摘要
北柴胡(Bupleurum chinense DC.)为伞形科柴胡属植物,是药用柴胡的主要来源之一,其中所含的主要有效成分柴胡皂苷类是一种齐墩果烷类型的三萜皂苷。目前,国内外对于柴胡皂苷的生物合成研究还很不深入。本文采用改良的trizol方法,成功的提取了北柴胡总RNA,然后以提取的总RNA为模板,采用RT-PCR方法,首次从北柴胡中克隆得到HMGR、IPPI、FPS和β-AS基因—4个柴胡皂苷合成途径中的基因。大小分别为470bp、532bp、466bp、1349bp,分别编码157、177、155、449个氨基酸多肽。对4个基因进行一系列序列分析(NCBI在线BLAST,特征性保守域分析,酶切位点分析,southern杂交分析,分子系统进化树分析)后表明克隆得到了4个基因保守性最强的核心片断。采用RT-PCR方法,在北柴胡中首次克隆得到6个适于实时定量RT-PCR的管家基因片段,片段大小分别为101bp(18S rRNA)、96bp(Cyclophilin)、128bp(EF1α)、121bp(L2)、101bp(β-tubulin)、99bp(Actin)。实时定量RT-PCR试验后,通过C_T值定量分析和genorm软件分析18S rRNA、Cyclophilin、EF1α、L2、β-tubulin、Actin基因在北柴胡各器官中表达的稳定性,得出β-tubulin基因是最稳定表达的内参基因,表达最不稳定的是18S rRNA基因。以β-tubulin为内参照,采用实时定量RT-PCR方法扩增,并用2~(-ΔΔCT)相对定量数据分析方法定量HMGR、IPPI、FPS、β-AS基因表达数据,结果显示,在柴胡皂苷合成途径中离终产物最远的HMGR基因和离终产物最近的β-AS基因均在根中表达量最高,根可能为合成柴胡皂苷的主要器官。本试验通过对柴胡皂苷合成相关酶的表达研究证实了北柴胡的各器官都有合成柴胡皂苷的能力,4个皂苷合成相关基因在北柴胡根、茎、叶、花、果实中均有表达,其中IPPI在茎中表达量最高,花中表达量最低;FPS在花中表达量最高,叶子中表达量最低。
Bupleurum chinense DC.(umbelliferae,bupleurum) is one of the main sources of medicinal bupleurum.The main effective component saikosaponin is oleanane-type triterpene saponin.At present, there were little research on the saikosaponin biosynthesis,cDNA fragments,encoding HMGR,IPPI,FPS andβ-AS which were involved in saikosaponin biosynthesis pathway in B.chinense were amplified by RT-PCR with the total RNA as a template,cDNA fragments of HMGR,IPPI,FPS andβ-AS gene were 470bp,532bp,466bp,1349bp in length encoding 157,177,155,449 amino acids respectively.The results of 4 genes sequence analysis(including blast analysis,conservative domain analysis,enzyme digestion sites analysis,southern hybridization analysis,phylogeneic tree analysis) indicated that the most conservative core fragments of 4 genes were cloned,cDNA fragments of six housekeeping genes were cloned by RT-PCR for the first time,the cDNA fragments were 101bp(18S rRNA),96bp(Cyclophilin), 128bp(EF1α),121bp(L2),101bp(β-tubulin),99bp(Actin) in length respectively.After the real time RT-PCR,expression stability of 18S rRNA,Cyclophilin,EF1α,L2,β-tubulin,Actin were analyzed with C_T value and genorm,the result indicated that the most stable gene isβ-tubulin,on the contrary,is 18S rRNA.Using theβ-tubulin as the internal reference standard,HMGR、IPPI、FPS、β-AS gene were amplified by real time RT-PCR.The data were analysed by 2~(-ΔΔCT).HMGR gene far from end product andβ-AS gene nearby end product showed the highest expression level on root.Root was the main organ for saikosaponin biosynthesis probably.The result of tissue expression of saikosaponin biosynthesis-related genes showed that saikosaponin can be synthesized in every organs from B. chinense.Four of the saikosaponin biosynthesis-related genes were all expressed in roots,stems,leaves, flowers and fruits from B.chinense.The highest expression level of IPPI was in stems and the lowest was in flowers.The highest expression level of FPS was in flowers and the lowest was in leaves.
