长春花萜类吲哚生物碱合成途径中关键酶基因的克隆、表达
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摘要
本文以长春花(Catharanthus roseus(L．)G．Don．)植物幼苗为材料，利用Gateway克隆技术对其萜类吲哚生物碱(Terpene indole alkaloids，TIAs)生物合成途径中编码关键酶的基因进行大规模克隆，获得相关基因的入门克隆和表达克隆，制备相应的抗体。并对这些基因的mRNA表达水平进行检测。所得结果如下：
1．以长春花植物幼苗中提取的总RNA为模板，反转录生成cDNA，经两步Gateway-PCR法获得长春花TIAs类化合物生物合成途径中编码关键酶DXS、DXR、G10H、SLS、TDC、STR和DAT的目的基因(dxs、dxr、g10h、sls、tdc、str和dat)的全长序列。
2．利用Gateway克隆技术通过BP重组反应将目的基因插入到入门载体(pDONR201)中，构建目的基因的入门质粒，筛选出阳性克隆并通过测序验证。
3．通过LR重组反应将目的基因从入门载体上转移到表达载体(pETG10A／20A／30A)，导入大肠杆菌表达菌株BL21(DE3)的感受态细胞中。通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达，经SDS-PAGE检测，表明重组的分别含有dxr、sls、str和g10h基因的表达质粒的大肠杆菌菌株均有目的蛋白的表达。
4．利用Ni-TED树脂分别对表达量较高的DXR、SLS和STR融合HIS标签的蛋白进行纯化，纯化后的蛋白经SDS-PAGE检测没有杂带，用Brad-ford法对纯化后的蛋白进行定量，结果表明所得蛋白已达到制备抗体的要求，目前抗体制备正在进行中。
5．对这些基因在长春花幼苗中mRNA表达水平的Northern杂交实验显示dxr、sls、str和tdc基因在根、茎、叶、花和果实中均有表达，并且从总体上看根和幼叶中的表达量相对高些，老叶中的表达量相对低一些；但是没有检测到目的基因g10h、dxs和dat相应的mRNA杂交信号，原因可能是与所选的植物材料的生长阶段和生长环境有关。
This thesis performed a large scale of cloning for genes encoding key enzymes in terpene indole alkaloids (TIAs) pathway from Catharanthus roseus (L.)G.Don.via Gateway technology. These genes' mRNA expression levels in C. roseus seedlings were also detected. The results are as follows:
1. The full length sequences of dxs, dxr, g10h, sls, tdc, str and dat which encoded key enzymes involved in terpenoid indole alkaloids (TIAs) biosynthesis pathway were obtained via two-step Gateway PCR method, with the transcripted cDNA from total RNA of Catharanthus roseus seedlings as templates.
2. After PCR amplification, these attB-flanked DNA fragments were inserted into attPcontaining donor vector (pDONR201) by BP clonase to generate entry clones, respectively. Positive clones were screened first by colony cracking, PCR and restriction enzyme analysis, and then these inserted genes were confirmed by gene sequencing.
3. In order to get prokaryotic expression systems, ORFs of dxr, g10h, sls and str have been transferred to their destination vectors: pETG10A, pETG20A and pETG30A, and been transformed into competent cells of E.cloi host strain BL21(DE3). SDS-PAGE analysis results indicated that those recombinant expression clones which encoded enzymes of DXR, SLS and STR were overexpressed after IPTG induction.
4. Fusion proteins DXR-HIS, DXR-HIS-TrxA, DXR-HIS-GST, SLS-HIS and STR-HIS were purified by Ni-TED resin. According to the results of SDS-PAGE and Bradford, these purified proteins were qualified for antisera preparation.
5. Northern blot results displayed that transcript levels of dxr, sls, str and tdc in root, stem, leave, flower and fruit from C roseus were detected, in general the transcript levels in root and young leave were higher than in other tissues. But there're no signals for Northern blot ofglOh, dxs and dat. For one reason maybe that there're no mRNA transcripts in the seedling stage we chosed for these genes, or maybe they're inclined to be under strict post-transcriptional regulation.
1．以长春花植物幼苗中提取的总RNA为模板，反转录生成cDNA，经两步Gateway-PCR法获得长春花TIAs类化合物生物合成途径中编码关键酶DXS、DXR、G10H、SLS、TDC、STR和DAT的目的基因(dxs、dxr、g10h、sls、tdc、str和dat)的全长序列。
2．利用Gateway克隆技术通过BP重组反应将目的基因插入到入门载体(pDONR201)中，构建目的基因的入门质粒，筛选出阳性克隆并通过测序验证。
3．通过LR重组反应将目的基因从入门载体上转移到表达载体(pETG10A／20A／30A)，导入大肠杆菌表达菌株BL21(DE3)的感受态细胞中。通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达，经SDS-PAGE检测，表明重组的分别含有dxr、sls、str和g10h基因的表达质粒的大肠杆菌菌株均有目的蛋白的表达。
4．利用Ni-TED树脂分别对表达量较高的DXR、SLS和STR融合HIS标签的蛋白进行纯化，纯化后的蛋白经SDS-PAGE检测没有杂带，用Brad-ford法对纯化后的蛋白进行定量，结果表明所得蛋白已达到制备抗体的要求，目前抗体制备正在进行中。
5．对这些基因在长春花幼苗中mRNA表达水平的Northern杂交实验显示dxr、sls、str和tdc基因在根、茎、叶、花和果实中均有表达，并且从总体上看根和幼叶中的表达量相对高些，老叶中的表达量相对低一些；但是没有检测到目的基因g10h、dxs和dat相应的mRNA杂交信号，原因可能是与所选的植物材料的生长阶段和生长环境有关。
This thesis performed a large scale of cloning for genes encoding key enzymes in terpene indole alkaloids (TIAs) pathway from Catharanthus roseus (L.)G.Don.via Gateway technology. These genes' mRNA expression levels in C. roseus seedlings were also detected. The results are as follows:
1. The full length sequences of dxs, dxr, g10h, sls, tdc, str and dat which encoded key enzymes involved in terpenoid indole alkaloids (TIAs) biosynthesis pathway were obtained via two-step Gateway PCR method, with the transcripted cDNA from total RNA of Catharanthus roseus seedlings as templates.
