人参皂苷含量与皂苷生物合成关键酶活力及基因表达量相关性研究

发布时间：2018-09-03 06:20

【摘要】：人参作为中国的道地药材,其主要有效成分为人参皂苷。目前,对于人参皂苷合成途径的研究越来越深入,而人参皂苷合成途径的关键酶的活力和关键酶的基因表达量与皂苷含量的相关性尚未见报道。深入研究这种关系,对人参的规范使用及鉴定、产地溯源以及利用生物工程手段提高人参皂苷含量具有重要的理论和实际应用价值。本研究选择人参皂苷主要合成途径中催化达玛烷二醇型人参皂苷合成途径的7个关键酶进行研究,研究了不同年份间以及不同地区间关键酶的基因相对表达量、酶活力与皂苷含量的相关性,分析出与人参生长时间和人参生长地域相关的酶。本研究中,以吉林省集安市(2-6年)、靖宇(5年)、抚松(5年)、汪清(5年)地区新鲜采摘的人参样本为研究对象,以人参发根为研究部位,分别利用分光光度法和高效液相色谱法对人参总皂苷和单体皂苷的含量进行测定,利用实时荧光定量PCR法对人参皂苷合成的关键酶3-羟基-3-甲基戊二酰CoA还原酶、法尼基焦磷酸合成酶、鲨烯合酶、鲨烯环氧酶、达玛烯二醇合酶、细胞色素P450酶、糖基转移酶的基因表达量进行测定,利用比色法、酶联免疫吸附测定法、高效液相色谱法、液相色谱-质谱联用、气相色谱-质谱联用等方法分别对7个关键酶的活力进行了测定,获得的实验结果采用SPSS软件结合R语言进行相关性分析。研究结果显示:不同年份中人参单体皂苷与总皂苷具有显著的相关性,人参单体皂苷Re的含量在不同地区的溯源的上具有重要意义,达玛烷二醇合酶和糖基转移酶的酶活力在人参的不同年份鉴别上具有重要意义,3-羟基-3-甲基戊二酰CoA还原酶以及达玛烷二醇合酶的基因表达量在人参的地域溯源方面将具有重要的参考价值。本论文的研究为人参皂苷生物合成途径的深入研究提供了生物化学及分子生物学的支持,也为人参的年份鉴定及地域溯源指标的建立提供了理论依据。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.51

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