引文
[1]程爱霞,娄永根,毛颖波,等.植物萜类化合物的生物合成与生态学功能[J].植物学报(英文版),2007,49(2):179.
[2]都晓伟,刘鸣远.柴胡皂甙的组织化学研究[J].中国中药杂志,1992,17:261-263.
[3]付佳,王洋,阎秀峰.萜类化合物的生理生态功能及经济价值[J].东北林业大学学报,2003,31(6):59-62.
[4]葛发欢,李莹,谢健鸣.超临界CO2从柴胡中萃取挥发油及其皂甙的研究[J].中国中药杂志,2000,25(3):149-153.
[5]李嵘,王喆之.植物萜类合成酶3-羟基-3-甲基戊二酰辅酶A还原酶的生物信息学分析[J].广西植物,2006,26(5):464-473.
[6]梁鸿,赵玉英,崔艳君,等.北柴胡中黄酮类化合物的分离鉴定[J].北京医科大学学报,2000,32(3):223-225.
[7]梁鸿,赵玉英,李德宇.柴胡属植物化学成分及药理活性研究进展[J].国外医药:植物药分册,1999,14(5):191-195.
[8]梁鸿,赵玉英,邱海蕴.北柴胡中新皂甙的结构鉴定[J].药学学报,1998,33(1):37-41.
[9]梁之桃,秦民坚,王峥涛.柴胡属植物皂苷成分研究进展[J].天然产物研究与开发,2001.13(6):67-72.
[10]刘沁舡,潭利,白焱晶.柴胡属植物皂苷近10年研究概况[J].中国中药杂志.2002,27(1):7-11.
[11]刘智,余龙江,栗茂腾,等.紫杉醇及其前体在中国红豆杉植株中合成和积累部位探讨[J].华中农业大学学报.2006,25(3):313-317.
[12]鲁守平,隋新霞,孙群,等.药用植物次生代谢的生物学作用及生态环境因子的影响[J].天然产物研究与开发,2006,18(6):1027-1032.
[13]罗永明,刘爱华,李琴,等.植物萜类化合物的生物合成途径及其关键酶的研究进展(续完)[J].江西中医学院学报,2003,15(2):46-49.
[14]谭玲玲,胡正海,蔡霞,等.北柴胡营养器官中主要化学成分的组织化学定位及其含量比较[J].分子细胞生物学报,2007,40(4):214-222.
[15]王英华,羽野芳生,野村太郎.小叶黑柴胡中皂甙成分的研究[J].中国中药杂志,1998,23(2):96-98.
[16]杨文修.柴胡总皂甙促胰腺腺泡酶分泌和升高[Ca2+]的动力学特征[J].中国学术期刊文摘(科技快报),1999,5(12):15-29.
[17]张相年,张相波.柴胡皂苷的生理活性及在治疗肾病中的作用[J].广东药学院学报,2000,16(2):121-124.
[18]邹凤莲,寿森炎,叶纨芝,等.类黄酮化合物在植物胁迫反应中作用的研究进展[J].细胞生物学杂志,2004,26(1):39-44.
[19]Abe I,Prestwich G D.Molecular Cloning,Characterization,and Functional Expression of Rat Oxidosqualene Cyclase cDNA[J].Proceedings of the National Academy of Sciences,1995,92(20):9274-9278.
[20]Bach T J.Hydroxymethylglutaryl-CoA reductase,a key enzyme in phytosterol synthesis?[J].Lipids,1986,21(1):82-88.
[21]Baldwin I T.An Ecologically Motivated Analysis of Plant-Herbivore Interactions in Native Tobacco[Z].Plant Physiology,2001,127:1449-1458.
[22]Bezier A,Lambert B,Baillieul F.Study of Defense-related Gene Expression in Grapevine Leaves and Berries Infected with Botrytis cinerea[J]. European Journal of Plant Pathology, 2002, 108(2):111-120.
[23] Bird D A, Franceschi V R, Facchini P J. A Tale of Three Cell Types: Alkaloid Biosynthesis Is Localized to Sieve Elements in Opium Poppy[J]. The Plant Cell, 2003, 15(11): 2626.