2. After PCR amplification, these attB-flanked DNA fragments were inserted into attPcontaining donor vector (pDONR201) by BP clonase to generate entry clones, respectively. Positive clones were screened first by colony cracking, PCR and restriction enzyme analysis, and then these inserted genes were confirmed by gene sequencing.
3. In order to get prokaryotic expression systems, ORFs of dxr, g10h, sls and str have been transferred to their destination vectors: pETG10A, pETG20A and pETG30A, and been transformed into competent cells of E.cloi host strain BL21(DE3). SDS-PAGE analysis results indicated that those recombinant expression clones which encoded enzymes of DXR, SLS and STR were overexpressed after IPTG induction.
4. Fusion proteins DXR-HIS, DXR-HIS-TrxA, DXR-HIS-GST, SLS-HIS and STR-HIS were purified by Ni-TED resin. According to the results of SDS-PAGE and Bradford, these purified proteins were qualified for antisera preparation.
5. Northern blot results displayed that transcript levels of dxr, sls, str and tdc in root, stem, leave, flower and fruit from C roseus were detected, in general the transcript levels in root and young leave were higher than in other tissues. But there're no signals for Northern blot ofglOh, dxs and dat. For one reason maybe that there're no mRNA transcripts in the seedling stage we chosed for these genes, or maybe they're inclined to be under strict post-transcriptional regulation.
引文
[1] O'Connor SE, Maresh JJ. Chemistry and biology of monoterpene indole alkaloid biosynthesis[J]. Nat Prod Rep, 2006, 23(4): 532-547
[2] Cragg GM, Newman DJ. Plants as a source of anti-cancer and anti-HIV agents[J]. Annals of applied biology, 2003, 143(2): 127-133
[3] Van der Heijden R, Jacobs DI, Snoeijer W, Hallard D, Verpoorte R. The Catharanthus alkaloids: pharmacognosy and biotec. hnoiogy[J]. Curr Med Chem, 2004, 11(5): 607-628
[4] Lorence A, Nessler C L. Camptothecin, over four decades of surprising findings[J]. Phytochemistry, 2004, 65(20): 2735-2749
[5] Blasko G, Cordell GA. Isolation, structure elucidation, and biosynthesis of the bisindole alkaloids of Catharanthus, in the alkaloids[M]. New York: Academic Press, 1990: 1-76
[6] Mathe G, Reizenstein P. Phase I pharmacologic study of a new Vinca alkaloid: navelbine[J]. Cancer letters, 1985, 27(3): 285-293
[7] Coltman C. Vinorelbine (Navelbine)-A new agent for the treatment of non-small cell lung cancer: A summary[J]. Lung cancer, 1995, 12(3): 312-316
[8] Moreno PRH, Van Der Heijiden R, Verpoorte R. Cell and tissures of Catharanthus roseus-A literature survery Ⅱ, Updating from 1988 to 1993[J]. Plant Cell Tiss and Org Cult, 1995, 42(1): 1-25
[9] 汪洪，孙敏，伍春莲，王颖．长春花生物碱生物合成途径中关键步骤与代谢调控研究进展[J]．中国中药杂志，2001，26(10)：656-659
[10] Jacobs DI, van der Heijden R, Verpoorte R. Proteomics in plant biotechnology and secondary metabolism research[J]. Phytochem Anal, 2000, 11(5): 277-287
[11] Shanks JV and Morgan J. Plant "hairy root" culture[J]. Biochemical Engineering, 1999, 23(6): 151-155
[12] Girl A and Lakshmi Narasu M. Transgenic hairy roots: recent trends and applications[J]. Biotcchnology Advances, 2000. 18(1): 1-22
[13] Sevon N and Oksman-Caldentey KM. Agrobacterium rhizogenes-mediatcd transformation: root cultures as a source of alkaloids[J]. Planta Med, 2002, 68(10): 859-868
[14] Sudha CG,. Obul Reddy B, Ravishankar GA, Seeni S. Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f a rare and endemic medicinal plant[J]. Biotechnol Letter, 2003, 25(8): 631-636
[15] Moyano E, Fomale S, Palazon J, Cusido RM, Bagni N, Pinol MT. Alkaloid Production in Duboisia Hybrid Hairy Root Cultures Overexpressing the Pmt Gene[J]. Phytochemistry, 2002, 59(7): 697-702
[16] Facchini PJ. Alkaloid biosynthesis in plants: biochemistry, cell biology, molecular regulation, and metabolic engineering applications[J]. Annu Rev Plant Physiol Plant Mol Biol, 2001, 52(1): 29—66
[17] Rischer H, Oresic M, Seppanen-Laakso T, Katajamaa M, Lammertyn F, Ardiles-Diaz W, Van Montagu MC, Inze D, Oksman-Caldentey KM, Goossens A. Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells[J]. Proceedings of the National Academy of Sceiences of the United States of America, 2006, 103(14): 5614-5619
[18] Zonneveld BJ, Leitch IJ, Bennett MD. First nuclear DNA amounts in more than 300 angiosperms[J]. Annals of Botany, 2005, 96(2): 229-244
[19] Murata J, Bienzle D, Brandle JE, Sensen CW, De Luca V. Expressed sequence tags from Madagascar periwinkle (Cataranthus roseus)[J]. FEBS Letters, 2006, 580(18): 4501-4507
[20] Amit KJ, Dubey PK, Rana RC. In vitro callus induction and biomass production of Catharanthus roseus[J]. Plant Arch. 2005, 5(1): 55—60
[21] Sreevalli Y, Baskaran K, Chandrashekara RS, Kulkami RN. Preliminary observations on the effeet of irrigation frequency and genotypes on yield and alkaloid concentration in periwinkle[J]. Journal of Medicinal and Aromatic Plant Sciences, 2001, 23 (1a): 356-358
