茉莉酸甲酯处理雷公藤悬浮细胞的基因差异表达分析
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摘要
雷公藤(Tripterygium wilfordii Hook.f.)作为一种具有很好应用和开发前景的农用资源植物,其市场需求日益加大。为利用现代生物技术调控雷公藤次生代谢产物生物碱的生物合成提供依据,本文以茉莉酸甲酯(MeJA)为诱导子,采用继代3周的雷公藤悬浮细胞为材料,通过cDNA-AFLP技术分析了MeJA诱导处理前后雷公藤悬浮细胞中差异基因的表达情况,主要得到了以下结论:
1 MeJA在50μM至400μM浓度范围内对雷公藤总生物碱含量的积累呈抑制作用。未用MeJA处理的悬浮细胞中总生物碱的含量达到了624.13μg·g~(-1) ,而不同浓度MeJA处理对总生物碱的积累影响不同,其中200μM浓度处理下总碱的含量略高于其他处理浓度,为478.76μg·g~(-1),仍然低于未用MeJA处理的。
2通过优化RNA的提取、酶切、连接、预扩增和选择性扩增等步骤,建立了适宜MeJA诱导下雷公藤悬浮细胞差异基因表达的cDNA-AFLP体系。
3利用64对AFLP扩增引物对处理和对照的cDNA进行AFLP分析和Blastn生物信息比对,共筛选到19条明显差异表达的cDNA片段。其中有7条找到同源序列,这些基因可能参与了细胞中的信号转导,转录调控和能量代谢等。12条未在数据库中找到同源信息,推测为未知基因,有待进一步验证。
4在比对出的7条TDFs中,A2T4C4片段与MYB蛋白基因同源(Blastn比对结果为100%)。MYB蛋白做为一类转录因子组成的一部分,对揭示次生代谢物转录调节机制有重要意义,推测MYB基因可能参与了茉莉酸甲酯调控的雷公藤悬浮细胞次生代谢物质的合成。对此,有待于获得A2T4C4片段其全长序列,并转入雷公藤悬浮细胞培养体系,在雷公藤悬浮细胞中做转化验证。
As an excellent application and development prospects botanical pesticides,the market demand of Tripterygiun wilfordii Hook. f is gradually increasing. In order to provide the basis for taking advantage of modern biological technology regulation tripterygium wilfordii secondary metabolite biosynthesis of alkaloids, cell suspension culture of tripterygium wilfordii was taken as tested plant material in this study. cDNA amplified fragment length polymorphism analysis (cDNA-AFLP) was used to display transcripts whose expression is rapidly altered during the treatment of Methyl Jasmonate (MeJA) to cell suspension culture of tripterygium wilfordii. Two sixty and four primer combinations were used for selective amplification. The main results were as follows:
1 The total alkaloids production was inhibited to some extent when MeJA was added into the medium. The total alkaloids production went to 624.13μg·g~(-1) (DW)in the control. The alkaloids production in cell suspension culture with 200μM MeJA treated reached 478.76μgg~(-1) (DW), lightly higher than the other concentrations, was still lower than control.
2 Through optimization experiment steps including: restriction enzymes cut, ligation, pre- amplified and amplification, an optimized cDNA– AFLP system of differentially expressed genes in the suspension cell culture of tripterygiun wilfordii Hook. f with elictor MeJA was established.
3 64 pairs of AFLP amplification primers were used to analyze the cDNA of the treated and control. Among the transcript derived fragments (TDFs), by further re-PCR amplification, cloning and sequencing,through blast analysis of the sequences we know that: seven fragments were found to be involved in the cells of the signal transduction, transcription regulation and energy metabolism in GenBank, however the other twelve fragments were found no significant homologous sequences in data base, probably they were new cDNA sequences specific to cell suspension culture of tripterygium wilfordii.
4 By blast of these sequences in GenBank, Homologous analysis indicated that No.A2T4C4 fragment was acquainted identical to MYB-like protein gene(Maxident 100%). MYB protein as a kind of transcription factors is critical to reveal the mechanism of secondary metabolites transcriptional regulation. Presumably, MYB protein gene may be involved in the regulation of MeJA to tripterygium wilfordii cells suspension culture secondary metabolites biosynthesis. So, No.A2T4C4 fragment is important for further understanding of the function of MYB genes and the molecular mechanism of tripterygium wilfordii total alkaloids biosynthesis.
