人参2,3-氧化角鲨烯代谢途径中关键酶的表达调控研究
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摘要
人参的主要药理活性成分为人参皂苷。研究表明，人参皂苷合成的前体物质为2,3－氧化角鲨烯，其主要代谢流向有两条，一条为合成皂苷类物质代谢途径（达玛烷型皂苷（dammarane-type ginsenosides），齐墩果烷型皂苷（oleanane-typeginsenosides）；另外一条是合成植物甾醇类物质，分别流向羊毛甾醇和环阿乔醇。在已报道的流向人参皂苷和植物甾醇两条支路中，达玛烯二醇合成酶（DS）是控制2,3-氧化角鲨烯流向人参皂苷合成途径的关键酶；环阿乔醇合成酶（CAS）控制2,3-氧化角鲨烯流向植物甾醇合成途径，本研究发现，在2,3-氧化角鲨烯流向植物甾醇合成途径中存在另一个关键酶——羊毛固醇合成酶（LAS），即，羊毛固醇合成酶（LAS）和环阿乔醇合成酶（CAS）共同控制2,3-氧化角鲨烯流向植物甾醇合成途径。由于人参栽培周期长，具有连作障碍，且容易受到病虫害干扰的缺点，本研究建立生长快速的人参发根无菌MS培养体系，并对人参发根提取物进行HPLC定量分析。
本研究通过RNA干扰技术分别调控人参发根中LAS、CAS和DS的表达，对人参皂苷生物合成途径中的关键酶基因LAS、CAS和DS进行功能鉴定，并在分子水平上调控人参皂苷的代谢途径，进而实现提高人参皂苷的含量。研究结果如下：
1.人参中存在2条植物甾醇合成途径，羊毛甾醇合成酶、环阿乔醇合成酶分别催化2，3-氧化角鲨烯产生植物甾醇合成前体（羊毛甾醇和环阿乔醇）。
2.建立适合于本研究人参发根继代培养体系，人参发根根尖的灭菌采用以2.0%NaClO灭菌90s，并以人参发根根尖1.0g/瓶，培养30d所形成的人参发根为本研究的植物试验材料。
3.分别选取LAS、DS和CAS基因的核心区域作为RNA干扰的siRNA片段，采用重组PCR两步法合成LAS、DS和CAS基因的RNAi元件；将RNAi元件与植物转化载体pBI121进行连接，得到含RNAi元件的重组质粒pBI121-LAS-RNAi、pBI121-DS-RNAi和pBI121-CAS-RNAi，并转化至发根农杆菌A4中；通过重组发根农杆菌转染新鲜的4年生人参根片外植体，从而成功诱导出分别含有LAS、DS和CAS基因相应RNAi载体的人参发根系。
4.通过检测人参对照组与LAS基因的RNAi载体转化组发根系中的总皂苷含量、单体皂苷Rg1、Re和Rb1含量、总甾醇的含量、羊毛甾醇的含量、达玛烯二醇的含量，以及环阿乔醇的含量，结果显示，LAS基因的RNAi载体转化组发根系中，LAS基因的表达受到明显的抑制，证明利用RNA干扰使基因沉默的方法切实可行；羊毛甾醇的含量和植物甾醇的含量同时受到显著的抑制，证明人参中存在羊毛甾醇合成酶催化2，3-氧化角鲨烯产生植物甾醇合成前体-羊毛甾醇的代谢流；人参总皂苷及单体皂苷的含量增加，即通过抑制羊毛甾醇合成酶的表达能够促进人参皂苷的合成。
5.通过检测人参对照组与DS基因的RNAi载体转化组发根系中的总皂苷含量、单体皂苷Rg1、Re和Rb1含量、总甾醇的含量、羊毛甾醇的含量、达玛烯二醇的含量，以及环阿乔醇的含量，结果显示，DS基因的RNAi载体转化组发根系中，达玛烯二醇含量、人参总皂苷和单体皂苷含量均受到明显的抑制，即DS基因是控制人参皂苷合成的关键酶基因；羊毛甾醇的含量、环阿乔醇和植物甾醇的含量均有显著提升，表明通过抑制达玛烯二醇合成酶的表达能够抑制人参皂苷的合成，并促进植物甾醇的合成。
6.通过检测人参发根对照组与LAS、DS、CAS基因相应的RNAi载体转化组人参发根系的总皂苷含量、单体皂苷Rg1、Re和Rb1含量、总甾醇的含量、羊毛甾醇的含量、达玛烯二醇的含量，以及环阿乔醇的含量，结果显示CAS基因的RNAi载体转化组发根系中，CAS基因的表达受到明显的抑制，即CAS基因的RNAi成功；人参总皂苷及单体皂苷的含量显著增加，表明通过抑制环阿乔醇合成酶的表达能够促进人参皂苷的合成。
7. RNAi使部分基因沉默，且对人参发根的外观形态不产生影响。
8.本研究分别获得人参皂苷含量提升54.93%的人参发根系和植物甾醇含量提升67.15%的人参发根系。
Ginsenosides are the key pharmacologically active ingredients in ginseng. Previousstudies have suggested that ginsenosides and phytosterols share the same precursor,2,3-oxidosqualene in Panax quinquefolium and Panax ginseng biosynthetic pathway.The main metabolic pathway of ginsenosides is composed of two branches. Onepathway leads to synthesis of ginsenosides contained dammarane-type ginsennosidesand oleanane-type ginsenosides, while the other produces lanosterols and cycloartenols.The enzymes cycloartenol synthase (CAS) and dammarenediol synthase (DS) are thekey enzymes responsible for ginsenoside and phytosterol biosynthesis from2,3-oxidosqualene, respectively. In this research, we found another key enzyme inphytosterol synthesis pathway——lanosterol synthase(LAS), which catalyzed2,3-oxidosqualene together with cycloartenol synthase. Traditional cultivation ofginseng has some inconvenient factors, such as long growth periods, continuousharvesting obstacles, and susceptibility to pests and pathogens. In this research, weestablished rapid growth ginseng hairy roots asepsis system with MS medium, andanalyzed ginseng hairy roots extracts with HPLC quantitatively.
Here, we first regulated and identified LAS、CAS and DS gene in ginseng hairyroots by RNAi technique, then regulated the metabolic pathways of ginsenosides atmolecular level in order to improve its production.
The key contents and conclusions are as follows:
1. There are two phytosterol synthesis pathways in ginseng, leading to synthesisof lanosterol and cycloartenols catalyzed by LAS and CAS respectively.
2. Sub-culture ginseng hairy roots system suited to this study was established. Theroot tips of ginseng hairy roots were sterilized by2.0%NaClO for90seconds, thendistributed into culture flasks (1.0g/flask). After30days cultivation, the experimentalmaterials for this research were obtained.
3. Selecting core regions of LAS、DS and CAS, composed their RNAi elementswith PT-PCR respectively. The recombinant plasmids——pBI121-LAS-RNAi、pBI121-DS-RNAi and pBI121-CAS-RNAi——were constructed by connecting theseelements with plant transformation carrier pBI121respectively. RNAi engineeredbacteria were constructed by transferred RNAi expression vectors to Agrobacteriumrhizogenes A4. Then new ginseng hairy roots were induced by these engineered bacteria respectively.
4. We compared contents of total ginsenoside、ginsenoside (Rg1、Re and Rb1)、total phytosterols、 lanosterols、 cycloartenols and dammarenediols between LASRNAi group and control group. Results of these comparisons indicated that RNAi coulddown-regulated expression of LAS. The results quantity of phytosterol decreased alongwith shrink of lanosterols indicated the existence of lanosterols metabolic pathway inginseng. While increasing production of total ginsenoside and ginsenoside indicated thatsuppression of LAS gene could promote yields of ginsenosides.
5. Contents of total ginsenoside and ginsenoside(Rg1、Re and Rb1)、 totalphytosterols、 lanosterols、 cycloartenols and dammarenediols between DS RNAigroup and control group were also compared. A sharp declined of dammarenediol、totalginsenoside and ginsenoside in DS RNAi group demonstrated that DS was the keyenzyme in biosynthesis of ginsenosides. On the contrary, production of totalphytosterol、 lanosterols and cycloartenols were increased in DS RNAi group. Thisphenomenon indicated that the biosynthesis of ginsenosides and phytosterols competeda common precursor——2,3-oxidosqualene.
6. We also compared contents of total ginsenoside and ginsenoside (Rg1、Re andRb1)、 total phytosterols、 lanosterols、 cycloartenols and dammarenediols betweenCAS RNAi group and control group. Reduction of cycloartenols in CAS RANi groupindicated the successful interference of CAS gene. While increase of total ginsenosideand ginsenoside illustrated that suppression of CAS gene could promote synthesis ofginsenosides.