[22] Jaleel CA, Gopi R, Lakshmanan GMA, Panneerselvam R. Triadimefon induced changes in the antioxidant metabolism and ajmalicine production in Catharanthus rosesus (L.) G. Don[J]. Plant Science, 2006, 171 (2): 271-276
[23] Gantet P and Memelink J. Transcription factors: tools to engineer the production of pharmacologically active plant metabolites[J]. TRENDS in Pharmacological Sciences, 2002, 23(12): 563-569
[24] Vaquez-Flota AF, Moreno-Valenzuela O, Miranda-Ham ML, Coello-Coello J, Loyola-Vargas VM. Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultues[J]. Plant Cell Tissue Organ Cult, 1994, 38(1): 273-279
[25] Aerts RJ, Gisi D, De Carolis E, De Luca V, Baumann TW. Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthus and Cinchona seedlings[J]. Plant Journal, 1994, 5(1): 635-643
[26] Yahia A, Kevers C, Gaspar T, Chenieux JC, Rideau M, Creche J. Cytokinins and ethylene stimulate indole alkaloid accumulation in cell suspension cultures of Catharanthus roseus by two distinct mechanisms[J]. Plant Science, 1998, 133(1): 9-15
[27] Papon N, Bremer J, Vansiri A, Andreu F, Rideau M. Creche J. Cytokinin and ethylene control indole alkaloid production at the level of the MEP/terpenoid pathway in Catharanthus roseus suspension cells[J]. Planta Medica, 2005, 71(6): 572-574
[28] Contin A, Heijden R, Lefeber AW, Verpoorte R. The iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate pathway in Catharanthus roseus cell culture[J]. FEBS Letter, 1998, 434(3): 413-416
[29] Zhengui Zheng, Madeline Wu. Cadmium treatment enhances the production of alkaloid secondary metabolites in Catharanthus roseus[J]. Plant Science, 2004, 166(22): 507-514
[30] Sreevalli Y, Kulkarni RN, Baskaran K, Chandrashekara RS. Increasing the content of leaf and root alkaloids of high alkaloid content mutants of periwinkle through nitrogen fertilization[J]. Industrial Crops and Products, 2004, 19(2): 191-195
[31] van der Fits L, Memelink J. ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[J]. Science, 2000, 289(5477): 295-297
[32] Vaquez-Flota F, Carrillo-Pech M, Minero-Garcia Y, Miranda-Ham ML. Alkaloid metabolism in wounded Catharanthus roseus seedlings[J]. Plant Physiology and Biochemistry, 2004, 42: 623-628
[33] Misra N, Gupta AK. Effect of salinity and different nitrogen sources on the activity of antioxidant enzymes and indole alkaloid content in Catharanthus roseus seedlings[J]. Journal of Plant Physiology, 2006, 163(1): 11-18
[34] Zarate R, Dirks C, van der Heijden R, Verpoorte R. Terpenoid indole alkloid profile changes in Catharanthus pusillus during development[J]. Plant Science, 2001, 160: 971-977
[35] Hong SB, Hughes EH, Shanks JV, San Ka-Yiu, Gibson SI. Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots[J]. Biotechnol Prog, 2003, 19(3): 1105-1108
[36] Mincheva Z, Courtois M, Creche J, Rideau M, Viaud-Massuard MC. One-pot synthesis of functionalized 4, 5-dihydroisoxazole derivatives via nitrile oxides and biological evaluation with plant cells[J]. Bioorganic and Medicinal Chemistry, 2004, 12(1): 191-197
[37] Singh RP, Jaiwal PK editors. Plant Genetic Engineering[M]. Houston: Sci Tech Publishing LLC, 2003: 1, 297-315
[38] Pasquali G, Porto DD, Fett-Neto AG. Metabolic Engineering of Cell Cultures versus Whole Plant Complexity in Production of Bioactive Monoterpene lndole Alkaloids: Recent Progress Related to Old Dilemma[J]. Journal of Bioscienee and Bioengineering, 2006, 101(4): 287-296
[39] Kuzuyama T, Shimiza T, Takahashi S. Fosmidomycin, a specific inhibitor of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for isoprenoid biosynethesis[J]. Tetrahedro Letter, 1998, 39: 7913-7916
[40] Proteau PJ. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase: an overview[J], Bioorganic Chemistry, 2004, 32(6): 483-493
[41] Xihou Yin, Proteau PJ. Characterization of native and histidine-tagged deoxyxylulose 5-phosphate reductoisomerase from the cyanobacterium Synechocystis sp. PCC6803[J]. Biochimica et Biophysica Acta, 2003, 1652: 75-81
[42] Veau B, Courtois M, Oudin A, Chenieux JC, Rideau M, Clastre M. Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus[J]. Biochinmica et Biophysica Acta, 2000, 1517(1): 159-163
[43] Markus LB, Rujan T, Martin W, Croteau R. Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes[J]. The proceedings of the National Academy of Sciences of the United States of American, 2000, 97(24): 13172-13177
[44] Irmler S, Sehr6der G, St-Pierre B, Crouch NP, Hotze M, Schmidt J, Strack D, Matem U, Schroder J. Indole alkaloid biosynthesis in Catharanthus roseus-new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase[J]. Plant Journal, 2000, 24(6): 797-804
[45] Veau B, Courtois M, Oudin A, Chenieux JC, Rideau M, Clastre M. Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus[J]. Bioehim Biophys Acta, 2000, 1517(1): 159-163