[24] Bohlmann J, Meyer-Gauen G, Croteau R. Plant terpenoid synthases: Molecular biology and phylogenetic analysis[Z]. National Acad Sciences, 1998,95: 4126-4133.
[25] Brunner A M, Yakovlev I A, Strauss S H. Validating internal controls for quantitative plant gene expression studies[J]. feedback, 2005,4(1): 14.
[26] Buchanan B B, Gruissem W, Jones R L. Biochemistry and Molecular Biology of Plants[J].Rockville, MD: American Society of Plant Physiologists, 2000,260.
[27] Burleigh S H. Relative quantitative RT-PCR to study the expression of plant nutrient transporters in arbuscular mycorrhizas[J]. Plant Science, 2001,160(5): 899-904.
[28] Campbell M, Hahn F M, Poulter C D, et al. Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis thaliana[J]. Plant Molecular Biology, 1998, 36(2): 323-328.
[29] Carretero-Paulet L, Cair(?) A, Botella-Pavia P, et al. Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase[J]. Plant Molecular Biology, 2006, 62(4): 683-695.
[30] Chen L R, Chen Y J, Lee C Y, et al. MeJA-induced transcriptional changes in adventitious roots of Bupleurum kaoi[J]. Plant Science, 2007, 173(1): 12-24.
[31] Choi D W, Jung J D, Ha Y I, et al. Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites[J]. Plant Cell Reports, 2005, 23(8): 557-566.
[32] Choi D, Ward B L, Bostock R M. Differential Induction and Suppression of Potato 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes in Response to Phytophthora infestans and to Its Elicitor Arachidonic Acid[J]. The Plant Cell Online, 1992,4(10): 1333-1344.
[33] Czechowski T, Bari R P, Stitt M, et al. Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root-and shoot-specific genes[J]. The Plant Journal, 2004, 38(2): 366-379.
[34] Davis E M, Tsuji J, Davis G D, et al. Purification of (+)-5-cadinene synthase, a sesquiterpene cyclase from bacteria-inoculated cotton foliar tissue[J]. Phytochemistry, 1996, 41(4): 1047-1055.
[35] De Luca V, Cutler A J. Subcellular Localization of Enzymes Involved in Indole Alkaloid Biosynthesis in Catharanthus roseus 1[J]. Plant Physiology, 1987, 85(4): 1099-1102.
[36] Denbow C J, Lang S, Cramer C L. The N-terminal Domain of Tomato 3-Hydroxy-3-methylglutaryl-CoA Reductases[J]. Journal of Biological Chemistry, 1996, 271(16):9710.
[37] Enyedi A J, Yalpani N, Silverman P, et al. Signal molecules in systemic plant resistance ot pathogens and pests[J]. Cell(Cambridge), 1992, 70(6): 879-886.
[38] Facchini P J, De Luca V. Phloem-Specific Expression of Tyrosine/Dopa Decarboxylase Genes and the Biosynthesis of Isoquinoline Alkaloids in Opium Poppy[J]. The Plant Cell Online, 1995, 7(11):1811-1821.
[39] Fahn A. Secretory tissues in plants[M]. Academic Press New York, 1979.
[40] Gallagher C, Cervantes-Cervantes M, Wurtzel E. Surrogate biochemistry: use of Escherichia coli to identify plant cDNAs that impact metabolic engineering of carotenoid accumulation[J]. Applied Microbiology and Biotechnology, 2003,60(6): 713-719.
[41] Gershenzon J, Croteau R. Terpenoid biosynthesis: the basic pathway and formation of monoterpenes, sesquiterpenes, and diterpenes[J]. Lipid Metabolism in Plants. CRC Press, Boca Raton, FL, 1993:339.
[42] Gonzalez M C, Echevarria C, Vidal J, et al. Isolation and characterisation of a wheat phosphoenolpyruvate carboxylase gene. Modelling of the encoded protein[J]. Plant science(Limerick), 2002, 162(2): 233-238.
[43] Hadacek F. Secondary Metabolites as Plant Traits: Current Assessment and Future Perspectives[J].Critical Reviews in Plant Sciences, 2002, 21(4): 273-322.