1 MeJA在50μM至400μM浓度范围内对雷公藤总生物碱含量的积累呈抑制作用。未用MeJA处理的悬浮细胞中总生物碱的含量达到了624.13μg·g~(-1) ,而不同浓度MeJA处理对总生物碱的积累影响不同,其中200μM浓度处理下总碱的含量略高于其他处理浓度,为478.76μg·g~(-1),仍然低于未用MeJA处理的。
2通过优化RNA的提取、酶切、连接、预扩增和选择性扩增等步骤,建立了适宜MeJA诱导下雷公藤悬浮细胞差异基因表达的cDNA-AFLP体系。
3利用64对AFLP扩增引物对处理和对照的cDNA进行AFLP分析和Blastn生物信息比对,共筛选到19条明显差异表达的cDNA片段。其中有7条找到同源序列,这些基因可能参与了细胞中的信号转导,转录调控和能量代谢等。12条未在数据库中找到同源信息,推测为未知基因,有待进一步验证。
4在比对出的7条TDFs中,A2T4C4片段与MYB蛋白基因同源(Blastn比对结果为100%)。MYB蛋白做为一类转录因子组成的一部分,对揭示次生代谢物转录调节机制有重要意义,推测MYB基因可能参与了茉莉酸甲酯调控的雷公藤悬浮细胞次生代谢物质的合成。对此,有待于获得A2T4C4片段其全长序列,并转入雷公藤悬浮细胞培养体系,在雷公藤悬浮细胞中做转化验证。
As an excellent application and development prospects botanical pesticides,the market demand of Tripterygiun wilfordii Hook. f is gradually increasing. In order to provide the basis for taking advantage of modern biological technology regulation tripterygium wilfordii secondary metabolite biosynthesis of alkaloids, cell suspension culture of tripterygium wilfordii was taken as tested plant material in this study. cDNA amplified fragment length polymorphism analysis (cDNA-AFLP) was used to display transcripts whose expression is rapidly altered during the treatment of Methyl Jasmonate (MeJA) to cell suspension culture of tripterygium wilfordii. Two sixty and four primer combinations were used for selective amplification. The main results were as follows:
1 The total alkaloids production was inhibited to some extent when MeJA was added into the medium. The total alkaloids production went to 624.13μg·g~(-1) (DW)in the control. The alkaloids production in cell suspension culture with 200μM MeJA treated reached 478.76μgg~(-1) (DW), lightly higher than the other concentrations, was still lower than control.
2 Through optimization experiment steps including: restriction enzymes cut, ligation, pre- amplified and amplification, an optimized cDNA– AFLP system of differentially expressed genes in the suspension cell culture of tripterygiun wilfordii Hook. f with elictor MeJA was established.
3 64 pairs of AFLP amplification primers were used to analyze the cDNA of the treated and control. Among the transcript derived fragments (TDFs), by further re-PCR amplification, cloning and sequencing,through blast analysis of the sequences we know that: seven fragments were found to be involved in the cells of the signal transduction, transcription regulation and energy metabolism in GenBank, however the other twelve fragments were found no significant homologous sequences in data base, probably they were new cDNA sequences specific to cell suspension culture of tripterygium wilfordii.