[46] Carretero-Paulet L, Cairo A, Botella-Pavia P. Besumbes O, Campos N, Boronat A, Rodriguez-Concepcion M. Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase[J]. Plant Mol Biol, 2006, 62(4): 683-695
[47] Khemvong S, Suvaehittanont W. Molecular cloning and expression of a cDNA encoding 1-deoxy-D-xylulose-5-phosphate synthase from oil palm Elaeis guineeensis Jaeq[J]. Plant Science, 2005, 169(3): 571-578
[48] Vetter HE Marigold U, Schroder G, Mamer FJ, Werck-Reichhart D, SchrGder J. Molecular analysis and heterologous expression of an inducible cytochrome P-450 protein from periwinkle (Catharanthus roseus L)[J]. Plant Physiology, 1992, 100(2): 998-1007
[49] Merillon JM, Ouelhazi L, Doireau P, Chenieux J C, Rideau M. Metabolic changes and alkaloid production in habituated and non-habituated cells of Catharanthus roseus growns in hormone-free medium: comparing hormone-deprived non-habituated cells with habituated cells[J]. Journal of Plant Physiology, 1989, 134(1): 54-60
[50] Moreno PRH. Influence of stress factors on the secondary metabolism in suspension cultured Catharanthus roseus cells[D]. Leiden: Leiden University PhD Thesis, 1994: 7-12
[51] McKnight TD, Bergey DR, Bumett RJ, Nessler CL. Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants[J]. Planta, 1991, 185(2): 148-152
[52] Kahn R, Durst F. Function and evolution of plant cytoehrome p450[J]. Recent Advance Phytochemistry, 2000, 34: 151-189
[53] Meijer AH, Verpoorte R, Hoge JHC. Regulation of enzymes and genes involved in terpenoid indole alkaloid biosynthesis in Catharanthus roseus[J]. J Plant Res, 1993, 3: 145-164
[54] Meehan TD, Coscia CJ. Hydroxylation of geraniol and nerol by a monooxygenase from Vinca rosea[J]. Biochem Biophys Res Commun, 1973, 53(4): 1043-1048
[55] Collu G, Unver N, Peltenburg-Looman A, Van der Heijden R, Verpoorte R, Memelink J. Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis[J]. FEBS Letter, 2001, 508(2): 215-220
[56] Canto-Canche BB, Meijer AH, Collu G, Verpoorte R, Loyola-Vargas VM. Characterization of a polyclonal antiserum against the monoterpene monooxygenase, geraniol 10-hydroxylase from Cantharanthus roseus[J]. Plant Physiology, 2005, 162(4): 393-402
[57] Moreno-Valenzuela OA, Galaz-Avalos RM. Minero-Garcia Y, Loyola-Vargas VM. Effect of differentiation on the regulation of indole alkaloid production in Catharanthus roseus hairy roots[J]. Plant Cell Reports, 1998, 18: 99-104
[58] Goddijn OJM, Pennings EJM, van der Helm P, Schilperoort RA, Verpoorte R. Hoge JHC. Overexpression of a tryptophan decarboxylase cDNA in Catharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production[J]. Transgenic Research, 1995, 4(5): 315-323
[59] Contin A, van der Heijden R, ten Hooen HJG, Verpoorte R. The inoculum size triggers tryptamine or secologanin biosynthesis in Catharanthus roseus cell culture[J]. Plant Science, 1998, 139(2): 205-211
[60] Stockigt J, Ruppert M. Strictosidine-the biosynthetic key to monoterpenoid indole alkaloids[J]. Comprehensive Natural Products Chemistry, 1999, 4: 109-138
[61] WANG Miao, LI Qiu-rong. Transient expression of strictosidine synthase in tobacco leaves by vacuum infiltration[J]. Acta biochimica et biophysica sinica, 2002, 34(6): 703-706
[62] Geerlings A, Hallard D, van der Heijden R, Verpoorte R. Alkaloid production by a Cinchona officinalis 'Ledgeriana' hairy root culture containing constitutive expression constructs of tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus[J]. Plant Cell Reports, 1999, 19: 191-196
[63] Dutta A, Batra J, Pandey-Rai S, Singh D, Kumar S, Sen J. Expression of terpenoid indole alkaloid corresponds to accumulation of related alkaloids in Catharanthus roseus[J]. Planta, 2005, 220(3): 376-383
[64] Memelink J, Verpoorte R, Kijne JW. ORCAnization of jasmonate responsive gene expression in alkaloid metabolism[J]. Trends in Plant Science, 2001, 6: 212-219
[65] Yamazaki Y, Urano A, Sudo H, Kitajima M, Takayama H, Yamazaki M, Aimi N, Saito K. Metabolite profiling of alkaloids and strictosidine synthase activity in camptothecin producing plants[J]. Phytochemistry, 2003, 62(3): 461-470
[66] Benoit SP, Vazquez-Flota FA, De Luca V. Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate[J]. Plant Cell, 1999, 11(5): 887-900
[67] Xue yan Ma, Koepke J, Fritzsch G, Diem R, Kutchan TM. Crystallization and preliminary Xray crystallographic ananlysis of strictosidine synthase from Rauvolfia: the first member of a novel enzyme family[J]. Biochimica et Biophysica Acta, 2004, 1702(1): 121-124
[68] Zarate R, Bonavia M, Geerlings A, van der Heijden R, Verpoorte R. Expression of strictosidine-D-glucosidase cDNA from Catharanthus roseus involved in the monoterpene indole alkaloid pathway in a transgenic suspension culture of Nicotiana tabacum[J]. Plant Physiol Biochem, 2001, 39(9): 763-769
[69] Stevens LH, Biom TJM, Verpoorte R. Subcellular localisation of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernaemontana divaricata[J]. Plant Cell Rep, 1993, 12: 573-576