7. RNA interference could not influence morphology of ginseng hairy roots.
8. By RNAi technique, we established two kinds of new ginseng hairy rootssystems. One possesses a54.93%increase of ginsenosides, while the other possesses a67.15%increase of phytosterols.
本研究通过RNA干扰技术分别调控人参发根中LAS、CAS和DS的表达，对人参皂苷生物合成途径中的关键酶基因LAS、CAS和DS进行功能鉴定，并在分子水平上调控人参皂苷的代谢途径，进而实现提高人参皂苷的含量。研究结果如下：
1.人参中存在2条植物甾醇合成途径，羊毛甾醇合成酶、环阿乔醇合成酶分别催化2，3-氧化角鲨烯产生植物甾醇合成前体（羊毛甾醇和环阿乔醇）。
2.建立适合于本研究人参发根继代培养体系，人参发根根尖的灭菌采用以2.0%NaClO灭菌90s，并以人参发根根尖1.0g/瓶，培养30d所形成的人参发根为本研究的植物试验材料。
3.分别选取LAS、DS和CAS基因的核心区域作为RNA干扰的siRNA片段，采用重组PCR两步法合成LAS、DS和CAS基因的RNAi元件；将RNAi元件与植物转化载体pBI121进行连接，得到含RNAi元件的重组质粒pBI121-LAS-RNAi、pBI121-DS-RNAi和pBI121-CAS-RNAi，并转化至发根农杆菌A4中；通过重组发根农杆菌转染新鲜的4年生人参根片外植体，从而成功诱导出分别含有LAS、DS和CAS基因相应RNAi载体的人参发根系。
4.通过检测人参对照组与LAS基因的RNAi载体转化组发根系中的总皂苷含量、单体皂苷Rg1、Re和Rb1含量、总甾醇的含量、羊毛甾醇的含量、达玛烯二醇的含量，以及环阿乔醇的含量，结果显示，LAS基因的RNAi载体转化组发根系中，LAS基因的表达受到明显的抑制，证明利用RNA干扰使基因沉默的方法切实可行；羊毛甾醇的含量和植物甾醇的含量同时受到显著的抑制，证明人参中存在羊毛甾醇合成酶催化2，3-氧化角鲨烯产生植物甾醇合成前体-羊毛甾醇的代谢流；人参总皂苷及单体皂苷的含量增加，即通过抑制羊毛甾醇合成酶的表达能够促进人参皂苷的合成。
5.通过检测人参对照组与DS基因的RNAi载体转化组发根系中的总皂苷含量、单体皂苷Rg1、Re和Rb1含量、总甾醇的含量、羊毛甾醇的含量、达玛烯二醇的含量，以及环阿乔醇的含量，结果显示，DS基因的RNAi载体转化组发根系中，达玛烯二醇含量、人参总皂苷和单体皂苷含量均受到明显的抑制，即DS基因是控制人参皂苷合成的关键酶基因；羊毛甾醇的含量、环阿乔醇和植物甾醇的含量均有显著提升，表明通过抑制达玛烯二醇合成酶的表达能够抑制人参皂苷的合成，并促进植物甾醇的合成。
6.通过检测人参发根对照组与LAS、DS、CAS基因相应的RNAi载体转化组人参发根系的总皂苷含量、单体皂苷Rg1、Re和Rb1含量、总甾醇的含量、羊毛甾醇的含量、达玛烯二醇的含量，以及环阿乔醇的含量，结果显示CAS基因的RNAi载体转化组发根系中，CAS基因的表达受到明显的抑制，即CAS基因的RNAi成功；人参总皂苷及单体皂苷的含量显著增加，表明通过抑制环阿乔醇合成酶的表达能够促进人参皂苷的合成。
7. RNAi使部分基因沉默，且对人参发根的外观形态不产生影响。
8.本研究分别获得人参皂苷含量提升54.93%的人参发根系和植物甾醇含量提升67.15%的人参发根系。
Ginsenosides are the key pharmacologically active ingredients in ginseng. Previousstudies have suggested that ginsenosides and phytosterols share the same precursor,2,3-oxidosqualene in Panax quinquefolium and Panax ginseng biosynthetic pathway.The main metabolic pathway of ginsenosides is composed of two branches. Onepathway leads to synthesis of ginsenosides contained dammarane-type ginsennosidesand oleanane-type ginsenosides, while the other produces lanosterols and cycloartenols.The enzymes cycloartenol synthase (CAS) and dammarenediol synthase (DS) are thekey enzymes responsible for ginsenoside and phytosterol biosynthesis from2,3-oxidosqualene, respectively. In this research, we found another key enzyme inphytosterol synthesis pathway——lanosterol synthase(LAS), which catalyzed2,3-oxidosqualene together with cycloartenol synthase. Traditional cultivation ofginseng has some inconvenient factors, such as long growth periods, continuousharvesting obstacles, and susceptibility to pests and pathogens. In this research, weestablished rapid growth ginseng hairy roots asepsis system with MS medium, andanalyzed ginseng hairy roots extracts with HPLC quantitatively.
Here, we first regulated and identified LAS、CAS and DS gene in ginseng hairyroots by RNAi technique, then regulated the metabolic pathways of ginsenosides atmolecular level in order to improve its production.
The key contents and conclusions are as follows:
1. There are two phytosterol synthesis pathways in ginseng, leading to synthesisof lanosterol and cycloartenols catalyzed by LAS and CAS respectively.
2. Sub-culture ginseng hairy roots system suited to this study was established. Theroot tips of ginseng hairy roots were sterilized by2.0%NaClO for90seconds, thendistributed into culture flasks (1.0g/flask). After30days cultivation, the experimentalmaterials for this research were obtained.
3. Selecting core regions of LAS、DS and CAS, composed their RNAi elementswith PT-PCR respectively. The recombinant plasmids——pBI121-LAS-RNAi、pBI121-DS-RNAi and pBI121-CAS-RNAi——were constructed by connecting theseelements with plant transformation carrier pBI121respectively. RNAi engineeredbacteria were constructed by transferred RNAi expression vectors to Agrobacteriumrhizogenes A4. Then new ginseng hairy roots were induced by these engineered bacteria respectively.
4. We compared contents of total ginsenoside、ginsenoside (Rg1、Re and Rb1)、total phytosterols、 lanosterols、 cycloartenols and dammarenediols between LASRNAi group and control group. Results of these comparisons indicated that RNAi coulddown-regulated expression of LAS. The results quantity of phytosterol decreased alongwith shrink of lanosterols indicated the existence of lanosterols metabolic pathway inginseng. While increasing production of total ginsenoside and ginsenoside indicated thatsuppression of LAS gene could promote yields of ginsenosides.
5. Contents of total ginsenoside and ginsenoside(Rg1、Re and Rb1)、 totalphytosterols、 lanosterols、 cycloartenols and dammarenediols between DS RNAigroup and control group were also compared. A sharp declined of dammarenediol、totalginsenoside and ginsenoside in DS RNAi group demonstrated that DS was the keyenzyme in biosynthesis of ginsenosides. On the contrary, production of totalphytosterol、 lanosterols and cycloartenols were increased in DS RNAi group. Thisphenomenon indicated that the biosynthesis of ginsenosides and phytosterols competeda common precursor——2,3-oxidosqualene.
6. We also compared contents of total ginsenoside and ginsenoside (Rg1、Re andRb1)、 total phytosterols、 lanosterols、 cycloartenols and dammarenediols betweenCAS RNAi group and control group. Reduction of cycloartenols in CAS RANi groupindicated the successful interference of CAS gene. While increase of total ginsenosideand ginsenoside illustrated that suppression of CAS gene could promote synthesis ofginsenosides.
7. RNA interference could not influence morphology of ginseng hairy roots.
8. By RNAi technique, we established two kinds of new ginseng hairy rootssystems. One possesses a54.93%increase of ginsenosides, while the other possesses a67.15%increase of phytosterols.
引文
1. Vogler B K, Pittler M H, Ernst E.1999. The efficacy of ginseng. A systematicreview of randomised clinical trials[J]. European Journal of ClinicalPharmacology.1999,55:567-575.
2. Shibata S. Chemistry and cancer preventing activities of ginseng saponins and somerelated triterpenoid compounds[J]. Journal of Korean Medical Science.2001,16:S28-S37.
3.郭志廷,王鲁,褚秀玲,等.浅谈人参皂甙Rh2对肿瘤细胞增殖周期和凋亡的影响[J].中兽医杂志.2006,(2):40-4.
4.高峰,潘晓鹏,于丽梅,等.人参皂甙Rh2对环磷酞胺(CY)诱发小鼠白细胞减少的影响[J].人参研究.1995,(4):37-38.
5.高瑞兰,金锦海,牛映平,等.人参总皂甙增加白血病细胞对化疗药物的敏感性[J].中国中西医结合杂志.1999,19(1):17-19.
6.文哗,裴玉萍,陈英杰.人参茎叶皂甙对醋酸泼尼松造成的高脂血症的影响[J].中国中药杂志.1996,21(7):430-432.
