生态因子对人参皂苷含量及其生物合成关键酶基因表达的影响

发布时间：2018-03-05 20:10

本文选题：人参　切入点：药材质量　出处：《吉林农业大学》2016年博士论文　论文类型：学位论文

【摘要】：人参(Panax ginseng C.A.Mey.)为五加科珍贵中药材,应用历史悠久。吉林省长白山区是人参的主产区,人参药材原料及其大量药品和保健品供应全国并出口众多的国家和地区。目前,人参主要药效成分人参皂苷合成与积累规律研究的不足,制约了药材质量调控技术的创新。从生态学视角开展人参皂苷合成及其关键酶基因表达与生态因子相关性研究,对阐明人参皂苷合成的生态调控机制及其主导因子,推动人参药材质量调控技术的科技进步具有重要意义。本文以吉林省栽培人参为研究对象,采样区覆盖人参道地产区15个县市,设127个研究样地,位于北纬40°58′1.2″~44°26′54.6″,东经125°20′43.8″~130°51′24.1″,跨3.4个纬度,5.5个经度;样地海拔高度在96m~1450m之间,高差1354m。研究中共获得人参总皂苷及8种单体皂苷含量、人参皂苷合成途径FPS、DS、β-AS和CYP716A47等4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原始数据13000余个。基于GIS地理信息技术,应用ArcGIS软件的空间插值功能,通过DEM的多元线性回归运算,获得127个样地位点的9个气象因子数据。采用小气候定位观测系统对抚松县老岭五年生人参栽培样地进行24小时全生长季连续观测。在此基础上,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研究,阐释了生态因子对人参药材质量的影响,提出了人参皂苷合成与积累的生态调控策略及相关主导因子,旨在为推进人参药材质量的生态调控提供科学依据。主要研究结果如下:(1)建立了人参Rb1、Rb2、Rb3、Rc、Rd、Rg1、Re、Rf等8种皂苷离子液体超声辅助提取技术,人参皂苷的提取率是H2O和乙醇提取的1.42倍和5.57倍,其最佳工艺条件为0.3mol/L溴化1-丁基-3-甲基咪唑盐离子液体([C3MIM]Br)为溶剂,以固液比(g:mL)1:10溶解人参粉末,超声波辅助提取20min。(2)成功克隆了人参皂苷合成FPS、DS、β-AS和CYP716A47等4个关键酶基因,建立了GAPDH基因实时荧光定量RT-PCR方法,该方法具有灵敏、特异、快速及重复性强等优点,为进行人参功能基因表达的定量分析奠定了基础。(3)吉林省15个主产区人参质量均符合《中国药典》标准,其中Rb1、Re+Rg1分别达到国标的2.10-5.90倍、2.68-5.10倍;人参总皂苷和单体皂苷Rb1、Rc、Rg1、Rf含量随着栽培年限增加明显增加,其中,Rb1、Rg1增幅最大,二者含量最多可增加到1.1796%、1.0510%,说明延长栽培年限将有利于Rb1、Rg1的合成积累;聚类分析结果表明人参品质分区逐渐由片状向带状转化,其带状分区呈现出明显的以长白山为中心距离梯度,原因值得进一步的深入研究。(4)海拔高度对人参总皂苷含量有显著影响,海拔600-1000m范围内栽培人参有利于总皂苷积累。8种单体皂苷与海拔变化的关系比较复杂,其中Rb1和Rc为“双峰型”,但高海拔大于低海拔峰值;而Rg1、Re、Rf、Rb2、Rb3、Rd为“单峰型”,其中,Rg1、Re、Rf、Rb2峰值出现在600-800m区域,而Rb3、Rd的峰值出现在800-1000m区域。海拔高度间单体皂苷含量差异显著性分析表明,Rb1、Rg1、Re的差异显著性均较差,特别是Rb1在300-1000m内无显著差异。(5)采用偏最小二乘法分析气候因子与人参总皂苷和8种单体皂苷的回归系数和VIP权重系数研究结果表明,影响人参皂苷合成积累的主导生态因子依次是年降水量、日照时数、年均温度。人参皂苷含量与年均温度变化呈明显负相关性,而与日照时数和年降水量呈明显的正相关,说明较高的年降水量、较长的日照时数和较低的年均温度条件有利用人参皂苷的合成与积累。(6)采用多元逐步回归分析土壤因子与人参总皂苷和8种单体皂苷的偏相关系数和通径分析决策系数研究结果表明,人参皂苷含量与土壤pH呈显著正相关,说明土壤酸化显著抑制人参皂苷合成;与土壤K、Ca、Mg元素含量呈一定的正相关,并且土壤中较高的Cu、Mn、Zn元素含量对人参单体皂苷合成有一定的促进作用,而Na元素含量的增加不利于人参皂苷合成。但是,土壤因子对人参皂苷含量的影响远不如气候因子明显。土壤生态因子比较复杂,需要通过控制性试验开发更加深入的研究。(7)FPS、DS、β-AS和CYP716A47基因表达研究结果表明,4种基因在人参展叶期、绿果期、红果期和果后根生长期及根、茎、叶和果中均有表达,β-AS在绿果期表达量明显增强,并且一直处于高位,果后根生长期达到相对最高值,其关键表达部位是根,且极显著高于其它部位;DS的表达峰值出现在果后根生长期,且显著高于其它时期,其关键表达部位是叶,且极显著高于其它部位;FPS的表达峰值出现在红果期,且显著高于其它时期,其关键表达部位是根和叶,以根为相对优势;CYP716A47的表达峰值亦出现在红果期,且极显著高于其它时期,其关键表达部位是叶和果,以叶为相对优势。DS的表达极显著增加Rb2、Rb3、Rc、Rd含量,而CYP716A47的表达极显著增加Re含量,二者的表达均显著增加总皂苷含量。(8)生态因子与FPS、DS、β-AS和CYP716A47基因表达量相关性研究结果表明,相对湿度和土壤含水量明显增强FPS、DS、β-AS和CYP716A47的表达,并且人参皂苷合成途径下游的DS、β-AS和CYP716A47的表达显著增加总皂苷的合成与积累,而处于中游的FPS的表达对总皂苷含量没有显著影响,说明水分因子调控人参皂苷合成关键部位可能是下游基因。DS的表达与海拔高度呈显著正相关,最适范围在700m~900m之间。在4个供试基因中,DS对生态因子变化的反映最敏感,特别是与年均温度呈显著负相关,这与宏观尺度气候因子分析结果一致,说明DS很可能是人参皂苷合成的重要生态调控基因。
[Abstract]:Ginseng (Panax ginseng C.A.Mey.) for medicinal ofaraliaceae precious, the application has a long history. Jilin District of Changbai Mountain province is the main producing areas of ginseng, ginseng medicinal raw materials and a large number of drugs and health products supply all over the country and exported to many countries and regions. At present, insufficient research on the synthesis and accumulation of ginseng major components of ginseng saponin, restricted innovative technology of medicinal quality control. From the angle of ecology and ecological factors to carry out the correlation between the expression of ginsenoside synthesis and key enzyme genes, to elucidate the ecological regulation mechanism of ginsenoside synthesis and dominant factor, plays an important role in promoting the quality control technology of ginseng. The progress of science and technology in Jilin province cultivated ginseng as the research object, sampling area ginseng covering real estate areas of 15 counties, 127 research plots, located at 40 degrees north latitude 58 degrees 26 '' 1.2 "~44 54.6", longitude 125 degrees 20 '4 3.8 "~130 degrees 51 '24.1", across 3.4 latitude 5.5, longitude; sample altitude between 96m~1450m, height 1354m. of the total saponins of Panax ginseng and 8 kinds of monomer saponin content of ginseng saponin synthesis route FPS, DS, -AS and CYP716A47 expression of beta 4 key enzyme genes, and soil pH, conductivity, salt concentration and soil elements of raw data of more than 13000. GIS geographic information technology based on spatial interpolation function, the application of ArcGIS software, through multiple linear regression of DEM operations, 9 meteorological factors data of 127 sample points. The position using the microclimate observation system of Fusong County, Laoling location the five year old ginseng cultivation plots for 24 hours of continuous observation of the whole growing season. On this basis, from the macro scale and micro scale, the content of ginsenosides, ginsenoside biosynthesis key enzyme gene expression of soil and climate, altitude. Analysis on the data of ecological factors such as high correlation, explains the effects of ecological factors on the quality of Radix ginseng, the ginsenoside synthesis and accumulation of ecological control strategies and related main factors to promote the quality of Chinese medicinal materials ginseng ecological control to provide a scientific basis. The main results are as follows: (1) the establishment of the ginseng Rb1 Rb2, Rb3, Rc, Rd, Rg1, Re, Rf, and other 8 kinds of technology of extracting saponin from ultrasonic assisted ionic liquid, the extraction rate of ginsenoside H2O and ethanol extraction for 1.42 times and 5.57 times, the optimum conditions for 0.3mol/L bromide 1- butyl -3- methyl imidazole ionic liquid ([C3MIM]Br) as solvent. The solid-liquid ratio (g:mL) 1:10 dissolved ginseng powder, ultrasonic assisted extraction (20min. 2) was successfully cloned. The synthesis of ginsenosides FPS, DS, -AS and CYP716A47 beta 4 key enzyme genes, established GAPDH gene real time fluorescence quantitative RT-PCR method, this method It is sensitive, specific, rapid and repeatable advantages for ginseng functional gene expression lays the foundation for the quantitative analysis. (3) the 15 ginseng producing areas in Jilin province are in line with the quality standard > < China Pharmacopoeia, Rb1, Re+Rg1 respectively, 2.10-5.90 times, 2.68-5.10 times of the national standard; ginsenoside and single Rc Rg1, ginsenoside Rb1, Rf content increased with cultivation time increased significantly, among them, Rb1 Rg1, the largest increase, two were up to 1.1796% and 1.0510%, indicating extension of cultivation years will be conducive to the synthesis and accumulation of Rb1, Rg1; cluster analysis results show that the quality of ginseng gradually from lamellar zonal partition transformation. The zonation shows in Changbai Mountain as the center distance gradient, the reason is worth further research. (4) altitude had significant influence on the total ginsenoside content, elevation in the range of 600-1000m to cultivated ginseng total saponin The relationship between.8 and anthocyanin accumulation saponins monomer elevation change is more complex, including Rb1 and Rc as the "Shuangfeng type", but the high altitude than at the lower peak; while Rg1, Re, Rf, Rb2, Rb3, Rd is "unimodal", among them, Rg1, Re, Rf, Rb2 peak in 600-800m area Rb3, the peak of Rd appeared in the 800-1000m region. Significant differences of monomer saponin content elevation analysis showed that Rb1, Rg1, Re were lower significantly difference, especially Rb1 had no significant difference in 300-1000m. (5) analysis of climatic factors and the total saponins of Panax ginseng and 8 kinds of monoginsenosides by using partial least squares regression coefficient and VIP coefficient results showed