油菜素内酯促进药用植物青蒿中青蒿素的生物合成

doi:10.7523/j.issn.2095-6134.2015.04.008

摘要/Abstract

摘要： 油菜素内酯是一种植物生长调节剂,它对启动子序列中含有E-box(CANNTG)元件的基因具有明显的调控作用.青蒿素生物合成的关键基因ADS、DBR 2和CYP71AV1 启动子中均含有此序列元件.研究油菜素内酯处理对青蒿中青蒿素生物合成的影响.结果表明,80 μmol/L的油菜素内酯是处理的最适浓度;80 μmol/L油菜素内酯处理4 d 后青蒿素含量比对照增加1倍多,在该处理条件下青蒿素生物合成的关键基因ADS, CYP71AV1和DBR2 的表达均被上调.本研究的结果表明,油菜素内酯通过促进青蒿素生物合成关键基因的表达而增加青蒿素的合成.这一研究结果对生产上提高青蒿素含量具有一定参考价值.

关键词: 青蒿, 青蒿素, 基因表达, 油菜素内酯

Abstract: As a plant growth regulator, brassinosteroid is able to activate the expression of the genes in their promoter sequences containing E-box (CANNTG) cis-element. Since the key enzyme genes of artemisinin biosynthesis pathway, including ADS (amorpha-4,11-diene synthase), DBR2 (artemisinic aldehyde Δ11(13) reductase), and CYP71AV1 (cytochrome P450 monooxygenase), all contain this element, the effect of brassinosteroid on artemisinin biosynthesis is investigated. Our results show that 80 μmol/L of brassinosteroid is the optimal concentration for foliar application of Artemisia annua L.; the artemisinin content increases by more than 100% compared to that of the control after 80 μmol/L brassinosteroid treatment for 4 days; and the expressions of artemisinin biosynthesis-related genes ADS, CYP71AV1, and DBR2 are all up-regulated after treatment. The above results indicate that brassinosteroid promotes the biosynthesis of artemisinin by up-regulating the expression of the key genes involved in artemisinin biosynthesis, which could be used as a possible way to increase artemisinin production of medicinal plant A. annua.

Key words: Artemisia annua L., artemisinin, gene expression, brassinosteroid

中图分类号:

TQ464

池剑亭, 申亚琳, 舒位恒, 王红. 油菜素内酯促进药用植物青蒿中青蒿素的生物合成[J]. 中国科学院大学学报, 2015, 32(4): 476-481.

CHI Jianting, SHEN Yalin, SHU Weiheng, WANG Hong. Artemisinin biosynthesis of Artemisia annua L. promoted by brassinosteroid[J]. , 2015, 32(4): 476-481.