7.杨燕,何康,吴铁,等.人参茎叶皂甙对高胆固醇饮食大鼠血脂的影响[J].实验生物学报.1999,32(4):349-352.
8.徐承水.人参降脂作用的实验研究[J].长春中医学院学报.2000,16(3):46.
9.李校坤,林灼锋,黄亚东,等.人参三醇组甙抗辐射作用的研究[J].中药材.2002,25(11):805-808.
10.刘丽波,孙晓玲,张海英,等.人参三醇组甙对小鼠骨髓细胞染色体的辐射防护作用[J].中华放射击医学与防护杂志.2002,22(2):114-115.
11.王红丽,吴铁,吴志华.人参皂甙抗皮肤衰老作用实验研究[J].广东药学院学报.2003,19(1):25-27.
12.张春枝,安利佳,金凤燮.人参皂苷生理活性的研究进展[J].食品与发酵工业.2002,04:70-74.
13. Zhao Z, Cao Y, Yuan S D. Effects of ginseng stem and leave saponin on prolactinand menstrual cycle of experimental hyperprolactinemia rats[J]. Chinese Journal ofApplied Physiology.2005,21(2):144-146.
14.任跃英丛林官秀芝姜喜同.人参资源现状及可持续发展战略[J].人参研究.2003,15(4):9-10.
15. Furuya T, Yoshikawa T, Ishii T, et al. Regulation of saponin production in calluscultures of Panax ginseng[J]. Planta Medica.1983,47(4):200-204.
16. Asaka I, Li I, Hirotani M, Asada Y, Furuya T. Production of ginsenoside saponinsby culturing ginseng (Panax ginseng) embryogenic tissues in bioreactors[J].Biotechnology Letters.1993,15:1259-1264.
17.刘长军,侯嵩生.真菌激发子对人参悬浮培养细胞的生长和人参皂甙生物合成的影响[J].实验生物学报.1999,02:169-174.
18. Thanh N T, Murthy H N, Yu K W, et al. Methyl jasmonate elicitation enhancedsynthesis of ginsenoside by cell suspension cultures of Panax ginseng in5-lballoon type bubble bioreactors[J]. Applied Microbiology and Biotechnology,2005,67(2):197-201.
19. Yoshikawa T, Furuya T. Saponin production by cultures of Panax ginsengtransformed with Agrobacterium rhizogens[J]. Plant Cell Reports.1987,6:449-453.
20. Mathur A, Shukla Y N, Pal, M. Saponin production in callus and cell suspensioncultures of Panax quinquefolium[J]. Phytochemistry.1994,351221-1225.
21. Son S. H., Choi S. M., S.J. Hyung S.R. et al. Induction and culture of mountainginseng adventitious roots and AFLP analysis for identifying mountain ginseng[J].Biotechnology Bioprocess Engineering.1999,4:119–123.
22. Yu J Y, DeRuiter S L, Turner D L. RNA interference by expression ofshort-interfering RNAs and hairpin RNAs in mammalian cells[J]. Proceedings ofthe National Academy of Sciences.2002,99:6047-6052.
23. Kim Y S, Hahn E J, Murthy H N, Paek K Y. Adventitious root growth andginsenoside accumulation in Panax ginseng cultures as affected by methyljasmonate[J]. Biotechnology. Letters.2004,26:1619-1622.
24. Kim Y S, Yeung E C, Hahn E J, et al. Combined effects of phytohormone,indole-3-butyric acid, and methyl jasmonate on root growth and ginsenosideproduction in adventitious root cultures of Panax ginseng C.A. Meyer[J].Biotechnology. Letters.2007,29(11):1789-1792.
25.赵寿经,杨振堂,李昌禹,等.发根农杆菌介导的人参遗传转化及人参皂甙工厂化生产[J].云南大学学报,1999,21(S3):142.
26. Richter L J, Thanavala Y, Arntzen C J, et al. Production of hepatitis B surfaceantigen in transgenic plants for oral immunization[J]. Nature Biotechnology.2000,18(11):1167-117
27. Chilton MD, Tepfer DA, Petit A et al. Agrobacterium rhizogenes insert T-DNAinto the genomes of the host plant root cell [J]. Nature.1982,295:432-434.
28.孙彬贤,杨光孝,汪沁琳,等.人参毛状根合成人参皂苷培养条件的优化[J].中成药.2003,25(9):746-748.
29. Liang Y, Zhao S. Progress in understanding of gin senoside biosynthesis[J]. PlantBiology.2008,10(4):415-421.
30. Sung-Sick Woo, Ji-Sook Song, Ju-Yeon Lee, et al. Selection of high ginsenosideproducing ginseng hairy root lines using targeted metabolic analysis[J].Phytochemistry.2004,65:2751–2761.
31. Javier P, Rosa M, CusidóM, et al. Elicitation of different Panax ginsengtransformed root phenotypes for an improved ginsenoside production[J]. PlantPhysiology and Biochemistry.2003,41:1019–1025.
32. Palazona J, Moyano E, Cusido RM, et al. Alkaloid production in Duboisia hybridhairy roots and plants overexpressing the h6h gene[J]. Plant Science.2003,165:1289-1295
33. Woo S S, Song J S, Lee J Y, et al. Selection of high ginsenoside producing ginsenghairy root lines using targeted metabolic analysis[J]. Phytochemistry.2004,65(20):2751-2761.
34.周倩耘,丁家宜,刘峻,等.植物激素对人参毛状根生长和皂甙含量的影响[J].植物资源与环境学报,2003,12(01):26-28.
35.周倩耘,周建民,刘峻,等.诱导子对人参毛状根中皂苷含量的影响[J].南京军医学院学报,2003,25(02):76-78.
36. Kosmas H, Miranda Tand Anne E O. Biosynthesis of Triterpenoid Saponins inPlant[J]. Biotechnology.2002,75:32-49.
37. Kushiro T, Ohno Y, Shibuya Y, Ebizuka Y. In vitro conversion of2,3-oxidosqualeneinto dammarenediol by Panax ginseng microsomes[J]. Biological&PharmaceuticalBulletin.1997,20(3):292-294.
38. Haralampidis K, Trojanowska M. Osbourn A E. Biosynthesis of triterpenoidsaponin in plants[J]. Advances in Biochemical Engineering/Biotechnology.2001,75:31-49.
39. Choi, D W, Jung J D, Ha Y I, Park H W, In D S, Chung H J. Liu J R. Analysis oftranscripts in methyl jasmonate-treated ginseng hairy roots to identify genesinvolved in the biosynthesis of ginsenosides and other secondary metabolites[J].Plant Cell Reports.2005,23:557-566.
40. Kushiro T, Shibuya M, Ebizuka Y. Beta-amyrin synthase--cloning ofoxidosqualene cyclase that catalyzes the formation of the most popular triterpeneamong higher plants[J]. European Journal of Biochemistry.1998,256(1):238-244.
41. Morita M, Shibuya M, Kushiro T, Masuda K, Ebizuka Y. Molecular cloning andfunctional expression of triterpene synthases from pea (Pisum sativum)-newalpha-amyrin-producing enzyme is a multifunctional triterpene synthase[J].European Journal of Biochemistry.2000,267:3453-3460.
42. Suzuki M., Xiang T., Ohyama K., et al. Lanosterol synthase in dicotyledonousplants[J]. Plant and Cell Physiology.2006,47(5):565-71.
43. Haralampidis K, Trojanowska M, Osbourn A. Biosynthesis of triterpenoid saponinsin plants[J]. Advances in Biochemical Engineering/Biotechnology.2002,75:31–49.
44. Lee M H, Jeong J H, Seo J W, et al. Enhanced triterpene and phytosterolbiosynthesis in Panax ginseng overexpressing squalene synthase gene[J].Plant&Cell Physiology.2004,45(8):976-984.
45.何景,曾沧江.中国植物志[M].北京：科学出版社.1978,54:180-181.
46.张树尘.中国人参[M].上海:科技教育出版社.1992,3.
47.顾学裘.药物制剂注解[M].北京:人民卫生出版社.1983,181
48.田建明,张志伟,叶金梅,等.人参皂甙RgZ对离体心脏的影响[J].长春中医学院学报.1999,5(6):43.
49.张文健,张文杰,李英骥,等.单体人参皂甙Rbl对成年大鼠心肌细胞凋亡的影响[J].白求恩医科大学学报.2001,27(6):598-600.
50.刘正湘,刘晓春.人参皂甙Rbl与Re对大鼠缺血再灌注心肌细胞凋亡的影响[J].中国组织化学与细胞化学杂志.2002,11(4):374-378.
51.王健春,计国义,孙文伟,等.人参茎叶皂甙对失血性休克犬血浆皮质醇、醛固酮及单胺递质变化的影响[J].白求恩医科大学学报.1998,4(6):580-582.
