔高度等生态因子相关性的大数据分析，提出了人参皂苷合成的生态调控因子，为推进人参药材质量调控技术的创新提供了科学依据。.（1）建立了人参8种皂苷离子液体超声辅助提取技术，提取率是H2O和乙醇的1.42倍和5.57倍。克隆了人参皂苷合成9个关键酶基因，建立了实时荧光定量PCR方法，为人参皂苷及功能基因表达的定量分析奠定了基础。.（2）偏最小二乘法分析气候因子与人参总皂苷和8种单体皂苷相关性研究表明，人参药材质量与温度变化呈明显负相关，与年降水量呈明显的正相关，说明较高的年降水量和较低的温度条件有利用人参皂苷的合成与积累。.（3）多元逐步回归分析土壤因子与人参总皂苷和8种单体皂苷的相关性研究表明，人参药材质量与土壤pH呈显著正相关，土壤酸化显著抑制人参皂苷合成，但土壤Na元素含量的增加不利于人参皂苷合成。.（4）9个关键酶基因在根、茎、叶和果中差异表达，表达高值多数出现在红果期和果后期。DS与Rb2、Rb3、Rc、Rd含量，CYP716A47与Re含量，β-AS和CYP716A52v2与Ro含量，CYP716A53v2与Rg1含量呈极显著正相关。.（5）生态因子与人参皂苷合成关键酶基因表达的相关性研究表明，9个基因均与土壤含水量呈显著或极显著正相关， FPS、β-AS和CYP716A47与相对湿度呈极显著正相关，而DS和CYP716A47与平均温度呈显著负相关，其余7个基因与平均温度呈显著正相关。.（6）水因子是人参皂苷合成的主导生态因子,灌溉有利于提高人参质量；温度因子是重要限制因子，主要通过DS基因调控；DS基因的表达部位是叶，峰值出现在果后期，因此，秋季保持叶功能有利于人参皂苷合成积累。.关键词：人参，药材质量，关键酶基因，生态因子，质量调控