that the main ecological factors influencing the synthesis of ginsenosides accumulation in annual precipitation, annual average temperature, sunshine time. There was a negative correlation between the content of ginsenoside and annual temperature change, and sunshine hours and annual precipitation is significantly positive Related, indicating a high annual rainfall, long sunshine hours and low temperature conditions using annual synthesis and accumulation of ginsenosides. (6) the coefficient of partial correlation and multiple stepwise regression analysis of soil factor and total saponins of Panax ginseng and 8 kinds of monoginsenosides the decision coefficient and path analysis results showed that ginsenoside content was significantly positively correlated with soil pH, soil acidification significantly inhibited the synthesis of ginsenosides; and soil K, Ca, were positively correlated with Mg content in soil, and the higher Cu, Mn, Zn elements have a certain role in promoting the ginsenoside synthesis, increased the content of Na is not conducive to the synthesis of ginsenosides. However, effects of soil factors on ginsenoside content than climate factors significantly. Soil ecological factors is more complex, need to study more in-depth development by controlling the test. (7) FPS, DS, -AS The expression of CYP716A47 gene and the results of the study show that 4 genes in ginseng leaf, green fruit stage, fruit stage and fruit growth period after root and root, stem, leaf and fruit were expressed in the expression of beta -AS in the green fruit stage was significantly enhanced, and has been at a high level, as the highest value on root growth of fruit after the key expression site is the root, and was significantly higher than that of other parts of the emergence of DS; the expression peak of root long-term survival in fruit, and significantly higher than the other periods, the key is the expression site of leaves, and was significantly higher than that of other parts of the FPS; the expression peak appeared in the red fruit period, and significantly higher than the other period. The key is the expression site of root and leaf, root of comparative advantage; the expression peak of CYP716A47 also appeared in the red fruit stage, and significantly higher than other period, the key is the expression site of leaf and fruit, the leaves are the relative expression of.DS significantly increased Rb2, Rb3, Rc, Rd and CYP content. The expression of 716A47 significantly increased the content of Re, the expression of the two genes significantly increased the content of total saponins. (8) the ecological factors and FPS, DS, -AS and CYP716A47 beta gene expression correlation research results show that the relative humidity and soil water content significantly increased FPS, DS, -AS and CYP716A47 expression of beta, and ginsenoside synthetic pathways downstream of DS, -AS expression and CYP716A47 were significantly increased the synthesis and accumulation of total saponins, and the expression is in the middle of the FPS had no significant effect on the content of total saponins, suggesting that water factors regulating the synthesis of Ginsenosides of key parts may be positively correlated with the expression of downstream gene of.DS high altitude, the optimum range 700m~900m. In the 4 tested genes, the most sensitive to DS changes of ecological factors, especially has significant negative correlation with annual average temperature, the macro scale and climate factor analysis results, indicating that DS is likely to It is an important ecological regulation gene for the synthesis of ginsenoside.

【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S567.51

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