[70] Madyastha KM, Ridgway JE, Dwyer JG, Coscia CJ. Subcellular localization of a cytochrome P-450-dependent monooxygenase in vesicles of the higher plant Catharnathus roseus[J]. J Cell Biol, 1999, 72: 303-313
[71] Geerlings A, Ibanez MML, Memelink J, van der Heijden R. Verpoorte R. Molecular cloning and analysis of strictosidine b-D-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus[J]. Journal of Biological Chemistry, 2000. 275(5): 3051-3056
[72] St-Pierre B, De Luca V. A cytochrome P-450 monooxygenase catalyzes the first step in the conversion of tabersonine to vindoline in Catharanthus roseus[J]. Plant Physiology, 1995, 109(1): 131-139
[73] De Luca V, Cutler AJ. Subcellular localization of enzymes involved in indole alkaloid biosynthesis in Catharanthusroseus[J]. Plant Physiology, 1987, 85(44): 1099-1102
[74] Dethier M, De Luca V. Partial purification of an N-methyltransferase involved in vindoline biosynthesis in Catharanthus roseus[J]. Phytochemistry, 1993, 32(3): 673-678
[75] De Carolis E, Chan F, Balsevich J, De Luca V. Isolation and characterization of a 2-oxoglutarate-dependent dioxygenase involved in the second-to-last step in vindoline biosynthesis[J]. Plant Physiology, 1990, 94(3): 1323-1329
[76] Sottomayor M, Lopez-Serrano M, Di-Cosmo F, Ros-Barcelo A. Purification and characterization of alpha-3', 4'-anhydrovinblastine synthase (peroxidase-like) from Catharanthus rosesus[J]. FEBS Letter, 1998, 428(3): 299-303
[77] Pasquali G, Goddijn OJ, de Waal A, Verpoorte R, Schilperoort RA. Coordinated regulation of two indole alkaloid biosynthetic genes from Catharanthus roseus by auxin and elicitors[J]. Plant Mol Biol, 1992, 18(6): 1121-1131
[78] Vazquez-Flota FA, De Luca V. Developmental and light regulation of desacetoxyvindoline 4-hydroxylase in Catharanthus roseus (L.) G. Don[J]. Plant Physiology, 1998, 117(4): 1351-1361
[79] De Luca V, St-Pierre B. The cell and developmental biology of alkaloid biosynthesis[J]. Trends Plant Science, 2000, 5(4): 168-173
[80] Meijer AH, Verpoorte R, Hoge JHC. Regulation and Enzymes and Genes Involed in Terpenoid Indole Alkaloid Biosynthesis In Catharanthus roseus[J]. J plant Res, 1993, 3: 145-151
[81] Landy A. Dynamic, structural, and regulatory aspects of Lambda site-specific recombination [J]. Annu Rev Biochem, 1989, 58: 913-949
[82] Hartley JL, Temple GF, Brasch MA. DNA cloning using in vitro site-specific recombination[J]. Genome Res, 2000, 10(11): 1788-1795
[83] 梅文倩，宋文强，潘怡，巩威，朱玉贤．利用Gateway克隆技术大规模克隆拟南芥转录因子[J]．分子植物育种，2004，2(3)：358-364
[84] 龙松华，张宁，邱德文，黎定军，黄炜．Gateway(?)技术构建交链孢菌JH505 cDNA文库[J]．微生物学报，2005，45(6)：963-965
[85] 陈其军，安瑞，周海梦，陈珈，王学臣．使用与Gateway技术兼容的T载体获得入门克隆[J]．生物化学与生物物理进展，2004，31(10)：951-954
[86] Shukla AK, Shasany AK, Gupta MM, Khanuja SPS. Transcriptome analysis in Catharanthus roseus leaves and roots for comparative terpenoid indole alkaloid rofiles[J]. Journal of Experimental Botany, 2006, 57(14): 3921-3932
[87] Riechmann JL and Ratcliffe OJ. A genomic perspective on plant transcription factors[J]. Curr Opin Plant Biol, 2000, 3(5): 423-434
[88] Murata J, De Luca V. Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus rosesus[J]. The Plant Journal, 2005, 44(4): 581-594
[2] Cragg GM, Newman DJ. Plants as a source of anti-cancer and anti-HIV agents[J]. Annals of applied biology, 2003, 143(2): 127-133
[3] Van der Heijden R, Jacobs DI, Snoeijer W, Hallard D, Verpoorte R. The Catharanthus alkaloids: pharmacognosy and biotec. hnoiogy[J]. Curr Med Chem, 2004, 11(5): 607-628
[4] Lorence A, Nessler C L. Camptothecin, over four decades of surprising findings[J]. Phytochemistry, 2004, 65(20): 2735-2749
[5] Blasko G, Cordell GA. Isolation, structure elucidation, and biosynthesis of the bisindole alkaloids of Catharanthus, in the alkaloids[M]. New York: Academic Press, 1990: 1-76
[6] Mathe G, Reizenstein P. Phase I pharmacologic study of a new Vinca alkaloid: navelbine[J]. Cancer letters, 1985, 27(3): 285-293
[7] Coltman C. Vinorelbine (Navelbine)-A new agent for the treatment of non-small cell lung cancer: A summary[J]. Lung cancer, 1995, 12(3): 312-316
[8] Moreno PRH, Van Der Heijiden R, Verpoorte R. Cell and tissures of Catharanthus roseus-A literature survery Ⅱ, Updating from 1988 to 1993[J]. Plant Cell Tiss and Org Cult, 1995, 42(1): 1-25
[9] 汪洪，孙敏，伍春莲，王颖．长春花生物碱生物合成途径中关键步骤与代谢调控研究进展[J]．中国中药杂志，2001，26(10)：656-659
[10] Jacobs DI, van der Heijden R, Verpoorte R. Proteomics in plant biotechnology and secondary metabolism research[J]. Phytochem Anal, 2000, 11(5): 277-287
[11] Shanks JV and Morgan J. Plant "hairy root" culture[J]. Biochemical Engineering, 1999, 23(6): 151-155
[12] Girl A and Lakshmi Narasu M. Transgenic hairy roots: recent trends and applications[J]. Biotcchnology Advances, 2000. 18(1): 1-22
[13] Sevon N and Oksman-Caldentey KM. Agrobacterium rhizogenes-mediatcd transformation: root cultures as a source of alkaloids[J]. Planta Med, 2002, 68(10): 859-868