4 By blast of these sequences in GenBank, Homologous analysis indicated that No.A2T4C4 fragment was acquainted identical to MYB-like protein gene(Maxident 100%). MYB protein as a kind of transcription factors is critical to reveal the mechanism of secondary metabolites transcriptional regulation. Presumably, MYB protein gene may be involved in the regulation of MeJA to tripterygium wilfordii cells suspension culture secondary metabolites biosynthesis. So, No.A2T4C4 fragment is important for further understanding of the function of MYB genes and the molecular mechanism of tripterygium wilfordii total alkaloids biosynthesis.
引文
曹华兴,胡凡. 2002-1-28.植物悬浮培养细胞生产雷公藤总生物碱的方法.中国发明专利,ZL02100774.8
陈昆昌,杨仁洲,王传宝.1986.湖北产雷公藤的内酯成分.中草药, 17(6): 242
陈列忠,王开金,陈建明,吕仲贤,郑许松,韩国兴. 2007.雷公藤提取物对小菜蛾的生物活性.中国生物学防治, 23(2): 174~179
黄永鑫,臧玲玲,蒋琛. 2011.水杨酸和茉莉酸甲酯对喜树种子中次生代谢产物含量的影响.价值工程, 3:299
井莉,柯昌强,李希强,叶阳.2008.雷公藤中倍半萜生物碱的分离与结构鉴定.中国药物化学杂志, 18(3): 120~125
蒋科技,皮妍,侯嵘,唐克轩. 2010.植物内源茉莉酸类物质的生物合成途径及其生物学意义.植物学报,45(2):137~148
李春玉,李援朝. 1999.雷公藤化学成分研究.药学学报, 34(8): 605~607
李修伟. 2009.摇蚊GSTs特性分析及雷公藤杀虫活性应用基础研究. [博士学位论文].陕西杨凌:西北农林科技大学
李琰,冯俊涛,王永宏,郭小虎,祁新帅,张兴. 2008.雷公藤愈伤组织诱导及杀虫活性研究.西北农林科技大学学报, 36(5): 103~108
李琰. 2008.雷公藤组织培养生产次生代谢产物及其代谢调控研究. [博士学位论文].陕西杨凌:西北农林科技大学
李琰,冯俊涛,史晓燕,吴勇延,张兴. 2009.雷公藤组培产物中雷公藤甲素和总生物碱含量的测定.农药学学报, 11(3): 367~372
林光美,张敏,侯长红. 2009.雷公藤研究进展.中国农学通报, 25(23): 90~93
林绥,李援朝,刘子皎,邓福孝.2000.雷公藤中木脂素类成分的研究.中国药学杂志, 35(4):231~232
林绥,李援朝,樱井信子,曹剑虹,邓思珊,夏志林,谢狄霖. 2002.雷公藤榕碱的结构与分离.药学学报, 37(2): 128~130
罗都强,张兴. 2002.雷公藤非生物碱对菜青虫杀虫活性研究.农药学学报, 2(4): 94~96
吕燮余,张崇璞,陈沄. 1987.雷公藤的化学成分.江苏医药, 12: 640~643
马哲,梁茂新,张颖. 2011.中药雷公藤化学成分及药理作用研究进展.亚太传统医药, 7(3):157~160
齐凤慧,詹亚光,景天忠. 2008.诱导子对植物细胞培养中次生代谢物的调控机制.天然产物研究与开发, 20: 568~573
秦国伟. 1982.雷公藤多苷三萜类成分的研究.化学学报, 1982(40): 637
莎姆布鲁克J. 2005.分子克隆实验指南.第三版.黄培堂译.科学出版社
舒孝顺,高中洪,杨祥良. 2009.雷公藤生物碱的化学和药理活性研究进展.中国农学通报,25(23):90~93
周平坤. 2007.辐射致癌效应与机理(吴德昌院士科技文选集2).北京:中国军事科学出版社:405
吴琼,周应群,孙超,陈士林. 2009.人参皂苷生物合成和次生代谢工程.中国生物工程杂志, 29(10): 102~108
杨广隶. 2010.雷公藤胚状体培养合成次生代谢产物初步研究. [硕士学位论文].陕西杨凌:西北农林科技大学
杨致荣,毛雪,李润植. 2005.植物次生代谢基因工程研究进展.植物生理与分子生物学学报, 31(1): 11~18
尹作鸿,朱蔚华. 1992.雷公藤愈伤组织的固体培养.中国药学杂志, 27(1): 3~6
岳跃冲,范燕萍. 2011.植物萜类合成酶及其代谢调控的研究进展.园艺学报, 38(2):379~388
战晴晴,隋春,张杰,魏建. 2009.药用植物次生代谢相关酶基因克隆方法综述中国现代应用药学杂志, 26(10):805~809
赵剑,朱蔚华,王文科. 1999.植物生理学通讯, 35(1):60~68
张兴,李琰,冯俊涛,王智辉,王永宏. 2008-08-28.一种雷公藤悬浮细胞诱导不定根的方法.中国发明专利, 200810017697.8
周琳,冯俊涛,马志卿,张兴. 2006.雷公藤总生物碱对粘虫的生物活性.植物保护学报, 33(4): 401~406
周琳. 2007.雷公藤总生物碱杀虫作用研究. [博士学位论文].陕西杨凌:西北农林科技大学
朱金荣,黄宝康,吴锦忠,等. 2008.茉莉酸甲酯胁迫处理对薄荷次生代谢产物的影响.时珍国医国药, 19(8): 2011-2012
朱文超. 2010. cDNA-AFLP分析辣椒抗寒性及CaAQP基因克隆与表达研究. [硕士学位论文].陕西杨凌:西北农林科技大学
Aerts R J, Gisi D, Deearolis E, Deluea V, Baumann T W. 1994. Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthus and Cinchonaseedlings. Plant joumal, 5:635~643
Aerts R J, Schafer A, Hesse M, Baumann T W, Slusarenko A. 1996.Signalling molecules and the synthesis of alkaloids in Catharanthus roseus seedlings. Phytochemistry, 42:417~422
Aharoni A, de Vos R.C.H, Wein M, Sun Z,et al. 2001. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. The Plant Journal, 28(3): 319-332
Bachem C W B, Van der Hoeven R S, de Bruijn S M, Vreugdenhil D, Zabeau M, Visser R G F. 1996.
Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber development. Plant Journal, 9:745~753
Beroza M. 1953.Alkaloids from Triterygium wilfordii Hook:the structure of wilforine , wilfordine , wilforgine and wilfotrine. Chemical Science, 75 : 44
Brinker A M, Ma J, Lipsky P E, Raskin L.2007. Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae). Phytochemistry, 68:732~776
Broun P, Sommerville C. 2001. Progress in plant metabolic engineering. Proc Natl Acad Sci USA: , 98:8925~8927
Cappelli K, Porceddu A, Verini-Supplizi A, Capomaccio S, De Marchis F, Falcinelli M, Gaiti A, Silvestrelli M. 2005. cDNA AFLP-based techniques for studying transcript profiles in horses. Research in Veterinary Science, 79: 105~112
Cosio E G, Frey T, Verduyn R, Boom J V, Ebel J .1990. High-affinity binding of a synthetic heptaglucoside and fungal glucan phytoalexin elicitors to soybean membranes. FEBS Lett. 271:223~226
Creelman R A, Mullet J E. Oligosaccharins, brassinolides, and jasmonates: non-traditional regulators of plant growth, development, and gene expression. Plant Cell, 9:1211~1223
Deyamat.1983. The constituents of Eucommia ulmoids Oliv.Chem Pharm Bull, 31:2993
Diatchenko L, LauY F, Campbell A P, et al. 1996. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proceedings of the National Academy of Sciences, 93(12):6025~6030
Duan H Q, Takaishia Y. 1998. Structures of sesquiterpene poly ol alkaloids from Tripterygium hypoglaucum. Phytochemistry, 49:2185~2189
Duan H Q, Takaishia Y, Jia Y F, Li D. 2001. Sesquiterpene polyol esters from Tripterygium wilfordii. Phytochemistry, 56: 341~346
Farmer E E, Almeras E, Krishnamurthy V. 2003. Jasmonates and related oxylipins in plant responses to pathogenesis and herbivory. Curr Opin Plant Biol, 6:372~ 8
Feng N, Li Y, Tang J, Wang Y, Guo Me. 2010. cDNA-AFLP analysis on transcripts associated with hydroxysaf?or yellow A(HSYA) biosynthetic pathway in Carthamus tinctorius. Biochemical Systematics and Ecology, 1~10
Fischer A, Saedler H and Theissen. 1995. Restriction fragment length polymorphism coupled domain-directed differential display a highly efficient technique for expression analysis of multigene families. Proceedings of the National Academy of Sciences, 270(33):5331~5335
Goossens A, Hakkinen S T, Laakso I, Seppanen T S. 2003. A functional genomics approach toward the understanding of secondary metabolism in plant cells. Proc Natl Acad Sci USA, 100(14): 8595~8600 Gundlach H, Muller M J, Kutchan M J, Zenk M H.1993. Jasmonic acid is a signal transducer in elicitor - induced plant cell cultures. Proc Natl Acad Sci U.S.A., 89 :2389~2393
Habu Y, Fukada-Tanaka S , Hisatomi Y, Iida S. 1997. Amplified restriction fragment length polymorphism-based mRNA fingerprinting using a single restriction enzyme that recognizes a 4-bp sequence. Biochemical and Biophysical Research Communications, 234 : 516～521
Hsu T W, Tsai W C , Wang D P, Lin S, Hsiao Y Y, Chen W H, Chen H H. 2008. Differential gene expression analysis by cDNA-AFLP between ?ower buds of Phalaenopsis Hsiang Fei cv. H.F. and its somaclonal variant. Plant science, 175:415~422
Ketchum R E B, Gibson D M, Croteau R B, Shuler M L.1999.The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate. Biotechnol Bioeng, 62: 97~105
Kim Y S, Yeung E C, Hahn E J, Paek K Y.2007. Combined effects of phytohormone, indole-3-butyric acid,
and methyl jasmonate on root growth and ginsenoside production in adventitious root cultures of Panax ginseng C.A. Meyer. Biotechnol Lett, 29:1789~1792
Kimiko N, Chaiki Y, Wakako F, Yoshihisa T, Kozo S. 1998. Terpenoids in Transformed Root Culture of Tripterygium wilfordii. Phytochemistry, 49(6):1821~1824
Koornneef A, Leon-Reyes A, Ritsema T, Adriaan V, Floor C, Otter D, Loon V. 2008. Kinetics of salicylate mediated suppression of Jasmonate signaling reveal a role for redox modulation. Plant Physiology,147(3): 1358~1368
