本文選題：人參　切入點：藥材質(zhì)量　出處：《吉林農(nóng)業(yè)大學》2016年博士論文　論文類型：學位論文

【摘要】：人參(Panax ginseng C.A.Mey.)為五加科珍貴中藥材,應(yīng)用歷史悠久。吉林省長白山區(qū)是人參的主產(chǎn)區(qū),人參藥材原料及其大量藥品和保健品供應(yīng)全國并出口眾多的國家和地區(qū)。目前,人參主要藥效成分人參皂苷合成與積累規(guī)律研究的不足,制約了藥材質(zhì)量調(diào)控技術(shù)的創(chuàng)新。從生態(tài)學視角開展人參皂苷合成及其關(guān)鍵酶基因表達與生態(tài)因子相關(guān)性研究,對闡明人參皂苷合成的生態(tài)調(diào)控機制及其主導(dǎo)因子,推動人參藥材質(zhì)量調(diào)控技術(shù)的科技進步具有重要意義。本文以吉林省栽培人參為研究對象,采樣區(qū)覆蓋人參道地產(chǎn)區(qū)15個縣市,設(shè)127個研究樣地,位于北緯40°58′1.2″~44°26′54.6″,東經(jīng)125°20′43.8″~130°51′24.1″,跨3.4個緯度,5.5個經(jīng)度;樣地海拔高度在96m~1450m之間,高差1354m。研究中共獲得人參總皂苷及8種單體皂苷含量、人參皂苷合成途徑FPS、DS、β-AS和CYP716A47等4個關(guān)鍵酶基因表達量,以及土壤pH、電導(dǎo)率、鹽濃度及主要土壤元素等原始數(shù)據(jù)13000余個�；贕IS地理信息技術(shù),應(yīng)用ArcGIS軟件的空間插值功能,通過DEM的多元線性回歸運算,獲得127個樣地位點的9個氣象因子數(shù)據(jù)。采用小氣候定位觀測系統(tǒng)對撫松縣老嶺五年生人參栽培樣地進行24小時全生長季連續(xù)觀測。在此基礎(chǔ)上,從宏觀空間尺度和微觀時間尺度上,開展了人參皂苷含量、人參皂苷合成關(guān)鍵酶基因表達與氣候、土壤、海拔高度等生態(tài)因子相關(guān)性的大數(shù)據(jù)分析研究,闡釋了生態(tài)因子對人參藥材質(zhì)量的影響,提出了人參皂苷合成與積累的生態(tài)調(diào)控策略及相關(guān)主導(dǎo)因子,旨在為推進人參藥材質(zhì)量的生態(tài)調(diào)控提供科學依據(jù)。主要研究結(jié)果如下:(1)建立了人參Rb1、Rb2、Rb3、Rc、Rd、Rg1、Re、Rf等8種皂苷離子液體超聲輔助提取技術(shù),人參皂苷的提取率是H2O和乙醇提取的1.42倍和5.57倍,其最佳工藝條件為0.3mol/L溴化1-丁基-3-甲基咪唑鹽離子液體([C3MIM]Br)為溶劑,以固液比(g:mL)1:10溶解人參粉末,超聲波輔助提取20min。(2)成功克隆了人參皂苷合成FPS、DS、β-AS和CYP716A47等4個關(guān)鍵酶基因,建立了GAPDH基因?qū)崟r熒光定量RT-PCR方法,該方法具有靈敏、特異、快速及重復(fù)性強等優(yōu)點,為進行人參功能基因表達的定量分析奠定了基礎(chǔ)。(3)吉林省15個主產(chǎn)區(qū)人參質(zhì)量均符合《中國藥典》標準,其中Rb1、Re+Rg1分別達到國標的2.10-5.90倍、2.68-5.10倍;人參總皂苷和單體皂苷Rb1、Rc、Rg1、Rf含量隨著栽培年限增加明顯增加,其中,Rb1、Rg1增幅最大,二者含量最多可增加到1.1796%、1.0510%,說明延長栽培年限將有利于Rb1、Rg1的合成積累;聚類分析結(jié)果表明人參品質(zhì)分區(qū)逐漸由片狀向帶狀轉(zhuǎn)化,其帶狀分區(qū)呈現(xiàn)出明顯的以長白山為中心距離梯度,原因值得進一步的深入研究。(4)海拔高度對人參總皂苷含量有顯著影響,海拔600-1000m范圍內(nèi)栽培人參有利于總皂苷積累。8種單體皂苷與海拔變化的關(guān)系比較復(fù)雜,其中Rb1和Rc為“雙峰型”,但高海拔大于低海拔峰值;而Rg1、Re、Rf、Rb2、Rb3、Rd為“單峰型”,其中,Rg1、Re、Rf、Rb2峰值出現(xiàn)在600-800m區(qū)域,而Rb3、Rd的峰值出現(xiàn)在800-1000m區(qū)域。海拔高度間單體皂苷含量差異顯著性分析表明,Rb1、Rg1、Re的差異顯著性均較差,特別是Rb1在300-1000m內(nèi)無顯著差異。(5)采用偏最小二乘法分析氣候因子與人參總皂苷和8種單體皂苷的回歸系數(shù)和VIP權(quán)重系數(shù)研究結(jié)果表明,影響人參皂苷合成積累的主導(dǎo)生態(tài)因子依次是年降水量、日照時數(shù)、年均溫度。人參皂苷含量與年均溫度變化呈明顯負相關(guān)性,而與日照時數(shù)和年降水量呈明顯的正相關(guān),說明較高的年降水量、較長的日照時數(shù)和較低的年均溫度條件有利用人參皂苷的合成與積累。(6)采用多元逐步回歸分析土壤因子與人參總皂苷和8種單體皂苷的偏相關(guān)系數(shù)和通徑分析決策系數(shù)研究結(jié)果表明,人參皂苷含量與土壤pH呈顯著正相關(guān),說明土壤酸化顯著抑制人參皂苷合成;與土壤K、Ca、Mg元素含量呈一定的正相關(guān),并且土壤中較高的Cu、Mn、Zn元素含量對人參單體皂苷合成有一定的促進作用,而Na元素含量的增加不利于人參皂苷合成。但是,土壤因子對人參皂苷含量的影響遠不如氣候因子明顯。土壤生態(tài)因子比較復(fù)雜,需要通過控制性試驗開發(fā)更加深入的研究。(7)FPS、DS、β-AS和CYP716A47基因表達研究結(jié)果表明,4種基因在人參展葉期、綠果期、紅果期和果后根生長期及根、莖、葉和果中均有表達,β-AS在綠果期表達量明顯增強,并且一直處于高位,果后根生長期達到相對最高值,其關(guān)鍵表達部位是根,且極顯著高于其它部位;DS的表達峰值出現(xiàn)在果后根生長期,且顯著高于其它時期,其關(guān)鍵表達部位是葉,且極顯著高于其它部位;FPS的表達峰值出現(xiàn)在紅果期,且顯著高于其它時期,其關(guān)鍵表達部位是根和葉,以根為相對優(yōu)勢;CYP716A47的表達峰值亦出現(xiàn)在紅果期,且極顯著高于其它時期,其關(guān)鍵表達部位是葉和果,以葉為相對優(yōu)勢。DS的表達極顯著增加Rb2、Rb3、Rc、Rd含量,而CYP716A47的表達極顯著增加Re含量,二者的表達均顯著增加總皂苷含量。(8)生態(tài)因子與FPS、DS、β-AS和CYP716A47基因表達量相關(guān)性研究結(jié)果表明,相對濕度和土壤含水量明顯增強FPS、DS、β-AS和CYP716A47的表達,并且人參皂苷合成途徑下游的DS、β-AS和CYP716A47的表達顯著增加總皂苷的合成與積累,而處于中游的FPS的表達對總皂苷含量沒有顯著影響,說明水分因子調(diào)控人參皂苷合成關(guān)鍵部位可能是下游基因。DS的表達與海拔高度呈顯著正相關(guān),最適范圍在700m~900m之間。在4個供試基因中,DS對生態(tài)因子變化的反映最敏感,特別是與年均溫度呈顯著負相關(guān),這與宏觀尺度氣候因子分析結(jié)果一致,說明DS很可能是人參皂苷合成的重要生態(tài)調(diào)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.