[14] Sudha CG,. Obul Reddy B, Ravishankar GA, Seeni S. Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f a rare and endemic medicinal plant[J]. Biotechnol Letter, 2003, 25(8): 631-636
[15] Moyano E, Fomale S, Palazon J, Cusido RM, Bagni N, Pinol MT. Alkaloid Production in Duboisia Hybrid Hairy Root Cultures Overexpressing the Pmt Gene[J]. Phytochemistry, 2002, 59(7): 697-702
[16] Facchini PJ. Alkaloid biosynthesis in plants: biochemistry, cell biology, molecular regulation, and metabolic engineering applications[J]. Annu Rev Plant Physiol Plant Mol Biol, 2001, 52(1): 29—66
[17] Rischer H, Oresic M, Seppanen-Laakso T, Katajamaa M, Lammertyn F, Ardiles-Diaz W, Van Montagu MC, Inze D, Oksman-Caldentey KM, Goossens A. Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells[J]. Proceedings of the National Academy of Sceiences of the United States of America, 2006, 103(14): 5614-5619
[18] Zonneveld BJ, Leitch IJ, Bennett MD. First nuclear DNA amounts in more than 300 angiosperms[J]. Annals of Botany, 2005, 96(2): 229-244
[19] Murata J, Bienzle D, Brandle JE, Sensen CW, De Luca V. Expressed sequence tags from Madagascar periwinkle (Cataranthus roseus)[J]. FEBS Letters, 2006, 580(18): 4501-4507
[20] Amit KJ, Dubey PK, Rana RC. In vitro callus induction and biomass production of Catharanthus roseus[J]. Plant Arch. 2005, 5(1): 55—60
[21] Sreevalli Y, Baskaran K, Chandrashekara RS, Kulkami RN. Preliminary observations on the effeet of irrigation frequency and genotypes on yield and alkaloid concentration in periwinkle[J]. Journal of Medicinal and Aromatic Plant Sciences, 2001, 23 (1a): 356-358
[22] Jaleel CA, Gopi R, Lakshmanan GMA, Panneerselvam R. Triadimefon induced changes in the antioxidant metabolism and ajmalicine production in Catharanthus rosesus (L.) G. Don[J]. Plant Science, 2006, 171 (2): 271-276
[23] Gantet P and Memelink J. Transcription factors: tools to engineer the production of pharmacologically active plant metabolites[J]. TRENDS in Pharmacological Sciences, 2002, 23(12): 563-569
[24] Vaquez-Flota AF, Moreno-Valenzuela O, Miranda-Ham ML, Coello-Coello J, Loyola-Vargas VM. Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultues[J]. Plant Cell Tissue Organ Cult, 1994, 38(1): 273-279
[25] Aerts RJ, Gisi D, De Carolis E, De Luca V, Baumann TW. Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthus and Cinchona seedlings[J]. Plant Journal, 1994, 5(1): 635-643
[26] Yahia A, Kevers C, Gaspar T, Chenieux JC, Rideau M, Creche J. Cytokinins and ethylene stimulate indole alkaloid accumulation in cell suspension cultures of Catharanthus roseus by two distinct mechanisms[J]. Plant Science, 1998, 133(1): 9-15
[27] Papon N, Bremer J, Vansiri A, Andreu F, Rideau M. Creche J. Cytokinin and ethylene control indole alkaloid production at the level of the MEP/terpenoid pathway in Catharanthus roseus suspension cells[J]. Planta Medica, 2005, 71(6): 572-574
[28] Contin A, Heijden R, Lefeber AW, Verpoorte R. The iridoid glucoside secologanin is derived from the novel triose phosphate/pyruvate pathway in Catharanthus roseus cell culture[J]. FEBS Letter, 1998, 434(3): 413-416
[29] Zhengui Zheng, Madeline Wu. Cadmium treatment enhances the production of alkaloid secondary metabolites in Catharanthus roseus[J]. Plant Science, 2004, 166(22): 507-514
[30] Sreevalli Y, Kulkarni RN, Baskaran K, Chandrashekara RS. Increasing the content of leaf and root alkaloids of high alkaloid content mutants of periwinkle through nitrogen fertilization[J]. Industrial Crops and Products, 2004, 19(2): 191-195
[31] van der Fits L, Memelink J. ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[J]. Science, 2000, 289(5477): 295-297
[32] Vaquez-Flota F, Carrillo-Pech M, Minero-Garcia Y, Miranda-Ham ML. Alkaloid metabolism in wounded Catharanthus roseus seedlings[J]. Plant Physiology and Biochemistry, 2004, 42: 623-628
[33] Misra N, Gupta AK. Effect of salinity and different nitrogen sources on the activity of antioxidant enzymes and indole alkaloid content in Catharanthus roseus seedlings[J]. Journal of Plant Physiology, 2006, 163(1): 11-18
[34] Zarate R, Dirks C, van der Heijden R, Verpoorte R. Terpenoid indole alkloid profile changes in Catharanthus pusillus during development[J]. Plant Science, 2001, 160: 971-977
[35] Hong SB, Hughes EH, Shanks JV, San Ka-Yiu, Gibson SI. Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots[J]. Biotechnol Prog, 2003, 19(3): 1105-1108
[36] Mincheva Z, Courtois M, Creche J, Rideau M, Viaud-Massuard MC. One-pot synthesis of functionalized 4, 5-dihydroisoxazole derivatives via nitrile oxides and biological evaluation with plant cells[J]. Bioorganic and Medicinal Chemistry, 2004, 12(1): 191-197
[37] Singh RP, Jaiwal PK editors. Plant Genetic Engineering[M]. Houston: Sci Tech Publishing LLC, 2003: 1, 297-315