52.吕文伟,刘洁,田建明,等.20-(S)和20-(R)型人参皂甙RgZ对心源性休克犬血流动力学和血氧含量的影响[J].中草药.2003,34(3):254-25.
53.姜正林,吴新民,陈云,等.人参皂甙的抗鼠缺氧损伤作用[J].中华航海医学杂志..2000,7(l):28-32.
54.任非,马俊义,任晓丹,等.人参抗癌活性研究进展[J].河北中医药学报.2005,20(1):39-41.
55.聂荣庆,李扣华,胡国柱,等.人参皂甙RbⅠ抗新生大鼠大脑皮层神经细胞缺氧性凋亡研究[J].中国康复理论与实践.2004,10(12):723-725.
56.李爱红,柯开富,吴小梅,等.人参皂甙Rbl,Rb3,Rgl对培养皮层神经细胞的抗缺血效应及其机制[J].中风与神经疾病杂志.2004,21(3):231-234.
57.张云峰,姜正林,曹茂虹,等.人参皂甙RbⅠ对模拟缺血环境中的大鼠神经元胞内游离钙的影响[J].临床神经病学杂志.2005,18(6):440-442.
58.沈洪妹,张志军,姜正林.人参皂甙Rb3对大鼠缺氧性损伤的脑组织神经递质γ-氨基丁酸的影响[J].临床神经病学杂志.2005,18(6):455-456.
59.田京伟,傅风华,杨建雄.20(S)人参皂甙Rg3对脑缺血大鼠脑线粒体损伤的保护作用[J].中国药理学通报.2006,22(2):216-220.
60.潘树义,刘大庸,钟世镇,等.9种人参皂甙对培养鼠胚脊髓神经元生长的影响[J].脑与神经疾病杂志.2000,8(6):331-33.
61.彭成,雷载权.人参皂甙健脾益气作用的实验研究[J].中药药理与临床.1997,13(5):17-19.
62.赵俊.人参多糖的化学与药理学研究进展[J].国外医学中医中药分册.2004,26(2):79-81.
63.覃秀川,唯大员,郭新文,等.人参Rb组皂甙对急性心肌梗死大鼠自由基代谢的影响[J].吉林中医药.2002,22(3):51-52.
64.夏明泽,乔伯英,誉文敏,等.人参提取液对犬心肌缺血再灌注损伤保护作用的观察[J].实验动物科学与管理.1996,l(1):24-27.
65.赵祯,曹宇,袁淑德.人参茎叶皂甙对实验性高催乳素血症大鼠催乳素和动情周期的影响[J].中国应用生理学杂志.2005,21(2):144-146.
66.王欣,洪敏,金岩,等.人参皂甙对垂体-性腺系统分子内分泌功能调节作用[J].白求恩医科大学学报.1993,19(1):7-9.
67.李新民.人参皂甙对大鼠离体腺垂体细胞促性腺激素分泌功能影响的研究[J].白求恩医科大学学报.1998,5(4):293-295.
68.李杰,宋淑霞,吕占军.人参皂苷抗肿瘤作用的研究进展[J].中国肿瘤生物治疗杂志.2004,11(1):61-63.
69.郭志廷,王鲁,褚秀玲,等.浅谈人参皂甙Rh2对肿瘤细胞增殖周期和凋亡的影响[J].中兽医杂志.2006,(2):40-4.
70.陈文真,欧阳学农.人参皂苷抗癌机制的研究进展[J].福建中医药.2005,36(02):52-53.
71.高船舟,曲淑贤,吕广艳,等.20(R)-人参皂甙Rg3对K562/ADM细胞凋亡诱导的研究[J].大连医科大学学报.2001,23(3):171-173.
72.李静波,王秀清,张鸿源.人参茎叶皂甙和西洋参茎叶皂甙对单纯疤疹毒感染细胞的保护作用[J].中草药.1992,23(5):249-250.
73. Osbourn A E. Preformed antimicrobial compounds and plant defense againts fungalattack[J]. Plant Cell.1996.8:1821-1831.
74. Washida D, Shimomura K, Nakajima Y, et al. Ginsenosides in hairy roots of aPanax hybrid[J]. Phytochemistry.1998,49:2331–2335.
75. Lichtenthaler H K, Schwender J, Disch, A,RohmerM. Biosynthesis of isoprenoidsin higher plants proceeds via amevalonate independent pathway[J]. FEBS Letters.1997,400:271–274.
76. Hecht S, Eisenreich W, Adam P, Amslinger S, et al. Studies on thenonmevalonatepathway to terpenes: the role of the GcpE (IspG) protein[J]. Proceedings of theNational Academy of Sciences.2001,98:14837–14842.
77.刘丽.利用抑制剂对人参皂苷生物合成途径MVA与MEP的研究[D].吉林大学.2012.
78. Chappell J, Wolf F, Proulx J, Cuellar R, Saunders C. Is the reaction catalysed by3-hydroxy-3-m ethylglutaryl coenzyme A reductase a rate limiting step forisoprenoid biosynthesis in plants?[J]. Plant Physiology.1995, T09:1337-1343.
79. Seemann M, Bui B T S, Wolff M, et al. Isoprenoid bioynthesis in plant chloroplastsvia the MEP pathway: Direct thylakoid/ferredoxin-dependent photoreduction ofGcpE/IspG[J]. Federation of European Biochemical Societies.2006,580(6):1547-1552.
80. Lewis M J, Pros serI M, Mohib A, et al. Cloning and characterisation of aprenyltransferase from the aphid myzus persicae with potential involvement inalarm pheromone biosynthesis[J]. Insect Molecular Biology.2008,17(4):437-443.
81. Shibuya M, Hoshino M, Katsube Y, Hayashi H, Kushiro T, Ebizuka Y.Identification of beta-amyrin and sophoradiol24-hydroxylase by expressedsequence tag mining and functional expression assay[J]. FEBS Letters.2006,273:948-959.
82. Haralampidis K, Trojanowska M. Osbourn A E. Biosynthesis of triterpenoidsaponin in plants[J]. Advances in Biochemical Engineering/Biotechnology.2001,75:31-49.
83. Choi, D W, Jung J D, Ha Y I, Park H W, In D S, Chung H J. Liu J R. Analysis oftranscripts in methyl jasmonate-treated ginseng hairy roots to identify genesinvolved in the biosynthesis of ginsenosides and other secondary metabolites[J].Plant Cell Reports.2005,23:557-566.
84. Muller T H, Schaller H. Benveniste P. Molecular cloning and expression in yeast of2，3-oxidosqualene triterpene cyclases from Arabidopsis thaliana[J]. PlantMolecular Biology.2001,45：75-92.
85. Jin Shi-kun, Zhao Shou-jing. Construction of P. ginseng LS gene RNA InterferencePlant Expression Vector[C]. Environment, Chemistry and Biology.2012,49(80).
86. Seo J W, Jeong J H, Shin C G, et al. Overexpression of squalene synthase inEleutherococcus senticosus increases phytosterol and triterpene accumulation[J].Phytochemistry.2005,66(8):869-877.
87. Han J Y, In J G, Kwon Y S, et al. Regulation of ginsenoside and phytosterolbiosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng[J].Phytochemistry.2010,71:36-46.
88.罗志勇,陆秋恒,刘水平等.人参植物皂苷生物合成相关新基因的筛选与鉴定[J].生物化学与生物物理学报.2003,35(6):554-560.
89. Xu R, Fazio G C, Matsuda S P T. On the origins of triterpenoid skeletal diversity[J].Phytochemistry.2004,65:261–291.
90. Han J Y, Kwon Y S, Yang D C, et al. Expression and RNA interference-inducedsilencing of the dammarenediol synthase gene in Panax ginseng[J]. Plant&CellPhysiology.2006,47(12):1653-1662.
91. Suzuki H, Achnine L, Xu R A, et al. A genomics approach to the early stages oftriterpene saponin biosynthesis in Medicago truncatula[J]. Journal of Plant.2002,32(6):1033-1048.
92. Sawai S, Shindo T, Sato S, Kaneko T, Tabata S, Ayabe S, Aoki T. Functional andstructural analysis of genes encoding oxidosqualene cyclases of Lotus japonicas[J].Plant Science.2006,170:247–257.
93. Erik Limpens, Javier Ramos, Carolien Franken, et a1. RNA interference inAgrobacterium rhizogenes-transformed roots of Arabidop sis and Medicagotruncatula[J]. Journal of Experimental Botany.2004,55:983-992.
94. Zhao M M, An D R, Huang G H, He Z H, Chen J Y. A viral protein suppressessiRNA-directed interference in tobacco mosaic virus infection[J]. Acta Biochimicaet Biophysica Sinica (Shanghai).2005,37(4):248-253.
95.赵寿经,曹豪杰,何沐阳,王乐.人参β-AS基因RNAi表达载体工程菌的构建[J].吉林大学学报.2012,1608-1612.