参考文献

[1] Klayman D L. Qinghaosu (artemisinin): an antimalarial drug from China [J]. Science, 1985, 228(4703): 1 049-1 055.
[2] Mutabingwa T K. Artemisinin-based combination therapies (ACTs): best hope for malaria treatment but inaccessible to the needy [J]. Acta Tropica, 2005, 95(3): 305-315.
[3] Bouwmeester H J, Wallaart T E, Janssen M H, et al. Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis [J]. Phytochemistry, 1999, 52(5): 843-854.
[4] Teoh K H, Polichuk D R, Reed D W, et al. Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin [J]. FEBS Letters, 2006, 580(5): 1 411-1 416.
[5] Zhang Y, Teoh K H, Reed D W, et al. The molecular cloning of artemisinic aldehydeΔ11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua L. [J]. Journal of Biological Chemistry, 2008, 283(31): 21 501-21 508.
[6] Duke M V, Paul R N, Elsohly H N, et al. Localization of artemisinin and artemisitene in foliar tissues of glanded and glandless biotypes of Artemisia annua L. [J]. International Journal of Plant Sciences, 1994, 155(3): 365-372.
[7] Ferreira J F, Simon J E, Janick J. Relationship of artemisinin content of tissue-cultured, greenhouse-grown, and field-grown plants of Artemisia annua L. [J]. Planta Medica, 1995, 61(4): 351-355.
[8] Paddon C J, Westfall P J, Pitera D J, et al. High-level semi-synthetic production of the potent antimalarial artemisinin [J]. Nature, 2013, 496(7446): 528-532.
[9] Ro D K, Paradise E M, Ouellet M, et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast [J]. Nature, 2006, 440(7086): 940-943.
[10] Zhang L, Jing F, Li F, et al. Development of transgenic Artemisia annua L (Chinese wormwood) plants with an enhanced content of artemisinin, an effective anti-malarial drug, by hairpin-RNA-mediated gene silencing [J]. Biotechnology and Applied Biochemistry, 2009, 52(3): 199-207.
[11] Alam P, Abdin M Z. Overexpression of HMG-CoA reductase and amorpha-4,11-diene synthase genes in Artemisia annua L. and its influence on artemisinin content [J]. Plant Cell Reports, 2011, 30(10): 1 919-1 928.
[12] Zhang Y S, Ye H C, Liu B Y, et al. Exogenous GA3 and flowering induce the conversion of artemisinic acid to artemisinin in Artemisia annua plants [J]. Russian Journal of Plant Physiology, 2005, 52(1): 58-62.
[13] Kapoor R, Chaudhary V, Bhatnagar A K. Effects of arbuscular mycorrhiza and phosphorus application on artemisinin concentration in Artemisia annua L.[J]. Mycorrhiza, 2007, 17(7): 581-587.
[14] Wang H H, Ma C F, Li Z Q, et al. Effects of exogenous methyl jasmonate on artemisinin biosynthesis and secondary metabolites in Artemisia annua L.[J]. Industrial Crops and Products, 2010, 31(2): 214-218.
[15] Pu G B, Ma D M, Chen J L, et al. Salicylic acid activates artemisinin biosynthesis in Artemisia annua L.[J]. Plant Cell Reports, 2009, 28(7): 1 127-1 135.
[16] Liu D H, Zhang L D, Li C X, et al. Effect of wounding on gene expression involved in artemisinin biosynthesis and artemisinin production in Artemisia annua L.[J]. Russian Journal of Plant Physiology, 2010, 57(6): 882-886.
[17] Jing F Y, Zhang L, Li M Y, et al. Abscisic acid (ABA) treatment increases artemisinin content in Artemisia annua L. by enhancing the expression of genes in artemisinin biosynthetic pathway [J]. Biologia, 2009, 64(2): 319-323.
[18] Lei C Y, Ma D M, Pu G B, et al. Foliar application of chitosan activates artemisinin biosynthesis in Artemisia annua L. [J]. Industrial Crops and Products, 2011, 33(1): 176-182.
[19] Clouse S D. Molecular genetic studies confirm the role of brassinosteroids in plant growth and development [J]. Plant Journal, 1996, 10(1): 1-8.
[20] Li J, Chory J. Brassinosteroid actions in plants [J]. Journal of Experimental Botany, 1999, 50(332): 275-282.
[21] Feldmann K A, Marks M D, Christianson M L, et al. A dwarf mutant of Arabidopsis generated by T-DNA insertion mutagenesis [J]. Science, 1989, 243(4896): 1 351-1 354.
[22] Chory J, Nagpal P, Peto C A. Phenotypic and genetic-analysis of det2, a new mutant that affects light-regulated seedling development in Arabidopsis [J]. Plant Cell, 1991, 3(5): 445-459.
[23] Mangelsdorf D J, Thummel C, Beato M, et al. The nuclear receptor superfamily: the second decade [J]. Cell, 1995, 83(6): 835-839.
[24] Friedrichsen D M, Joazeiro C A, Li J, et al. Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase [J]. Plant Physiology, 2000, 123(4): 1 247-1 256.
[25] Li J, Chory J. A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction [J]. Cell, 1997, 90(5): 929-938.
[26] Wang Z Y, Nakano T, Gendron J, et al. Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroids biosynthesis [J]. Developmental Cell, 2002, 2(4): 505-513.
[27] Yin Y, Wang Z Y, Mora-Garcia S, et al. BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation [J]. Cell, 2002, 109(2): 181-191.
[28] He J X, Gendron J M, Sun Y, et al. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses [J]. Science, 2005, 307(5715): 1 634-1 638.
[29] Yin Y, Vafeados D, Tao Y, et al. A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis [J]. Cell, 2005, 120(2): 249-259.
[30] Sun Y, Fan X Y, Cao D M, et al. Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis [J]. Developmental Cell, 2010, 19(5): 765-777.
[31] Yu X, Li L, Zola J, et al. A brassinosteroid transcriptional network revealed by genome-wide identification of BES1 target genes in Arabidopsis thaliana [J]. Plant Journal, 2011, 65(4): 634-646.
[32] Yin Y H, Vafeados D, Tao Y, et al. A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis [J]. Cell, 2005, 120(2): 249-259.
[33] Zhao S S, Zeng M Y. Application of precolumn reaction to high-performance liquid chromatography of qinghaosu in animal plasma [J]. Analytical Chemistry, 1986, 58(2): 289-292.
[34] Ma D M, Pu G B, Lei C Y, et al. Isolation and characterization of AaWRKY1, an Artemisia annua L. transcription factor that regulates the amorpha-4,11-diene synthase gene, a key gene of artemisinin biosynthesis [J]. Plant Cell Physiology, 2009, 50(12): 2 146-2 161.