[38] Pasquali G, Porto DD, Fett-Neto AG. Metabolic Engineering of Cell Cultures versus Whole Plant Complexity in Production of Bioactive Monoterpene lndole Alkaloids: Recent Progress Related to Old Dilemma[J]. Journal of Bioscienee and Bioengineering, 2006, 101(4): 287-296
[39] Kuzuyama T, Shimiza T, Takahashi S. Fosmidomycin, a specific inhibitor of 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for isoprenoid biosynethesis[J]. Tetrahedro Letter, 1998, 39: 7913-7916
[40] Proteau PJ. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase: an overview[J], Bioorganic Chemistry, 2004, 32(6): 483-493
[41] Xihou Yin, Proteau PJ. Characterization of native and histidine-tagged deoxyxylulose 5-phosphate reductoisomerase from the cyanobacterium Synechocystis sp. PCC6803[J]. Biochimica et Biophysica Acta, 2003, 1652: 75-81
[42] Veau B, Courtois M, Oudin A, Chenieux JC, Rideau M, Clastre M. Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus[J]. Biochinmica et Biophysica Acta, 2000, 1517(1): 159-163
[43] Markus LB, Rujan T, Martin W, Croteau R. Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes[J]. The proceedings of the National Academy of Sciences of the United States of American, 2000, 97(24): 13172-13177
[44] Irmler S, Sehr6der G, St-Pierre B, Crouch NP, Hotze M, Schmidt J, Strack D, Matem U, Schroder J. Indole alkaloid biosynthesis in Catharanthus roseus-new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase[J]. Plant Journal, 2000, 24(6): 797-804
[45] Veau B, Courtois M, Oudin A, Chenieux JC, Rideau M, Clastre M. Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus[J]. Bioehim Biophys Acta, 2000, 1517(1): 159-163
[46] Carretero-Paulet L, Cairo A, Botella-Pavia P. Besumbes O, Campos N, Boronat A, Rodriguez-Concepcion M. Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase[J]. Plant Mol Biol, 2006, 62(4): 683-695
[47] Khemvong S, Suvaehittanont W. Molecular cloning and expression of a cDNA encoding 1-deoxy-D-xylulose-5-phosphate synthase from oil palm Elaeis guineeensis Jaeq[J]. Plant Science, 2005, 169(3): 571-578
[48] Vetter HE Marigold U, Schroder G, Mamer FJ, Werck-Reichhart D, SchrGder J. Molecular analysis and heterologous expression of an inducible cytochrome P-450 protein from periwinkle (Catharanthus roseus L)[J]. Plant Physiology, 1992, 100(2): 998-1007
[49] Merillon JM, Ouelhazi L, Doireau P, Chenieux J C, Rideau M. Metabolic changes and alkaloid production in habituated and non-habituated cells of Catharanthus roseus growns in hormone-free medium: comparing hormone-deprived non-habituated cells with habituated cells[J]. Journal of Plant Physiology, 1989, 134(1): 54-60
[50] Moreno PRH. Influence of stress factors on the secondary metabolism in suspension cultured Catharanthus roseus cells[D]. Leiden: Leiden University PhD Thesis, 1994: 7-12
[51] McKnight TD, Bergey DR, Bumett RJ, Nessler CL. Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants[J]. Planta, 1991, 185(2): 148-152
[52] Kahn R, Durst F. Function and evolution of plant cytoehrome p450[J]. Recent Advance Phytochemistry, 2000, 34: 151-189
[53] Meijer AH, Verpoorte R, Hoge JHC. Regulation of enzymes and genes involved in terpenoid indole alkaloid biosynthesis in Catharanthus roseus[J]. J Plant Res, 1993, 3: 145-164
[54] Meehan TD, Coscia CJ. Hydroxylation of geraniol and nerol by a monooxygenase from Vinca rosea[J]. Biochem Biophys Res Commun, 1973, 53(4): 1043-1048
[55] Collu G, Unver N, Peltenburg-Looman A, Van der Heijden R, Verpoorte R, Memelink J. Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis[J]. FEBS Letter, 2001, 508(2): 215-220
[56] Canto-Canche BB, Meijer AH, Collu G, Verpoorte R, Loyola-Vargas VM. Characterization of a polyclonal antiserum against the monoterpene monooxygenase, geraniol 10-hydroxylase from Cantharanthus roseus[J]. Plant Physiology, 2005, 162(4): 393-402
[57] Moreno-Valenzuela OA, Galaz-Avalos RM. Minero-Garcia Y, Loyola-Vargas VM. Effect of differentiation on the regulation of indole alkaloid production in Catharanthus roseus hairy roots[J]. Plant Cell Reports, 1998, 18: 99-104
[58] Goddijn OJM, Pennings EJM, van der Helm P, Schilperoort RA, Verpoorte R. Hoge JHC. Overexpression of a tryptophan decarboxylase cDNA in Catharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production[J]. Transgenic Research, 1995, 4(5): 315-323
[59] Contin A, van der Heijden R, ten Hooen HJG, Verpoorte R. The inoculum size triggers tryptamine or secologanin biosynthesis in Catharanthus roseus cell culture[J]. Plant Science, 1998, 139(2): 205-211
[60] Stockigt J, Ruppert M. Strictosidine-the biosynthetic key to monoterpenoid indole alkaloids[J]. Comprehensive Natural Products Chemistry, 1999, 4: 109-138
[61] WANG Miao, LI Qiu-rong. Transient expression of strictosidine synthase in tobacco leaves by vacuum infiltration[J]. Acta biochimica et biophysica sinica, 2002, 34(6): 703-706