96. Itoh T, Jeong M T, Hirano Y, Tamura T, Matsumoto T. Occurrence of lanosterol andlanostenol in seeds of red pepper (Capsicum annuum). Steroids.1977,29:569-577.
97. Rees H H, Goad L J, Goodwin T W.2,3-oxidosqualene cycloartenol cyclase fromOchromonas malhamensis. Biochim. Biophys. Acta.1969,176(4):892-4.
98. Corey, E. J., Matsuda, S. P. T.&Bartel, B. Isolation of an Arabidopsis thaliana geneencoding cycloartenol synthase by functional expression in a yeast mutant lackinglanosterol synthase by the use of a chromatographic screen[J]. Proceedings of theNational Academy of Sciences.1993,90:11628-11632.
99. Goad L J. How is sterol synthesis regulated in higher plants?[J]. BiochemicalSociety Transactions.1983,11(5):548-552.
100. Richter L J, Thanavala Y, Arntzen C J, et al. Production of hepatitis B surfaceantigen in transgenic plants for oral immunization[J].Nature Biotechnology.2000,18(11):1167-117
101. Chilton MD, Tepfer DA, Petit A et al. Agrobacterium rhizogenes insert T-DNAinto the genomes of the host plant root cell [J]. Nature.1982,295:432-434.
102.何沐阳.干扰人参CS基因的表达对人参三萜成分含量的影响[D].吉林大学.2013.
103.曹豪杰.人参βAS基因的表达调控与功能分析[D].吉林大学.2013.
104. Jung J D, Park H W, Hahn Y, et al. Discovery of genes for ginsenoside biosynthesisby analysis of ginseng expressed sequence tags[J]. Plant cell reports.2003,22(3):224-30.
105.李用芳,周延清.发根农杆菌及其应用[J].生物学杂志.2000,17(6).
106. Assaad F F, Tucker K L, Signer E R. Epigenetic repeat-induced gene silencing(RIGS) in Arabidopsis[J]. Plant Molecular Biology.1993,22:1067–1085.
107. Abe I, Rohmer M, Prestwich G D. Enzymatic cyclization of squalene andoxidosqualene to sterols and triterpenes[J].Chemical Reviews.1993,93(6):2189-2206.
108. Andreas Henschel, Frank Buchholz, Bianca Habermann. DEQOR: a web-basedtool for the design and quality control of siRNAs[J]. Nucleic Acids Research.2004,32(Web Server Issue): W113-120.
109. Besso H, Saruwatari Y, Futamura K, Kunihiro K, Fuwa T, Tanaka O.High-performance liquid chromatographic determination of ginseng saponin byultraviolet derivatization[J]. Planta Medica.1979,37(3):226-233.
110. Castanotto D, Li H, Rossi J J. Functional siRNA expression from transfected PCRproducts[J]. RNA.2002,8:1454-1460.
111. Chan T W D, But P P H, Cheng S W, Kwork I M Y, Lau F W, Xu H X.Differentiation and Authentication of Panax ginseng, Panax quinquefolius, andGinseng Products by Using HPLC/MS[J]. Analytical Chemistry.2000,72:1281-1287.
112. Guo H S, Fei J F, Xie Q, Chua N H. A chemical-regulated inducible RNAi systemin plants[J]. Plant.2003,34:383-392.
113. Haralampidis K, Trojanowska M, Osbourn A. Biosynthesis of triterpenoid saponinsin plants[J]. Advances in Biochemical Engineering/Biotechnology.2002,75:31–49.
114. Higuchi R, Krummel B, Saiki RK. A general of in vitro preparation and specificmutagenesis of DNA fragments: study of protein and DNA interactions[J]. NucleicAcids Reseach.1988,16(15):7351–7367.
115. Helliwell C, Waterhouse P. Constructs and methods of highthrough put genesilencing in plants[J]. Methods.2003,30:289-295.
116. Hayashi H, Huang P, Kirakosyan A, Inoue K, Hiraoka N, Ikeshiro Y, Kushiro T,Shibuya M. Ebizuka Y. Cloning and characterization of a cDNA encodingb-amyrin synthase involved in glycyrrhizin and soyasaponin biosynthesis inlicorice[J]. Biological&Pharmaceutical Bulletin.2001,24:912-916.
117. Daisuke Miki, Ko Shimamoto. Simple RNAi vectors for stable and transientsuppression of gene function in rice[J]. Plant and Cell Physiology.2004,5:490–
495.
118. Deming Gou, Nili Jin, Lin Liu. Gene silencing in mammaliancells by PCR basedshort hairpin RNA[J]. FEBS letters.2003,548:113-118.
119. Iwagami Shozo, Sawabe Yoshiyuki, Nakagawa Terumichi. Micellar electrokineticchromatography for the analysis of crude drugs (III): Separation of ginsenosides inGinseng Radix[J]. Shoyakugaku Zasshi.1992,46(4):339-347.
120. Kosmas H, Miranda Tand Anne E O. Biosynthesis of Triterpenoid Saponins inPlant[J]. Biotechnology.2002,75:32-49.
121. Massaki Shibuya H Z, Aki Endo. Two branches of the lupeol synthase gene in themolecular evolution of plant oxidosqualene cyclases[J]. Eur J Bioche.1999，266：302-307.
122.梁彦龙.反义抑制环阿乔醇合成酶基因对人参发根皂苷合成的影响[D].吉林大学.2009.
123. Elkin Yuri N, Makhankov V V, Uvarova Nina L,Bondarenko Pavel V, ZubarevRoman A,Knysh Anatoly N. Combination of HPLC and californium-252plasmadesorption mass spectrometry for identifying composition of ginseng tinctures[J].Acta Plarmacologica Sinica.1993,14(2):97-100.
124. Liu Z M,Liu Z Z,Bai Q W, et al. Agroinfiltration a useful technique in plantmolecular biology research[J]. Chinses Journal of Biotechnology.2002,18(4):411-414.
125. Tansakul P, Shibuya M, Kushiro T, Ebizuka Y. Dammarenediol-II synthase, thefirst dedicated enzyme for ginsenoside biosynthesis, in Panax ginseng[J]. FEBSLetters.2006,580(22):5143-5149
126. Wesley S V, Helliwell C A, Smith N A, et al. Construct design for efficient,effective and high-throughput gene silencing in plants[J]. Plant.2001,27:581-590.
127. Waterhouse P M, Graham M W, Wang M B. Virus resistance and gene silencing inplants can be induced by simultaneous expression of sense and antisense RNA [J].Proceedings of the National Academy of Sciences.1998,95:13959–13964.
128. Zeynep Arziman, Thomas Horn, Michael Boutros. E-RNAi: a web application todesign optimized RNAi constructs[J]. Nucleic Acids Research.2005, W582-588.
129. Tenllado F, Barajas D, Vargas M, Atencio F A, Gonzalez-Jara P, Diaz-Ruiz J R.Transient expression of homologous hairpin RNA causes interference with plantvirus infection and is overcome by a virus encoded suppressor of gene silencing[J].Molecular Plant-microbe Interaction.2003,16(2):149-58.
130. Tenllado F, Diaz-Ruiz J R. Double-stranded RNA-mediated interference with plantvirus infection[J]. Virolgy.2001,75(24):12288-12297.
131.李同芬,王宝琴.薄层扫描法测定复方人参町中的人参皂甙Re的含量[J].中成药.1991,13(4):15-16.
132.王幕邹,高凤英,章观德,张淑蓉.人参的分析I人参皂甙元的提取、分离和比色测定[J].药学学报.1979,14(5):309-315.
133.颜玉贞.人参西洋参及其制剂总皂甙紫外分光光度法含量测定方法研究[J].中药新药与临床药理.1994,5(4):37-40.
134. Glockl Ingmar, Veit Markus, Blaschke Gottfried. Determination of ginsenosidesfrom Panax ginseng using micellar electrokinetic chromatography[J]. PlantaMedica.2002,68(2):158-161.
135. Iturbe-Ormaetxe I, Haralampidis K, Papadopoulou K, Osbourn A E. Molecularcloning and characterization of triterpene synthases from Medicago truncatula andLotus japonicus[J]. Plant Molecular Biology.2003,51:731-743.
136.张蕾.丹参牻牛儿基牻牛儿基焦磷酸合酶基因的克隆与功能研究[D].中国人民解放军军事医学科学院.2009.
2. Shibata S. Chemistry and cancer preventing activities of ginseng saponins and somerelated triterpenoid compounds[J]. Journal of Korean Medical Science.2001,16:S28-S37.
3.郭志廷,王鲁,褚秀玲,等.浅谈人参皂甙Rh2对肿瘤细胞增殖周期和凋亡的影响[J].中兽医杂志.2006,(2):40-4.
