【摘要】：人參作為中國(guó)的道地藥材,其主要有效成分為人參皂苷。目前,對(duì)于人參皂苷合成途徑的研究越來越深入,而人參皂苷合成途徑的關(guān)鍵酶的活力和關(guān)鍵酶的基因表達(dá)量與皂苷含量的相關(guān)性尚未見報(bào)道。深入研究這種關(guān)系,對(duì)人參的規(guī)范使用及鑒定、產(chǎn)地溯源以及利用生物工程手段提高人參皂苷含量具有重要的理論和實(shí)際應(yīng)用價(jià)值。本研究選擇人參皂苷主要合成途徑中催化達(dá)瑪烷二醇型人參皂苷合成途徑的7個(gè)關(guān)鍵酶進(jìn)行研究,研究了不同年份間以及不同地區(qū)間關(guān)鍵酶的基因相對(duì)表達(dá)量、酶活力與皂苷含量的相關(guān)性,分析出與人參生長(zhǎng)時(shí)間和人參生長(zhǎng)地域相關(guān)的酶。本研究中,以吉林省集安市(2-6年)、靖宇(5年)、撫松(5年)、汪清(5年)地區(qū)新鮮采摘的人參樣本為研究對(duì)象,以人參發(fā)根為研究部位,分別利用分光光度法和高效液相色譜法對(duì)人參總皂苷和單體皂苷的含量進(jìn)行測(cè)定,利用實(shí)時(shí)熒光定量PCR法對(duì)人參皂苷合成的關(guān)鍵酶3-羥基-3-甲基戊二酰CoA還原酶、法尼基焦磷酸合成酶、鯊烯合酶、鯊烯環(huán)氧酶、達(dá)瑪烯二醇合酶、細(xì)胞色素P450酶、糖基轉(zhuǎn)移酶的基因表達(dá)量進(jìn)行測(cè)定,利用比色法、酶聯(lián)免疫吸附測(cè)定法、高效液相色譜法、液相色譜-質(zhì)譜聯(lián)用、氣相色譜-質(zhì)譜聯(lián)用等方法分別對(duì)7個(gè)關(guān)鍵酶的活力進(jìn)行了測(cè)定,獲得的實(shí)驗(yàn)結(jié)果采用SPSS軟件結(jié)合R語言進(jìn)行相關(guān)性分析。研究結(jié)果顯示:不同年份中人參單體皂苷與總皂苷具有顯著的相關(guān)性,人參單體皂苷Re的含量在不同地區(qū)的溯源的上具有重要意義,達(dá)瑪烷二醇合酶和糖基轉(zhuǎn)移酶的酶活力在人參的不同年份鑒別上具有重要意義,3-羥基-3-甲基戊二酰CoA還原酶以及達(dá)瑪烷二醇合酶的基因表達(dá)量在人參的地域溯源方面將具有重要的參考價(jià)值。本論文的研究為人參皂苷生物合成途徑的深入研究提供了生物化學(xué)及分子生物學(xué)的支持,也為人參的年份鑒定及地域溯源指標(biāo)的建立提供了理論依據(jù)。
[Abstract]:Ginsenoside is the main effective component of ginseng as a Chinese medicinal material. At present, the research on ginsenoside synthesis pathway is more and more in-depth, but the activity of the key enzyme and the gene expression of the key enzyme in the ginsenoside synthesis pathway have not been reported. It is of great theoretical and practical value to study the relationship between ginsenoside and ginsenoside in standard use and identification, to trace the origin of ginseng, and to increase the content of ginsenoside by biological engineering. In this study, seven key enzymes in the main synthesis pathway of ginsenosides were selected to study the relative expression of key enzymes in different years and regions. The relationship between enzyme activity and ginsenoside content was analyzed. In this study, the fresh collected ginseng samples from Ji'an City, Jilin Province (2-6 years), Jingyu (5 years), Fusong (5 years) and Wangqing (5 years) were studied. The contents of ginsenoside and monomer saponins were determined by spectrophotometry and high performance liquid chromatography respectively. The key enzyme of ginsenoside synthesis, 3-hydroxy-3-methyl-glutaryl CoA reductase, was determined by real-time fluorescence quantitative PCR. The gene expression of farinyl pyrophosphate synthase, squalene cyclooxygenase, damarenediol synthase, cytochrome P450 enzyme and glycosyltransferase were determined by colorimetry, enzyme-linked immunosorbent assay (Elisa), high performance liquid chromatography (HPLC). The activities of seven key enzymes were determined by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) respectively. The experimental results were analyzed by SPSS software and R language. The results showed that ginsenoside and total saponins had significant correlation in different years, and the content of ginsenoside Re was of great significance in tracing the source of ginsenoside in different regions. The enzyme activity of Dammaranediol synthase and glycosyltransferase has important significance in the identification of ginseng in different years. The gene expression of 3-hydroxy-3-methyl-glutaryl CoA reductase and Dama diol synthase can be traced to the regional origin of ginseng. Aspect will have important reference value. The research in this paper provides the support of biochemistry and molecular biology for the further study of ginsenoside biosynthesis, and also provides the theoretical basis for the identification of ginseng year and the establishment of regional traceability index.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S567.51

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