[62] Geerlings A, Hallard D, van der Heijden R, Verpoorte R. Alkaloid production by a Cinchona officinalis 'Ledgeriana' hairy root culture containing constitutive expression constructs of tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus[J]. Plant Cell Reports, 1999, 19: 191-196
[63] Dutta A, Batra J, Pandey-Rai S, Singh D, Kumar S, Sen J. Expression of terpenoid indole alkaloid corresponds to accumulation of related alkaloids in Catharanthus roseus[J]. Planta, 2005, 220(3): 376-383
[64] Memelink J, Verpoorte R, Kijne JW. ORCAnization of jasmonate responsive gene expression in alkaloid metabolism[J]. Trends in Plant Science, 2001, 6: 212-219
[65] Yamazaki Y, Urano A, Sudo H, Kitajima M, Takayama H, Yamazaki M, Aimi N, Saito K. Metabolite profiling of alkaloids and strictosidine synthase activity in camptothecin producing plants[J]. Phytochemistry, 2003, 62(3): 461-470
[66] Benoit SP, Vazquez-Flota FA, De Luca V. Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate[J]. Plant Cell, 1999, 11(5): 887-900
[67] Xue yan Ma, Koepke J, Fritzsch G, Diem R, Kutchan TM. Crystallization and preliminary Xray crystallographic ananlysis of strictosidine synthase from Rauvolfia: the first member of a novel enzyme family[J]. Biochimica et Biophysica Acta, 2004, 1702(1): 121-124
[68] Zarate R, Bonavia M, Geerlings A, van der Heijden R, Verpoorte R. Expression of strictosidine-D-glucosidase cDNA from Catharanthus roseus involved in the monoterpene indole alkaloid pathway in a transgenic suspension culture of Nicotiana tabacum[J]. Plant Physiol Biochem, 2001, 39(9): 763-769
[69] Stevens LH, Biom TJM, Verpoorte R. Subcellular localisation of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernaemontana divaricata[J]. Plant Cell Rep, 1993, 12: 573-576
[70] Madyastha KM, Ridgway JE, Dwyer JG, Coscia CJ. Subcellular localization of a cytochrome P-450-dependent monooxygenase in vesicles of the higher plant Catharnathus roseus[J]. J Cell Biol, 1999, 72: 303-313
[71] Geerlings A, Ibanez MML, Memelink J, van der Heijden R. Verpoorte R. Molecular cloning and analysis of strictosidine b-D-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus[J]. Journal of Biological Chemistry, 2000. 275(5): 3051-3056
[72] St-Pierre B, De Luca V. A cytochrome P-450 monooxygenase catalyzes the first step in the conversion of tabersonine to vindoline in Catharanthus roseus[J]. Plant Physiology, 1995, 109(1): 131-139
[73] De Luca V, Cutler AJ. Subcellular localization of enzymes involved in indole alkaloid biosynthesis in Catharanthusroseus[J]. Plant Physiology, 1987, 85(44): 1099-1102
[74] Dethier M, De Luca V. Partial purification of an N-methyltransferase involved in vindoline biosynthesis in Catharanthus roseus[J]. Phytochemistry, 1993, 32(3): 673-678
[75] De Carolis E, Chan F, Balsevich J, De Luca V. Isolation and characterization of a 2-oxoglutarate-dependent dioxygenase involved in the second-to-last step in vindoline biosynthesis[J]. Plant Physiology, 1990, 94(3): 1323-1329
[76] Sottomayor M, Lopez-Serrano M, Di-Cosmo F, Ros-Barcelo A. Purification and characterization of alpha-3', 4'-anhydrovinblastine synthase (peroxidase-like) from Catharanthus rosesus[J]. FEBS Letter, 1998, 428(3): 299-303
[77] Pasquali G, Goddijn OJ, de Waal A, Verpoorte R, Schilperoort RA. Coordinated regulation of two indole alkaloid biosynthetic genes from Catharanthus roseus by auxin and elicitors[J]. Plant Mol Biol, 1992, 18(6): 1121-1131
[78] Vazquez-Flota FA, De Luca V. Developmental and light regulation of desacetoxyvindoline 4-hydroxylase in Catharanthus roseus (L.) G. Don[J]. Plant Physiology, 1998, 117(4): 1351-1361
[79] De Luca V, St-Pierre B. The cell and developmental biology of alkaloid biosynthesis[J]. Trends Plant Science, 2000, 5(4): 168-173
[80] Meijer AH, Verpoorte R, Hoge JHC. Regulation and Enzymes and Genes Involed in Terpenoid Indole Alkaloid Biosynthesis In Catharanthus roseus[J]. J plant Res, 1993, 3: 145-151
[81] Landy A. Dynamic, structural, and regulatory aspects of Lambda site-specific recombination [J]. Annu Rev Biochem, 1989, 58: 913-949
[82] Hartley JL, Temple GF, Brasch MA. DNA cloning using in vitro site-specific recombination[J]. Genome Res, 2000, 10(11): 1788-1795
[83] 梅文倩，宋文强，潘怡，巩威，朱玉贤．利用Gateway克隆技术大规模克隆拟南芥转录因子[J]．分子植物育种，2004，2(3)：358-364
[84] 龙松华，张宁，邱德文，黎定军，黄炜．Gateway(?)技术构建交链孢菌JH505 cDNA文库[J]．微生物学报，2005，45(6)：963-965
[85] 陈其军，安瑞，周海梦，陈珈，王学臣．使用与Gateway技术兼容的T载体获得入门克隆[J]．生物化学与生物物理进展，2004，31(10)：951-954
[86] Shukla AK, Shasany AK, Gupta MM, Khanuja SPS. Transcriptome analysis in Catharanthus roseus leaves and roots for comparative terpenoid indole alkaloid rofiles[J]. Journal of Experimental Botany, 2006, 57(14): 3921-3932
[87] Riechmann JL and Ratcliffe OJ. A genomic perspective on plant transcription factors[J]. Curr Opin Plant Biol, 2000, 3(5): 423-434
[88] Murata J, De Luca V. Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus rosesus[J]. The Plant Journal, 2005, 44(4): 581-594