4.高峰,潘晓鹏,于丽梅,等.人参皂甙Rh2对环磷酞胺(CY)诱发小鼠白细胞减少的影响[J].人参研究.1995,(4):37-38.
5.高瑞兰,金锦海,牛映平,等.人参总皂甙增加白血病细胞对化疗药物的敏感性[J].中国中西医结合杂志.1999,19(1):17-19.
6.文哗,裴玉萍,陈英杰.人参茎叶皂甙对醋酸泼尼松造成的高脂血症的影响[J].中国中药杂志.1996,21(7):430-432.
7.杨燕,何康,吴铁,等.人参茎叶皂甙对高胆固醇饮食大鼠血脂的影响[J].实验生物学报.1999,32(4):349-352.
8.徐承水.人参降脂作用的实验研究[J].长春中医学院学报.2000,16(3):46.
9.李校坤,林灼锋,黄亚东,等.人参三醇组甙抗辐射作用的研究[J].中药材.2002,25(11):805-808.
10.刘丽波,孙晓玲,张海英,等.人参三醇组甙对小鼠骨髓细胞染色体的辐射防护作用[J].中华放射击医学与防护杂志.2002,22(2):114-115.
11.王红丽,吴铁,吴志华.人参皂甙抗皮肤衰老作用实验研究[J].广东药学院学报.2003,19(1):25-27.
12.张春枝,安利佳,金凤燮.人参皂苷生理活性的研究进展[J].食品与发酵工业.2002,04:70-74.
13. Zhao Z, Cao Y, Yuan S D. Effects of ginseng stem and leave saponin on prolactinand menstrual cycle of experimental hyperprolactinemia rats[J]. Chinese Journal ofApplied Physiology.2005,21(2):144-146.
14.任跃英丛林官秀芝姜喜同.人参资源现状及可持续发展战略[J].人参研究.2003,15(4):9-10.
15. Furuya T, Yoshikawa T, Ishii T, et al. Regulation of saponin production in calluscultures of Panax ginseng[J]. Planta Medica.1983,47(4):200-204.
16. Asaka I, Li I, Hirotani M, Asada Y, Furuya T. Production of ginsenoside saponinsby culturing ginseng (Panax ginseng) embryogenic tissues in bioreactors[J].Biotechnology Letters.1993,15:1259-1264.
17.刘长军,侯嵩生.真菌激发子对人参悬浮培养细胞的生长和人参皂甙生物合成的影响[J].实验生物学报.1999,02:169-174.
18. Thanh N T, Murthy H N, Yu K W, et al. Methyl jasmonate elicitation enhancedsynthesis of ginsenoside by cell suspension cultures of Panax ginseng in5-lballoon type bubble bioreactors[J]. Applied Microbiology and Biotechnology,2005,67(2):197-201.
19. Yoshikawa T, Furuya T. Saponin production by cultures of Panax ginsengtransformed with Agrobacterium rhizogens[J]. Plant Cell Reports.1987,6:449-453.
20. Mathur A, Shukla Y N, Pal, M. Saponin production in callus and cell suspensioncultures of Panax quinquefolium[J]. Phytochemistry.1994,351221-1225.
21. Son S. H., Choi S. M., S.J. Hyung S.R. et al. Induction and culture of mountainginseng adventitious roots and AFLP analysis for identifying mountain ginseng[J].Biotechnology Bioprocess Engineering.1999,4:119–123.
22. Yu J Y, DeRuiter S L, Turner D L. RNA interference by expression ofshort-interfering RNAs and hairpin RNAs in mammalian cells[J]. Proceedings ofthe National Academy of Sciences.2002,99:6047-6052.
23. Kim Y S, Hahn E J, Murthy H N, Paek K Y. Adventitious root growth andginsenoside accumulation in Panax ginseng cultures as affected by methyljasmonate[J]. Biotechnology. Letters.2004,26:1619-1622.
24. Kim Y S, Yeung E C, Hahn E J, et al. Combined effects of phytohormone,indole-3-butyric acid, and methyl jasmonate on root growth and ginsenosideproduction in adventitious root cultures of Panax ginseng C.A. Meyer[J].Biotechnology. Letters.2007,29(11):1789-1792.
25.赵寿经,杨振堂,李昌禹,等.发根农杆菌介导的人参遗传转化及人参皂甙工厂化生产[J].云南大学学报,1999,21(S3):142.
26. Richter L J, Thanavala Y, Arntzen C J, et al. Production of hepatitis B surfaceantigen in transgenic plants for oral immunization[J]. Nature Biotechnology.2000,18(11):1167-117
27. Chilton MD, Tepfer DA, Petit A et al. Agrobacterium rhizogenes insert T-DNAinto the genomes of the host plant root cell [J]. Nature.1982,295:432-434.
28.孙彬贤,杨光孝,汪沁琳,等.人参毛状根合成人参皂苷培养条件的优化[J].中成药.2003,25(9):746-748.
29. Liang Y, Zhao S. Progress in understanding of gin senoside biosynthesis[J]. PlantBiology.2008,10(4):415-421.
30. Sung-Sick Woo, Ji-Sook Song, Ju-Yeon Lee, et al. Selection of high ginsenosideproducing ginseng hairy root lines using targeted metabolic analysis[J].Phytochemistry.2004,65:2751–2761.
31. Javier P, Rosa M, CusidóM, et al. Elicitation of different Panax ginsengtransformed root phenotypes for an improved ginsenoside production[J]. PlantPhysiology and Biochemistry.2003,41:1019–1025.
32. Palazona J, Moyano E, Cusido RM, et al. Alkaloid production in Duboisia hybridhairy roots and plants overexpressing the h6h gene[J]. Plant Science.2003,165:1289-1295
33. Woo S S, Song J S, Lee J Y, et al. Selection of high ginsenoside producing ginsenghairy root lines using targeted metabolic analysis[J]. Phytochemistry.2004,65(20):2751-2761.
34.周倩耘,丁家宜,刘峻,等.植物激素对人参毛状根生长和皂甙含量的影响[J].植物资源与环境学报,2003,12(01):26-28.
35.周倩耘,周建民,刘峻,等.诱导子对人参毛状根中皂苷含量的影响[J].南京军医学院学报,2003,25(02):76-78.
36. Kosmas H, Miranda Tand Anne E O. Biosynthesis of Triterpenoid Saponins inPlant[J]. Biotechnology.2002,75:32-49.
37. Kushiro T, Ohno Y, Shibuya Y, Ebizuka Y. In vitro conversion of2,3-oxidosqualeneinto dammarenediol by Panax ginseng microsomes[J]. Biological&PharmaceuticalBulletin.1997,20(3):292-294.
38. Haralampidis K, Trojanowska M. Osbourn A E. Biosynthesis of triterpenoidsaponin in plants[J]. Advances in Biochemical Engineering/Biotechnology.2001,75:31-49.
39. Choi, D W, Jung J D, Ha Y I, Park H W, In D S, Chung H J. Liu J R. Analysis oftranscripts in methyl jasmonate-treated ginseng hairy roots to identify genesinvolved in the biosynthesis of ginsenosides and other secondary metabolites[J].Plant Cell Reports.2005,23:557-566.
40. Kushiro T, Shibuya M, Ebizuka Y. Beta-amyrin synthase--cloning ofoxidosqualene cyclase that catalyzes the formation of the most popular triterpeneamong higher plants[J]. European Journal of Biochemistry.1998,256(1):238-244.
41. Morita M, Shibuya M, Kushiro T, Masuda K, Ebizuka Y. Molecular cloning andfunctional expression of triterpene synthases from pea (Pisum sativum)-newalpha-amyrin-producing enzyme is a multifunctional triterpene synthase[J].European Journal of Biochemistry.2000,267:3453-3460.
42. Suzuki M., Xiang T., Ohyama K., et al. Lanosterol synthase in dicotyledonousplants[J]. Plant and Cell Physiology.2006,47(5):565-71.
43. Haralampidis K, Trojanowska M, Osbourn A. Biosynthesis of triterpenoid saponinsin plants[J]. Advances in Biochemical Engineering/Biotechnology.2002,75:31–49.
44. Lee M H, Jeong J H, Seo J W, et al. Enhanced triterpene and phytosterolbiosynthesis in Panax ginseng overexpressing squalene synthase gene[J].Plant&Cell Physiology.2004,45(8):976-984.
45.何景,曾沧江.中国植物志[M].北京：科学出版社.1978,54:180-181.
46.张树尘.中国人参[M].上海:科技教育出版社.1992,3.
47.顾学裘.药物制剂注解[M].北京:人民卫生出版社.1983,181
48.田建明,张志伟,叶金梅,等.人参皂甙RgZ对离体心脏的影响[J].长春中医学院学报.1999,5(6):43.
49.张文健,张文杰,李英骥,等.单体人参皂甙Rbl对成年大鼠心肌细胞凋亡的影响[J].白求恩医科大学学报.2001,27(6):598-600.