[44] Haralampidis K, Bryan G, Qi X, et al. A new class of oxidosqualene cyclases directs synthesis of antimicrobial phytoprotectants in monocots[J]. Proceedings of the National Academy of Sciences,2001,98(23): 13431.
[45] Haralampidis K, Trojanowska M, Osbourn A E. Biosynthesis of Triterpenoid Saponins in Plants[J].Advances in Biochemical Engineering Biotechnology, 2002, 75: 31-50.
[46] Hayashi H, Huang P, Inoue K. Up-regulation of Soyasaponin Biosynthesis by Methyl Jasmonate in Cultured Cells of Glycyrrhiza glabra[J]. Plant & Cell Physiology, 2003,44(4): 404-411.
[47] Hemmerlin A, Rivera S B, Erickson H K, et al. Enzymes Encoded by the Farnesyl Diphosphate Synthase Gene Family in the Big Sagebrush Artemisia tridentata ssp. spiciformis[J]. Journal of Biological Chemistry, 2003, 278(34): 32132-32140.
[48] Hibi N, Fujita T, Hatano M, et al. Putrescine N-Methyltransferase in Cultured Roots of Hyoscyamus albus 1 n-Butylamine as a Potent Inhibitor of the Transferase both in Vitro and in Vivo[J]. Plant Physiology, 1992. 100(2): 826-835.
[49] Irmler S. Schroder G, St-Pierre B, et al. Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase[J].The Plant Journal. 2000, 24(6): 797-804.
[50] Joost O, Bianchini G, Bell A A, et al. Differential Induction of 3-Hydroxy-3-methylglutaryI CoA Reductase in Two Cotton Species Following Inoculation with Verticillium[J]. Molecular Plant Microbe Interactions, 1995, 8: 880-885.
[51] Kajiwara S, Fraser P D, Kondo K, et al. Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli[J]. Biochemical Journal, 1997,324(2): 421-426.
[52] Kanazawa H, Nagata Y, Matsushima Y. et al. Determination of acidic saponins in crude drugs by highperformance liquid chromatography on octadecylsilyl porous glass[J]. Journal of Chromatography, 1993, 630:408-414.
[53] Karst F, Lacroute F. Ergosterol biosynthesis inSaccharomyces cerevisiae[J]. Molecular Genetics and Genomics, 1977, 154(3): 269-277.
[54] Kim Y S, Cho J H, Ahn J, et al. Upregulation of Isoprenoid Pathway Genes During Enhanced Saikosaponin Biosynthesis in the Hairy Roots of Bupleurum falcatum[J]. Moleculars and Cells,2006, 22(3): 269-274.
[55] Klok E J, Wilson I W, Wilson D, et al. Expression Profile Analysis of the Low-Oxygen Response in Arabidopsis Root Cultures[J]. The Plant Cell, 2002, 14(10): 2481-2494.
[56] Korth K L, Stermer B A, Bhattacharyya M K, et al. HMG-CoA reductase gene families that differentially accumulate transcripts in potato tubers are developmentally expressed in floral tissues[J]. Plant Molecular Biology, 1997, 33(3): 545-551.
[57] Kuntz M, Romer S, Suire C, et al. Identification of a cDNA for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicum annuum: correlative increase in enzyme activity and transcript level during fruit ripening[J]. The Plant Journal, 1992,2(1): 25-34.
[58] Langer K, Ache P, Geiger D, et al. Poplar potassium transporters capable of controlling K+ homeostasis and K+-dependent xylogenesis[J]. The Plant Journal, 2002, 32(6): 997-1009.
[59] Leivar P, Gonzalez V M, Castel S, et al. Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase 1[J]. Plant Physiology, 2005, 137(1): 57-69.
[60] Letondal C. A Web interface generator for molecular biology programs in Unix[Z]. Oxford Univ Press, 2001,17:73-82.
[61] Lin C C, Yen M H, Chen J Y, et al. Anatomical And Histological Studies Of Bupleuri Radix[J].Am J Chin Med, 1991, 19(3-4): 265-274.
[62] Loguercio L L, Scott H C, Trolinder N L, et al. Hmg-coA Reductase Gene Family in Cotton (Gossypium hirsutum L.): Unique Structural Features and Differential Expression of hmg2 Potentially Associated with Synthesis of Specific Isoprenoids in Developing Embryos[J]. Plant and Cell Physiology, 1999,40(7): 750-761.