【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S567.51

【參考文獻】

相關(guān)期刊論文 前10條

1 徐茂軍;;藥用植物細胞次生代謝產(chǎn)物合成信號轉(zhuǎn)導(dǎo)機制研究進展[J];細胞生物學雜志;2009年05期

2 肖小河;陳士林;黃璐琦;肖培根;;中國道地藥材研究20年概論[J];中國中藥雜志;2009年05期

3 黎陽;張鐵軍;劉素香;陳常青;;人參化學成分和藥理研究進展[J];中草藥;2009年01期

4 關(guān)萍;馬丹煒;王貴俠;石建明;陳放;;利用ISSR標記對天麻的貴州種群遺傳多樣性分析[J];北京林業(yè)大學學報;2007年06期

5 楊生超;趙昶靈;文國松;蕭鳳回;;植物藥材道地性的分子機制研究與應(yīng)用[J];中草藥;2007年11期

6 徐茂軍;董菊芳;;一氧化氮通過依賴和不依賴活性氧的信號途徑介導(dǎo)桔青霉細胞壁誘導(dǎo)子促進紅豆杉懸浮細胞中紫杉醇生物合成[J];科學通報;2006年14期

7 徐茂軍;董菊芳;朱睦元;;NO通過水楊酸(SA)或者茉莉酸(JA)信號途徑介導(dǎo)真菌誘導(dǎo)子對粉葛懸浮細胞中葛根素生物合成的促進作用[J];中國科學C輯:生命科學;2006年01期

8 王洋,戴紹軍,閻秀峰;光強對喜樹幼苗葉片次生代謝產(chǎn)物喜樹堿的影響[J];生態(tài)學報;2004年06期

9 閻秀峰,王洋,尚辛亥,郭盛磊,于濤;光強和光質(zhì)對野外栽培高山紅景天生物量和紅景天甙含量的影響[J];生態(tài)學報;2004年04期

10 劉菊福,盧長安,廖福龍,劉岱,崔淑蓮,楊立新,馮學鋒,楊京玉,胡世林;不同產(chǎn)地黃芩提取物主要藥效作用的比較[J];中國中醫(yī)藥信息雜志;2001年03期

，

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