50.刘正湘,刘晓春.人参皂甙Rbl与Re对大鼠缺血再灌注心肌细胞凋亡的影响[J].中国组织化学与细胞化学杂志.2002,11(4):374-378.
51.王健春,计国义,孙文伟,等.人参茎叶皂甙对失血性休克犬血浆皮质醇、醛固酮及单胺递质变化的影响[J].白求恩医科大学学报.1998,4(6):580-582.
52.吕文伟,刘洁,田建明,等.20-(S)和20-(R)型人参皂甙RgZ对心源性休克犬血流动力学和血氧含量的影响[J].中草药.2003,34(3):254-25.
53.姜正林,吴新民,陈云,等.人参皂甙的抗鼠缺氧损伤作用[J].中华航海医学杂志..2000,7(l):28-32.
54.任非,马俊义,任晓丹,等.人参抗癌活性研究进展[J].河北中医药学报.2005,20(1):39-41.
55.聂荣庆,李扣华,胡国柱,等.人参皂甙RbⅠ抗新生大鼠大脑皮层神经细胞缺氧性凋亡研究[J].中国康复理论与实践.2004,10(12):723-725.
56.李爱红,柯开富,吴小梅,等.人参皂甙Rbl,Rb3,Rgl对培养皮层神经细胞的抗缺血效应及其机制[J].中风与神经疾病杂志.2004,21(3):231-234.
57.张云峰,姜正林,曹茂虹,等.人参皂甙RbⅠ对模拟缺血环境中的大鼠神经元胞内游离钙的影响[J].临床神经病学杂志.2005,18(6):440-442.
58.沈洪妹,张志军,姜正林.人参皂甙Rb3对大鼠缺氧性损伤的脑组织神经递质γ-氨基丁酸的影响[J].临床神经病学杂志.2005,18(6):455-456.
59.田京伟,傅风华,杨建雄.20(S)人参皂甙Rg3对脑缺血大鼠脑线粒体损伤的保护作用[J].中国药理学通报.2006,22(2):216-220.
60.潘树义,刘大庸,钟世镇,等.9种人参皂甙对培养鼠胚脊髓神经元生长的影响[J].脑与神经疾病杂志.2000,8(6):331-33.
61.彭成,雷载权.人参皂甙健脾益气作用的实验研究[J].中药药理与临床.1997,13(5):17-19.
62.赵俊.人参多糖的化学与药理学研究进展[J].国外医学中医中药分册.2004,26(2):79-81.
63.覃秀川,唯大员,郭新文,等.人参Rb组皂甙对急性心肌梗死大鼠自由基代谢的影响[J].吉林中医药.2002,22(3):51-52.
64.夏明泽,乔伯英,誉文敏,等.人参提取液对犬心肌缺血再灌注损伤保护作用的观察[J].实验动物科学与管理.1996,l(1):24-27.
65.赵祯,曹宇,袁淑德.人参茎叶皂甙对实验性高催乳素血症大鼠催乳素和动情周期的影响[J].中国应用生理学杂志.2005,21(2):144-146.
66.王欣,洪敏,金岩,等.人参皂甙对垂体-性腺系统分子内分泌功能调节作用[J].白求恩医科大学学报.1993,19(1):7-9.
67.李新民.人参皂甙对大鼠离体腺垂体细胞促性腺激素分泌功能影响的研究[J].白求恩医科大学学报.1998,5(4):293-295.
68.李杰,宋淑霞,吕占军.人参皂苷抗肿瘤作用的研究进展[J].中国肿瘤生物治疗杂志.2004,11(1):61-63.
69.郭志廷,王鲁,褚秀玲,等.浅谈人参皂甙Rh2对肿瘤细胞增殖周期和凋亡的影响[J].中兽医杂志.2006,(2):40-4.
70.陈文真,欧阳学农.人参皂苷抗癌机制的研究进展[J].福建中医药.2005,36(02):52-53.
71.高船舟,曲淑贤,吕广艳,等.20(R)-人参皂甙Rg3对K562/ADM细胞凋亡诱导的研究[J].大连医科大学学报.2001,23(3):171-173.
72.李静波,王秀清,张鸿源.人参茎叶皂甙和西洋参茎叶皂甙对单纯疤疹毒感染细胞的保护作用[J].中草药.1992,23(5):249-250.
73. Osbourn A E. Preformed antimicrobial compounds and plant defense againts fungalattack[J]. Plant Cell.1996.8:1821-1831.
74. Washida D, Shimomura K, Nakajima Y, et al. Ginsenosides in hairy roots of aPanax hybrid[J]. Phytochemistry.1998,49:2331–2335.
75. Lichtenthaler H K, Schwender J, Disch, A,RohmerM. Biosynthesis of isoprenoidsin higher plants proceeds via amevalonate independent pathway[J]. FEBS Letters.1997,400:271–274.
76. Hecht S, Eisenreich W, Adam P, Amslinger S, et al. Studies on thenonmevalonatepathway to terpenes: the role of the GcpE (IspG) protein[J]. Proceedings of theNational Academy of Sciences.2001,98:14837–14842.
77.刘丽.利用抑制剂对人参皂苷生物合成途径MVA与MEP的研究[D].吉林大学.2012.
78. Chappell J, Wolf F, Proulx J, Cuellar R, Saunders C. Is the reaction catalysed by3-hydroxy-3-m ethylglutaryl coenzyme A reductase a rate limiting step forisoprenoid biosynthesis in plants?[J]. Plant Physiology.1995, T09:1337-1343.
79. Seemann M, Bui B T S, Wolff M, et al. Isoprenoid bioynthesis in plant chloroplastsvia the MEP pathway: Direct thylakoid/ferredoxin-dependent photoreduction ofGcpE/IspG[J]. Federation of European Biochemical Societies.2006,580(6):1547-1552.
80. Lewis M J, Pros serI M, Mohib A, et al. Cloning and characterisation of aprenyltransferase from the aphid myzus persicae with potential involvement inalarm pheromone biosynthesis[J]. Insect Molecular Biology.2008,17(4):437-443.
81. Shibuya M, Hoshino M, Katsube Y, Hayashi H, Kushiro T, Ebizuka Y.Identification of beta-amyrin and sophoradiol24-hydroxylase by expressedsequence tag mining and functional expression assay[J]. FEBS Letters.2006,273:948-959.
82. Haralampidis K, Trojanowska M. Osbourn A E. Biosynthesis of triterpenoidsaponin in plants[J]. Advances in Biochemical Engineering/Biotechnology.2001,75:31-49.
83. Choi, D W, Jung J D, Ha Y I, Park H W, In D S, Chung H J. Liu J R. Analysis oftranscripts in methyl jasmonate-treated ginseng hairy roots to identify genesinvolved in the biosynthesis of ginsenosides and other secondary metabolites[J].Plant Cell Reports.2005,23:557-566.
84. Muller T H, Schaller H. Benveniste P. Molecular cloning and expression in yeast of2，3-oxidosqualene triterpene cyclases from Arabidopsis thaliana[J]. PlantMolecular Biology.2001,45：75-92.
85. Jin Shi-kun, Zhao Shou-jing. Construction of P. ginseng LS gene RNA InterferencePlant Expression Vector[C]. Environment, Chemistry and Biology.2012,49(80).
86. Seo J W, Jeong J H, Shin C G, et al. Overexpression of squalene synthase inEleutherococcus senticosus increases phytosterol and triterpene accumulation[J].Phytochemistry.2005,66(8):869-877.
87. Han J Y, In J G, Kwon Y S, et al. Regulation of ginsenoside and phytosterolbiosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng[J].Phytochemistry.2010,71:36-46.
88.罗志勇,陆秋恒,刘水平等.人参植物皂苷生物合成相关新基因的筛选与鉴定[J].生物化学与生物物理学报.2003,35(6):554-560.
89. Xu R, Fazio G C, Matsuda S P T. On the origins of triterpenoid skeletal diversity[J].Phytochemistry.2004,65:261–291.
90. Han J Y, Kwon Y S, Yang D C, et al. Expression and RNA interference-inducedsilencing of the dammarenediol synthase gene in Panax ginseng[J]. Plant&CellPhysiology.2006,47(12):1653-1662.
91. Suzuki H, Achnine L, Xu R A, et al. A genomics approach to the early stages oftriterpene saponin biosynthesis in Medicago truncatula[J]. Journal of Plant.2002,32(6):1033-1048.
92. Sawai S, Shindo T, Sato S, Kaneko T, Tabata S, Ayabe S, Aoki T. Functional andstructural analysis of genes encoding oxidosqualene cyclases of Lotus japonicas[J].Plant Science.2006,170:247–257.
93. Erik Limpens, Javier Ramos, Carolien Franken, et a1. RNA interference inAgrobacterium rhizogenes-transformed roots of Arabidop sis and Medicagotruncatula[J]. Journal of Experimental Botany.2004,55:983-992.