[63] Lossos I S, Czerwinski D K, Wechser M A, et al. Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies[J]. Leukemia, 2003,17(4): 789-795.
[64] Mahroug S, Burlat V, St-Pierre B. Cellular and sub-cellular organisation of the monoterpenoid indole alkaloid pathway in Catharanthus roseus[J]. Phytochemistry Reviews, 2007, 6(2): 363-381.
[65] Marchler-Bauer A, Bryant S H. CD-Search: protein domain annotations on the fly[J]. Nucleic Acids Research, 2004, 32(Web Server Issue): 327-331.
[66] Mcgarvey D J, Croteau R. Terpenoid Metabolism[Z]. Am Soc Plant Biol, 1995: 7, 1015-1026.
[67] Mcknight T D, Bergey D R, Burnett R J, et al. Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants[J]. Planta, 1991,185(2): 148-152.
[68] Miller B, Heuser T, Zimmer W. Functional involvement of a deoxy-D-xylulose 5-phosphate reductoisomerase gene harboring locus of Synechococcus leopoliensis in isoprenoid biosynthesis[J]. FEBS Letters, 2000, 481(3): 221-226.
[69] Nakano Y, Matsunaga H, Saita T, et al. Antiproliferative Constituents in Umbelliferae Plants II.Screening for Polyacetylenes in Some Umbelliferae Plants, and Isolation of Panaxynol and Falcarindiol from the Root of Heracleum moellendorffii[J]. Biological and Pharmaceutical Bulletin,1998, 21(3): 257-261.
[70] Nicot N, Hausman J F, Hoffmann L, et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress[J]. Journal of Experimental Botany, 2005,56(421): 2907-2914.
[71] Ohnuma S, Hirooka K, Hemmi H, et al. Conversion of Product Specificity of Archaebacterial Geranylgeranyl-diphosphate Synthase. IDENTIFICATION OF ESSENTIAL AMINO ACID RESIDUES FOR CHAIN LENGTH DETERMINATION OF PRENYLTRANSFERASE REACTION[J]. Journal of Biological Chemistry, 1996, 271(18): 831-837.
[72] Ono M, Yoshida A, Ito Y, et al. Phenethyl alcohol glycosides and isopentenol glycoside from fruit of Bupleurum falcatum[J]. Phytochemistry, 1999, 51(6): 819-823.
[73] Pasquali G, Goddijn O, Waal A, et al. Coordinated regulation of two indole alkaloid biosynthetic genes from Catharanthus roseus by auxin and elicitors[J]. Plant Molecular Biology, 1992, 18(6):1121-1131.
[74] Pichersky E, Gershenzon J. The formation and function of plant volatiles: perfumes for pollinator attraction and defense[J]. Current Opinion in Plant Biology, 2002, 5(3): 237-243.
[75] Prisic S, Xu M, Wilderman P R, et al. Rice Contains Two Disparate ent-Copalyl Diphosphate Synthases with Distinct Metabolic Functions 1[J]. Plant Physiology, 2004,136(4): 4228-4236.
[76] Ramos-Valdivia A C, Heijden R, Verpoorte R. Isopentenyl diphosphate isomerase: a core enzyme in isoprenoid biosynthesis. A review of its biochemistry and function[J]. Natural Product Reports,1997, 14(6): 591-603.
[77] Rance I, Fournier J, Esquerre-Tugaye M T. The incompatible interaction between Phytophthora parasitica var. nicotianae race 0 and tobacco is suppressed in transgenic plants expressing antisense lipoxygenase sequences[Z]. National Acad Sciences, 1998, 95: 6554-6559.
[78] Rodriguez-Concepcion M, Gruissem W. Arachidonic Acid Alters Tomato HMG Expression and Fruit Growth and Induces 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase-Independent Lycopene Accumulation[J]. Plant Physiology, 1999,119(1): 41-48.
[79] Salto A, Rilling H C. Farnersyl diphosphate synthases and cycloase[J]. Arch. Biochem. Biophys,1981,208:508-511.