Kutney J P, Hewitt G M, Kurihara T, Salisbury P J, Sindelar R D, Stuart K L, Townsley P M, Chalmer W T, Jacoli G G. 1981. Cytotoxic diterpenes triptolide, tripdiolide, and cytotoxic triterpenes from tissue cultures of Tripterygiumwilfordii. Canadian Journal of Chemistry, 59(17) : 2677~2683
Kutney J P, Hewitt G M, Lee G, Piotrowska K, Roberts M, Rettig S J. 1992. Studies with tissue cultures of the Chinese herbal plant, Tripterygiumwilfordii. Isolation of metabolites of interest in rheumatoid arthritis, immunosuppression, and male contraceptive activity. Canadian Journal of Chemistry, 70(5) : 1455~ 1480
Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C, Weisshaar B, Martin C. 2000. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J, 19:6150~6161
Lee J, Vogt T, Schmidt J, Parthier B, Lobler M. 1997. Methyljasmonate-induced accumulation of coumaroyl conjugates in barley leaf segments. Phytochemistry, 44: 589~592
Liang P, Pardee A B. 1992. Differential differential display of eukaryotic messenger RNA by means of the polymerase chain reaction .Science, 257:967~971
Liang P, Averboukh L, Pardee A B . 1993. Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization. Nucleic Acids Res. 21:3269~3275
Menke F L , Parchmann S , Mueller M J , Kijne J W , Memelink J.1999.Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthelic gene in Catharanthus roseus. Plant Physiol, 119: 1289 ~1296
Mizukami H, Tavira Y, Ellis B E. 1993. Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures. Plant Cell Rep,12:706~709
Moyano E, Martinez-Garcia J F, and Martin C. 1996. Apparent redundancy in myb gene function provides gearing for the control of flavonoid biosynthesis in antirrhinum flowers. Plant Cell, 8(9): 1519-1532
Muller M J, Brodschelm W, Spannagl E, Zenk M H. 1993. Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid. Proc Natl Acad Sci USA, 90:7490~7494
Nuccio M L, Thomas T L. 1999. ATS1 and ATS3: two novel embryo-specific genes in Arabidopsis thaliana. Plant Molecular Biology, 39(6): 1153~1163
Reeves S A, Rubio M P, Louis D N, et al .1995. General method for PCR amplification and direct sequencing of mRNA differential display products. Bin Techniques, 18(1):18~20
Reijans M, Lascaris R, Groeneger A O, Wittenberg A, Wesselink E, Oeveren J V, Wit E D, Boorsma A, Voetdijk B, Spek H V D, Grivell L A, Simons G. 2003. Quantitative comparison of cDNA-AFLP, microarrays, and GeneChip expression data in Saccharomyces cerevisiae. Genomics, 82 :606–618
Rischer H, Oresic M, Seooanen-Laakso T, Katajamaa M, Lammertyn F, Ardiles-Diza W, Van Montagu M C E. 2006. Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells. Proceedings of the National Academy of Sciences, 103(14): 5614~5619
Saenz-Carbonell L, Loyola-Vargas V M.1996. Elicitation of tropane alkaloid biosynthesis in transformed root cultures of Datura stramonium. Appl Biochem Biotechnol, 61 :321
Schmitt J, Petersen M. 2002. Influence of methl Jasmonate and coniferyl alcohol on pinoresionl and matairesionl accumulation in a Forsyt hia intermedia suspension culture. Plant Cell Reports, 20(9): 885~890
Sharan M, Taguchi G, Gonda K, Jouke T, Shimosaka M, Hayashida N, Okazaki M. 1998. Effects of methyl jasmonate and elicitor on the activation of phenylalanine ammonia-lyase and the accumulation of scopoletin and scopolin in tobacco cell cultures. Plant Sci, 132: 13~19
Swingle W T, Haller H L, Siegler E H, Swingle M C. 1941. A Chinese Insecticidal Plant Tripterygium witfordii Introduced into the United State.Science, 93(2403):60~61
Takaishi Y. 1987. The chemical constituents of Tripterygium. Phytochemistry, 26(8):2325~2329
Van der Fits L,Memelink J. 2000. ORCA3,a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science, 289:295~297