94. Zhao M M, An D R, Huang G H, He Z H, Chen J Y. A viral protein suppressessiRNA-directed interference in tobacco mosaic virus infection[J]. Acta Biochimicaet Biophysica Sinica (Shanghai).2005,37(4):248-253.
95.赵寿经,曹豪杰,何沐阳,王乐.人参β-AS基因RNAi表达载体工程菌的构建[J].吉林大学学报.2012,1608-1612.
96. Itoh T, Jeong M T, Hirano Y, Tamura T, Matsumoto T. Occurrence of lanosterol andlanostenol in seeds of red pepper (Capsicum annuum). Steroids.1977,29:569-577.
97. Rees H H, Goad L J, Goodwin T W.2,3-oxidosqualene cycloartenol cyclase fromOchromonas malhamensis. Biochim. Biophys. Acta.1969,176(4):892-4.
98. Corey, E. J., Matsuda, S. P. T.&Bartel, B. Isolation of an Arabidopsis thaliana geneencoding cycloartenol synthase by functional expression in a yeast mutant lackinglanosterol synthase by the use of a chromatographic screen[J]. Proceedings of theNational Academy of Sciences.1993,90:11628-11632.
99. Goad L J. How is sterol synthesis regulated in higher plants?[J]. BiochemicalSociety Transactions.1983,11(5):548-552.
100. Richter L J, Thanavala Y, Arntzen C J, et al. Production of hepatitis B surfaceantigen in transgenic plants for oral immunization[J].Nature Biotechnology.2000,18(11):1167-117
101. Chilton MD, Tepfer DA, Petit A et al. Agrobacterium rhizogenes insert T-DNAinto the genomes of the host plant root cell [J]. Nature.1982,295:432-434.
102.何沐阳.干扰人参CS基因的表达对人参三萜成分含量的影响[D].吉林大学.2013.
103.曹豪杰.人参βAS基因的表达调控与功能分析[D].吉林大学.2013.
104. Jung J D, Park H W, Hahn Y, et al. Discovery of genes for ginsenoside biosynthesisby analysis of ginseng expressed sequence tags[J]. Plant cell reports.2003,22(3):224-30.
105.李用芳,周延清.发根农杆菌及其应用[J].生物学杂志.2000,17(6).
106. Assaad F F, Tucker K L, Signer E R. Epigenetic repeat-induced gene silencing(RIGS) in Arabidopsis[J]. Plant Molecular Biology.1993,22:1067–1085.
107. Abe I, Rohmer M, Prestwich G D. Enzymatic cyclization of squalene andoxidosqualene to sterols and triterpenes[J].Chemical Reviews.1993,93(6):2189-2206.
108. Andreas Henschel, Frank Buchholz, Bianca Habermann. DEQOR: a web-basedtool for the design and quality control of siRNAs[J]. Nucleic Acids Research.2004,32(Web Server Issue): W113-120.
109. Besso H, Saruwatari Y, Futamura K, Kunihiro K, Fuwa T, Tanaka O.High-performance liquid chromatographic determination of ginseng saponin byultraviolet derivatization[J]. Planta Medica.1979,37(3):226-233.
110. Castanotto D, Li H, Rossi J J. Functional siRNA expression from transfected PCRproducts[J]. RNA.2002,8:1454-1460.
111. Chan T W D, But P P H, Cheng S W, Kwork I M Y, Lau F W, Xu H X.Differentiation and Authentication of Panax ginseng, Panax quinquefolius, andGinseng Products by Using HPLC/MS[J]. Analytical Chemistry.2000,72:1281-1287.
112. Guo H S, Fei J F, Xie Q, Chua N H. A chemical-regulated inducible RNAi systemin plants[J]. Plant.2003,34:383-392.
113. Haralampidis K, Trojanowska M, Osbourn A. Biosynthesis of triterpenoid saponinsin plants[J]. Advances in Biochemical Engineering/Biotechnology.2002,75:31–49.
114. Higuchi R, Krummel B, Saiki RK. A general of in vitro preparation and specificmutagenesis of DNA fragments: study of protein and DNA interactions[J]. NucleicAcids Reseach.1988,16(15):7351–7367.
115. Helliwell C, Waterhouse P. Constructs and methods of highthrough put genesilencing in plants[J]. Methods.2003,30:289-295.
116. Hayashi H, Huang P, Kirakosyan A, Inoue K, Hiraoka N, Ikeshiro Y, Kushiro T,Shibuya M. Ebizuka Y. Cloning and characterization of a cDNA encodingb-amyrin synthase involved in glycyrrhizin and soyasaponin biosynthesis inlicorice[J]. Biological&Pharmaceutical Bulletin.2001,24:912-916.
117. Daisuke Miki, Ko Shimamoto. Simple RNAi vectors for stable and transientsuppression of gene function in rice[J]. Plant and Cell Physiology.2004,5:490–
495.
118. Deming Gou, Nili Jin, Lin Liu. Gene silencing in mammaliancells by PCR basedshort hairpin RNA[J]. FEBS letters.2003,548:113-118.
119. Iwagami Shozo, Sawabe Yoshiyuki, Nakagawa Terumichi. Micellar electrokineticchromatography for the analysis of crude drugs (III): Separation of ginsenosides inGinseng Radix[J]. Shoyakugaku Zasshi.1992,46(4):339-347.
120. Kosmas H, Miranda Tand Anne E O. Biosynthesis of Triterpenoid Saponins inPlant[J]. Biotechnology.2002,75:32-49.
121. Massaki Shibuya H Z, Aki Endo. Two branches of the lupeol synthase gene in themolecular evolution of plant oxidosqualene cyclases[J]. Eur J Bioche.1999，266：302-307.
122.梁彦龙.反义抑制环阿乔醇合成酶基因对人参发根皂苷合成的影响[D].吉林大学.2009.
123. Elkin Yuri N, Makhankov V V, Uvarova Nina L,Bondarenko Pavel V, ZubarevRoman A,Knysh Anatoly N. Combination of HPLC and californium-252plasmadesorption mass spectrometry for identifying composition of ginseng tinctures[J].Acta Plarmacologica Sinica.1993,14(2):97-100.
124. Liu Z M,Liu Z Z,Bai Q W, et al. Agroinfiltration a useful technique in plantmolecular biology research[J]. Chinses Journal of Biotechnology.2002,18(4):411-414.
125. Tansakul P, Shibuya M, Kushiro T, Ebizuka Y. Dammarenediol-II synthase, thefirst dedicated enzyme for ginsenoside biosynthesis, in Panax ginseng[J]. FEBSLetters.2006,580(22):5143-5149
126. Wesley S V, Helliwell C A, Smith N A, et al. Construct design for efficient,effective and high-throughput gene silencing in plants[J]. Plant.2001,27:581-590.
127. Waterhouse P M, Graham M W, Wang M B. Virus resistance and gene silencing inplants can be induced by simultaneous expression of sense and antisense RNA [J].Proceedings of the National Academy of Sciences.1998,95:13959–13964.
128. Zeynep Arziman, Thomas Horn, Michael Boutros. E-RNAi: a web application todesign optimized RNAi constructs[J]. Nucleic Acids Research.2005, W582-588.
129. Tenllado F, Barajas D, Vargas M, Atencio F A, Gonzalez-Jara P, Diaz-Ruiz J R.Transient expression of homologous hairpin RNA causes interference with plantvirus infection and is overcome by a virus encoded suppressor of gene silencing[J].Molecular Plant-microbe Interaction.2003,16(2):149-58.
130. Tenllado F, Diaz-Ruiz J R. Double-stranded RNA-mediated interference with plantvirus infection[J]. Virolgy.2001,75(24):12288-12297.
131.李同芬,王宝琴.薄层扫描法测定复方人参町中的人参皂甙Re的含量[J].中成药.1991,13(4):15-16.
132.王幕邹,高凤英,章观德,张淑蓉.人参的分析I人参皂甙元的提取、分离和比色测定[J].药学学报.1979,14(5):309-315.
133.颜玉贞.人参西洋参及其制剂总皂甙紫外分光光度法含量测定方法研究[J].中药新药与临床药理.1994,5(4):37-40.
134. Glockl Ingmar, Veit Markus, Blaschke Gottfried. Determination of ginsenosidesfrom Panax ginseng using micellar electrokinetic chromatography[J]. PlantaMedica.2002,68(2):158-161.
135. Iturbe-Ormaetxe I, Haralampidis K, Papadopoulou K, Osbourn A E. Molecularcloning and characterization of triterpene synthases from Medicago truncatula andLotus japonicus[J]. Plant Molecular Biology.2003,51:731-743.
136.张蕾.丹参牻牛儿基牻牛儿基焦磷酸合酶基因的克隆与功能研究[D].中国人民解放军军事医学科学院.2009.
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