[80] Samanani N, Park S U, Facchini P J. Cell Type-Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis[J]. The Plant Cell Online,2005, 17(3): 915-926.
[81] Samanani N, Park S U, Facchini P J. Cell Type-Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis[J]. The Plant Cell Online,2005. 17(3): 915-926.
[82] Saslowsky D. Winkel-Shirley B. Localization of flavonoid enzymes in Arabidopsis roots[J]. The Plant Journal. 2001. 27(1): 37-48.
[83] Seo J W, Jeong J H. Shin C G. et al. Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation[J]. Phytochemistry, 2005. 66(8):869-877.
[84] Siddiqui M A, Yamanaka A, Hirooka K, et al. Enzymatic and structural characterization of type II isopentenyl diphosphate isomerase from hyperthermophilic archaeon Thermococcus kodakaraensis[J]. Biochemical and Biophysical Research Communications, 2005, 331(4):1127-1136.
[85] Sottomayor M, Ros Barcelo A. Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of α -3', 4'-anhydrovinblastine: a specific role for a multifunctional enzyme[J].Protoplasma, 2003, 222(1): 97-105.
[86] Stevens L H, Blom T, Verpoorte R. Subcellular localization of tryptophan decarboxylase,strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernaemontana divaricata[J]. Plant Cell Reports, 1993, 12(10): 573-576.
[87] St-Pierre B, Vazquez-Flota F A, De Luca V. Multicellular Compartmentation of Catharanthus roseus Alkaloid Biosynthesis Predicts Intercellular Translocation of a Pathway Intermediate[J].The Plant Cell, 1999, 11(5): 887.
[88] Suzuki H, Achnine L, Xu R, et al. A genomics approach to the early stages of triterpene saponin biosynthesis in Medicago truncatula[J]. The Plant Journal, 2002, 32(6): 1033-1048.
[89] Suzuki K, Yamada Y, Hashimoto T. Expression of Atropa belladonna Putrescine N-Methyltransferase Gene in Root Pericycle[J]. Plant and Cell Physiology, 1999,40(3): 289-297.
[90] Swain T. Secondary Compounds as Protective Agents[J]. Annual Reviews in Plant Physiology,1977, 28(1): 479-501.
[91] Tattini M, Gravano E, Pinelli P, et al. Flavonoids accumulate in leaves and glandular trichomes of Phillyrea latifolia exposed to excess solar radiation[J]. New Phytologist, 2000, 148(1): 69-77.
[92] Thomas C, Meyer D, Wolff M, et al. Molecular characterization and spatial expression of the sunflower ABP1 gene[J]. Plant Molecular Biology, 2003, 52(5): 1025-1036.
[93] Tricarico C, Pinzani P, Bianchi S, et al. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies[J]. Analytical Biochemistry, 2002, 309(2): 293-300.
[94] Tsai Y J, Chen 1 L, Horng L Y, et al. Induction of Differentiation in Rat C6 Glioma Cells with Saikosaponins[J]. Phytotherapy Research, 2002,16(2): 117-121.
[95] Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol, 2002,3(7): 34.
[96] Waller G R, Jurzysta M, Thorne R. Allelopathic activity of root saponins from alfalfa (Medicago sativa L.) on weeds and wheat[J]. Bot. Bull. Acad. Sin, 1993, 34(1).
[97] Wang K, Ohnuma S. Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecular evolution[J]. Trends in Biochemical Sciences, 1999, 24(11):445-451.
[98] Wink M. Site of lupanine and sparteine biosynthesis in intact plants and in vitro organ cultures[J].Zeitschrift fur Naturforschung, C: Journal of Biosciences, 1987,42: 868.
[99] Xiaohua L, Jianying H. Saikosaponins from Bupleurum Falcatum L[J]. Chemical Research, 2000,11(1):8.
[100] Yang Z, Park H, Lacy G H, et al. Differential Activation of Potato 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Genes by Wounding and Pathogen Challenge[J]. The Plant Cell Online,1991. 3(4): 397-405.
[101] Zhang C, Liu L, Xu H, et al. Crystal Structures of Human IPP Isomerase: New Insights into the Catalytic Mechanism[J]. Journal of Molecular Biology, 2007, 366(5): 1437-1446.