Vom-Endt D, Kijne J W, Memelink J. 2002. Transcription factors controlling plant secondary metabolism: what regulates the regulators? Phytochemistry, 61: 107~114
William A, John G, Hendel J.1996. Reversed-phase high-performance liquid chromatographic determination of ginsenosides of Panax quinquefolium.J. Chromatogr, 775 :11~17
Wu C M, Zhou L M, Chai Y F, Wu Y T, Fan G R.2010. Three new sesquiterpene alkaloids from the root of Tripterygium wilfordii. Chinese Chemical Letters, 21: 830–833
Xu J Z, Lu J, Sun F, Zhu H Z, Wang L J, Zhang X Y, Ma Z J. 2011. Terpenoids from Tripterygium wilfordii. Phytochemistry, 3(18):1~6
Yamamoto S, Nakano T, Suzuki K, Shinshi H. 2004. Elicitor-induced activation of transcription via W box-related cis-acting elements from a basic chitinase gene by WRKY transcription factors in tobacco. Biochimica et Biophysica Acta, 1679: 279~287
Zabetakis I, Edwards R, O’Hagan D. 1999. Elicitation of tropane alkaloid biosynthesis in transformed root cultures of Datura stramonium. Phytochemistry, 50: 53~56
Zegzouti H, Marty C, Jones B, Bouquin T, Latch′e A, Pech J C, Bouzayen1 M. 1997. Plant Molecular Biology Reporter, 15: 236~245
Zhao J, Davis L C, Verpoorte R. 2005. Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances, 23(1):283~333
Zhao J, Hu Q, Guo Y Q, Zhu W H. 2001. Elicitor-induced indole alkaloid biosynthesis in Catharanthus roseus cell cultures is related to Ca2+ in?ux and the oxidative burst. Plant Sci, 161:423~431
陈昆昌,杨仁洲,王传宝.1986.湖北产雷公藤的内酯成分.中草药, 17(6): 242
陈列忠,王开金,陈建明,吕仲贤,郑许松,韩国兴. 2007.雷公藤提取物对小菜蛾的生物活性.中国生物学防治, 23(2): 174~179
黄永鑫,臧玲玲,蒋琛. 2011.水杨酸和茉莉酸甲酯对喜树种子中次生代谢产物含量的影响.价值工程, 3:299
井莉,柯昌强,李希强,叶阳.2008.雷公藤中倍半萜生物碱的分离与结构鉴定.中国药物化学杂志, 18(3): 120~125
蒋科技,皮妍,侯嵘,唐克轩. 2010.植物内源茉莉酸类物质的生物合成途径及其生物学意义.植物学报,45(2):137~148
李春玉,李援朝. 1999.雷公藤化学成分研究.药学学报, 34(8): 605~607
李修伟. 2009.摇蚊GSTs特性分析及雷公藤杀虫活性应用基础研究. [博士学位论文].陕西杨凌:西北农林科技大学
李琰,冯俊涛,王永宏,郭小虎,祁新帅,张兴. 2008.雷公藤愈伤组织诱导及杀虫活性研究.西北农林科技大学学报, 36(5): 103~108
李琰. 2008.雷公藤组织培养生产次生代谢产物及其代谢调控研究. [博士学位论文].陕西杨凌:西北农林科技大学
李琰,冯俊涛,史晓燕,吴勇延,张兴. 2009.雷公藤组培产物中雷公藤甲素和总生物碱含量的测定.农药学学报, 11(3): 367~372
林光美,张敏,侯长红. 2009.雷公藤研究进展.中国农学通报, 25(23): 90~93
林绥,李援朝,刘子皎,邓福孝.2000.雷公藤中木脂素类成分的研究.中国药学杂志, 35(4):231~232
林绥,李援朝,樱井信子,曹剑虹,邓思珊,夏志林,谢狄霖. 2002.雷公藤榕碱的结构与分离.药学学报, 37(2): 128~130
罗都强,张兴. 2002.雷公藤非生物碱对菜青虫杀虫活性研究.农药学学报, 2(4): 94~96
吕燮余,张崇璞,陈沄. 1987.雷公藤的化学成分.江苏医药, 12: 640~643
马哲,梁茂新,张颖. 2011.中药雷公藤化学成分及药理作用研究进展.亚太传统医药, 7(3):157~160
齐凤慧,詹亚光,景天忠. 2008.诱导子对植物细胞培养中次生代谢物的调控机制.天然产物研究与开发, 20: 568~573
秦国伟. 1982.雷公藤多苷三萜类成分的研究.化学学报, 1982(40): 637
莎姆布鲁克J. 2005.分子克隆实验指南.第三版.黄培堂译.科学出版社
舒孝顺,高中洪,杨祥良. 2009.雷公藤生物碱的化学和药理活性研究进展.中国农学通报,25(23):90~93
周平坤. 2007.辐射致癌效应与机理(吴德昌院士科技文选集2).北京:中国军事科学出版社:405
吴琼,周应群,孙超,陈士林. 2009.人参皂苷生物合成和次生代谢工程.中国生物工程杂志, 29(10): 102~108
杨广隶. 2010.雷公藤胚状体培养合成次生代谢产物初步研究. [硕士学位论文].陕西杨凌:西北农林科技大学
杨致荣,毛雪,李润植. 2005.植物次生代谢基因工程研究进展.植物生理与分子生物学学报, 31(1): 11~18
尹作鸿,朱蔚华. 1992.雷公藤愈伤组织的固体培养.中国药学杂志, 27(1): 3~6
岳跃冲,范燕萍. 2011.植物萜类合成酶及其代谢调控的研究进展.园艺学报, 38(2):379~388
战晴晴,隋春,张杰,魏建. 2009.药用植物次生代谢相关酶基因克隆方法综述中国现代应用药学杂志, 26(10):805~809
赵剑,朱蔚华,王文科. 1999.植物生理学通讯, 35(1):60~68
张兴,李琰,冯俊涛,王智辉,王永宏. 2008-08-28.一种雷公藤悬浮细胞诱导不定根的方法.中国发明专利, 200810017697.8
周琳,冯俊涛,马志卿,张兴. 2006.雷公藤总生物碱对粘虫的生物活性.植物保护学报, 33(4): 401~406
周琳. 2007.雷公藤总生物碱杀虫作用研究. [博士学位论文].陕西杨凌:西北农林科技大学
朱金荣,黄宝康,吴锦忠,等. 2008.茉莉酸甲酯胁迫处理对薄荷次生代谢产物的影响.时珍国医国药, 19(8): 2011-2012
朱文超. 2010. cDNA-AFLP分析辣椒抗寒性及CaAQP基因克隆与表达研究. [硕士学位论文].陕西杨凌:西北农林科技大学
Aerts R J, Gisi D, Deearolis E, Deluea V, Baumann T W. 1994. Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthus and Cinchonaseedlings. Plant joumal, 5:635~643
Aerts R J, Schafer A, Hesse M, Baumann T W, Slusarenko A. 1996.Signalling molecules and the synthesis of alkaloids in Catharanthus roseus seedlings. Phytochemistry, 42:417~422
Aharoni A, de Vos R.C.H, Wein M, Sun Z,et al. 2001. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. The Plant Journal, 28(3): 319-332
Bachem C W B, Van der Hoeven R S, de Bruijn S M, Vreugdenhil D, Zabeau M, Visser R G F. 1996.
Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber development. Plant Journal, 9:745~753
Beroza M. 1953.Alkaloids from Triterygium wilfordii Hook:the structure of wilforine , wilfordine , wilforgine and wilfotrine. Chemical Science, 75 : 44
Brinker A M, Ma J, Lipsky P E, Raskin L.2007. Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae). Phytochemistry, 68:732~776
Broun P, Sommerville C. 2001. Progress in plant metabolic engineering. Proc Natl Acad Sci USA: , 98:8925~8927
Cappelli K, Porceddu A, Verini-Supplizi A, Capomaccio S, De Marchis F, Falcinelli M, Gaiti A, Silvestrelli M. 2005. cDNA AFLP-based techniques for studying transcript profiles in horses. Research in Veterinary Science, 79: 105~112
Cosio E G, Frey T, Verduyn R, Boom J V, Ebel J .1990. High-affinity binding of a synthetic heptaglucoside and fungal glucan phytoalexin elicitors to soybean membranes. FEBS Lett. 271:223~226
Creelman R A, Mullet J E. Oligosaccharins, brassinolides, and jasmonates: non-traditional regulators of plant growth, development, and gene expression. Plant Cell, 9:1211~1223
Deyamat.1983. The constituents of Eucommia ulmoids Oliv.Chem Pharm Bull, 31:2993
Diatchenko L, LauY F, Campbell A P, et al. 1996. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proceedings of the National Academy of Sciences, 93(12):6025~6030
Duan H Q, Takaishia Y. 1998. Structures of sesquiterpene poly ol alkaloids from Tripterygium hypoglaucum. Phytochemistry, 49:2185~2189
Duan H Q, Takaishia Y, Jia Y F, Li D. 2001. Sesquiterpene polyol esters from Tripterygium wilfordii. Phytochemistry, 56: 341~346
Farmer E E, Almeras E, Krishnamurthy V. 2003. Jasmonates and related oxylipins in plant responses to pathogenesis and herbivory. Curr Opin Plant Biol, 6:372~ 8
Feng N, Li Y, Tang J, Wang Y, Guo Me. 2010. cDNA-AFLP analysis on transcripts associated with hydroxysaf?or yellow A(HSYA) biosynthetic pathway in Carthamus tinctorius. Biochemical Systematics and Ecology, 1~10
Fischer A, Saedler H and Theissen. 1995. Restriction fragment length polymorphism coupled domain-directed differential display a highly efficient technique for expression analysis of multigene families. Proceedings of the National Academy of Sciences, 270(33):5331~5335
Goossens A, Hakkinen S T, Laakso I, Seppanen T S. 2003. A functional genomics approach toward the understanding of secondary metabolism in plant cells. Proc Natl Acad Sci USA, 100(14): 8595~8600 Gundlach H, Muller M J, Kutchan M J, Zenk M H.1993. Jasmonic acid is a signal transducer in elicitor - induced plant cell cultures. Proc Natl Acad Sci U.S.A., 89 :2389~2393
Habu Y, Fukada-Tanaka S , Hisatomi Y, Iida S. 1997. Amplified restriction fragment length polymorphism-based mRNA fingerprinting using a single restriction enzyme that recognizes a 4-bp sequence. Biochemical and Biophysical Research Communications, 234 : 516～521
Hsu T W, Tsai W C , Wang D P, Lin S, Hsiao Y Y, Chen W H, Chen H H. 2008. Differential gene expression analysis by cDNA-AFLP between ?ower buds of Phalaenopsis Hsiang Fei cv. H.F. and its somaclonal variant. Plant science, 175:415~422
Ketchum R E B, Gibson D M, Croteau R B, Shuler M L.1999.The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate. Biotechnol Bioeng, 62: 97~105
Kim Y S, Yeung E C, Hahn E J, Paek K Y.2007. Combined effects of phytohormone, indole-3-butyric acid,
and methyl jasmonate on root growth and ginsenoside production in adventitious root cultures of Panax ginseng C.A. Meyer. Biotechnol Lett, 29:1789~1792
Kimiko N, Chaiki Y, Wakako F, Yoshihisa T, Kozo S. 1998. Terpenoids in Transformed Root Culture of Tripterygium wilfordii. Phytochemistry, 49(6):1821~1824
Koornneef A, Leon-Reyes A, Ritsema T, Adriaan V, Floor C, Otter D, Loon V. 2008. Kinetics of salicylate mediated suppression of Jasmonate signaling reveal a role for redox modulation. Plant Physiology,147(3): 1358~1368
Kutney J P, Hewitt G M, Kurihara T, Salisbury P J, Sindelar R D, Stuart K L, Townsley P M, Chalmer W T, Jacoli G G. 1981. Cytotoxic diterpenes triptolide, tripdiolide, and cytotoxic triterpenes from tissue cultures of Tripterygiumwilfordii. Canadian Journal of Chemistry, 59(17) : 2677~2683
Kutney J P, Hewitt G M, Lee G, Piotrowska K, Roberts M, Rettig S J. 1992. Studies with tissue cultures of the Chinese herbal plant, Tripterygiumwilfordii. Isolation of metabolites of interest in rheumatoid arthritis, immunosuppression, and male contraceptive activity. Canadian Journal of Chemistry, 70(5) : 1455~ 1480
Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C, Weisshaar B, Martin C. 2000. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J, 19:6150~6161
Lee J, Vogt T, Schmidt J, Parthier B, Lobler M. 1997. Methyljasmonate-induced accumulation of coumaroyl conjugates in barley leaf segments. Phytochemistry, 44: 589~592
Liang P, Pardee A B. 1992. Differential differential display of eukaryotic messenger RNA by means of the polymerase chain reaction .Science, 257:967~971
Liang P, Averboukh L, Pardee A B . 1993. Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization. Nucleic Acids Res. 21:3269~3275
Menke F L , Parchmann S , Mueller M J , Kijne J W , Memelink J.1999.Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthelic gene in Catharanthus roseus. Plant Physiol, 119: 1289 ~1296
Mizukami H, Tavira Y, Ellis B E. 1993. Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures. Plant Cell Rep,12:706~709
Moyano E, Martinez-Garcia J F, and Martin C. 1996. Apparent redundancy in myb gene function provides gearing for the control of flavonoid biosynthesis in antirrhinum flowers. Plant Cell, 8(9): 1519-1532
Muller M J, Brodschelm W, Spannagl E, Zenk M H. 1993. Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid. Proc Natl Acad Sci USA, 90:7490~7494
Nuccio M L, Thomas T L. 1999. ATS1 and ATS3: two novel embryo-specific genes in Arabidopsis thaliana. Plant Molecular Biology, 39(6): 1153~1163
Reeves S A, Rubio M P, Louis D N, et al .1995. General method for PCR amplification and direct sequencing of mRNA differential display products. Bin Techniques, 18(1):18~20
Reijans M, Lascaris R, Groeneger A O, Wittenberg A, Wesselink E, Oeveren J V, Wit E D, Boorsma A, Voetdijk B, Spek H V D, Grivell L A, Simons G. 2003. Quantitative comparison of cDNA-AFLP, microarrays, and GeneChip expression data in Saccharomyces cerevisiae. Genomics, 82 :606–618
Rischer H, Oresic M, Seooanen-Laakso T, Katajamaa M, Lammertyn F, Ardiles-Diza W, Van Montagu M C E. 2006. Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells. Proceedings of the National Academy of Sciences, 103(14): 5614~5619
Saenz-Carbonell L, Loyola-Vargas V M.1996. Elicitation of tropane alkaloid biosynthesis in transformed root cultures of Datura stramonium. Appl Biochem Biotechnol, 61 :321
Schmitt J, Petersen M. 2002. Influence of methl Jasmonate and coniferyl alcohol on pinoresionl and matairesionl accumulation in a Forsyt hia intermedia suspension culture. Plant Cell Reports, 20(9): 885~890
Sharan M, Taguchi G, Gonda K, Jouke T, Shimosaka M, Hayashida N, Okazaki M. 1998. Effects of methyl jasmonate and elicitor on the activation of phenylalanine ammonia-lyase and the accumulation of scopoletin and scopolin in tobacco cell cultures. Plant Sci, 132: 13~19
Swingle W T, Haller H L, Siegler E H, Swingle M C. 1941. A Chinese Insecticidal Plant Tripterygium witfordii Introduced into the United State.Science, 93(2403):60~61
Takaishi Y. 1987. The chemical constituents of Tripterygium. Phytochemistry, 26(8):2325~2329
Van der Fits L,Memelink J. 2000. ORCA3,a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science, 289:295~297
Vom-Endt D, Kijne J W, Memelink J. 2002. Transcription factors controlling plant secondary metabolism: what regulates the regulators? Phytochemistry, 61: 107~114
William A, John G, Hendel J.1996. Reversed-phase high-performance liquid chromatographic determination of ginsenosides of Panax quinquefolium.J. Chromatogr, 775 :11~17
Wu C M, Zhou L M, Chai Y F, Wu Y T, Fan G R.2010. Three new sesquiterpene alkaloids from the root of Tripterygium wilfordii. Chinese Chemical Letters, 21: 830–833
Xu J Z, Lu J, Sun F, Zhu H Z, Wang L J, Zhang X Y, Ma Z J. 2011. Terpenoids from Tripterygium wilfordii. Phytochemistry, 3(18):1~6
Yamamoto S, Nakano T, Suzuki K, Shinshi H. 2004. Elicitor-induced activation of transcription via W box-related cis-acting elements from a basic chitinase gene by WRKY transcription factors in tobacco. Biochimica et Biophysica Acta, 1679: 279~287
Zabetakis I, Edwards R, O’Hagan D. 1999. Elicitation of tropane alkaloid biosynthesis in transformed root cultures of Datura stramonium. Phytochemistry, 50: 53~56
Zegzouti H, Marty C, Jones B, Bouquin T, Latch′e A, Pech J C, Bouzayen1 M. 1997. Plant Molecular Biology Reporter, 15: 236~245
Zhao J, Davis L C, Verpoorte R. 2005. Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances, 23(1):283~333
Zhao J, Hu Q, Guo Y Q, Zhu W H. 2001. Elicitor-induced indole alkaloid biosynthesis in Catharanthus roseus cell cultures is related to Ca2+ in?ux and the oxidative burst. Plant Sci, 161:423~431