药学学报, 2014, 49(8): 1194-1199
引用本文:
刘娟, 韩晓敏, 梁良, 刘庆昌, 徐艳红, 杨成民, 张争, 孙晶, 魏建和. 濒危南药白木香悬浮细胞体系的建立[J]. 药学学报, 2014, 49(8): 1194-1199.
LIU Juan, HAN Xiao-min, LIANG Liang, LIU Qing-chang, XU Yan-hong, YANG Cheng-min, ZHANG Zheng, SUN Jing, WEI Jian-he. Establishment of a cell suspension culture system of endangered Aquilaria sinensis (Lour.) Gilg[J]. Acta Pharmaceutica Sinica, 2014, 49(8): 1194-1199.

濒危南药白木香悬浮细胞体系的建立
刘娟1, 韩晓敏3, 梁良4, 刘庆昌5, 徐艳红1, 杨成民1, 张争1,2, 孙晶6, 魏建和1,2
1. 中国医学科学院、北京协和医学院药用植物研究所, 北京 100193;
2. 中国医学科学院、北京协和医学院药用植物研究所海南分所, 海南省南药资源保护与开发重点实验室, 海南 万宁 571533;
3. 燕山大学, 河北 秦皇岛 066004;
4. 山东中医药大学, 山东 济南 250355;
5. 中国农业大学, 北京 100094;
6. 东北林业大学生命科学院, 黑龙江 哈尔滨 150040
摘要:
以白木香(Aquilaria sinensis) 茎尖为材料,利用诱导形成的愈伤组织构建悬浮细胞体系。结果表明,诱导愈伤组织最适培养基为MS + 2.0 mg·L-1 NAA + 1.0 mg·L-1 6-BA;经12次继代培养的愈伤组织生长旺盛、质地疏松适于悬浮培养;将其置于液体培养基MS + 2.0 mg·L-1 NAA + 1.0 mg·L-1 6-BA + 500.0 mg·L-1 CH中震荡培养,建成悬浮细胞体系。悬浮细胞生长曲线呈S型,初期增长缓慢,4~6天为对数增长期,7~12天进入平台期,12天以后细胞生长速度及活力迅速下降;GC-MS结果显示,培养0、3、6、9、12天悬浮细胞不含沉香倍半萜,使用100 μmol·L-1 MeJA处理24 h可检测到倍半萜。本实验构建的白木香悬浮细胞体系均一稳定、细胞活力良好,可作为研究伤害诱导白木香形成倍半萜的离体体系,同时也具备离体生产沉香倍半萜的潜力。
关键词:    白木香      愈伤组织      悬浮细胞培养      沉香倍半萜     
Establishment of a cell suspension culture system of endangered Aquilaria sinensis (Lour.) Gilg
LIU Juan1, HAN Xiao-min3, LIANG Liang4, LIU Qing-chang5, XU Yan-hong1, YANG Cheng-min1, ZHANG Zheng1,2, SUN Jing6, WEI Jian-he1,2
1. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China;
2. Hainan Branch Institute of Medicinal Plant, Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Wanning 571533, China;
3. Yanshan University, Qinhuangdao 066004, China;
4. Shandong University of Traditional Chinese Medicine, Jinan 250355, China;
5. China Agricultural Univesity, Beijing 100094, China;
6. College of Life Science, Northeast Forestry University, Harbin 150040, China
Abstract:
Aquilaria sinensis callus induced by stem tips were used to establish the suspension cell system. The results showed that the most suitable medium for callus induction and subculture is MS + 2.0 mg·L-1 NAA + 1.0 mg·L-1 6-BA. After 12 times of subculture, the energetic and loose callus, which were appropriate for cell suspension culture, were cultured and shook in liquid medium MS + 2.0 mg·L-1 NAA + 1.0 mg·L-1 6-BA + 500.0 mg·L-1 casein hydrolysate (CH) to establish the suspension cell system. The growth curve of suspension cells showed a "S" type. At the beginning of the culture, cell density increased slowly; during 4 to 6 days, suspension cells reached logarithmic growth period; during 7 to 12 days, suspension cells were in the platform period; but after 12 days, cell density and activity went down obviously. Agarwood sesquiterpenes were not detected in the suspension cells during the growth period, however, they could be detected in MeJA treated suspension cells. In this study, a stable and active growing suspension cell system was established, which was a proper system to study the mechanism of agarwood sesquiterpene formation, and additionally provided a potential way to generate agarwood sesquiterpenes through application of cell culture.
Key words:    Aquilaria sinensis    callus    suspension cell culture    agarwood sesquiterpene   
收稿日期: 2014-01-20
基金项目: 国家自然科学基金资助项目(81173481,31100220,31000136);北京协和医学院“新兴与交叉学科科研团队”项目(2012N07);科技部创新人才计划重点领域创新团队(2013),2013北京协和医学院研究生创新基金(2013-1007-07).
通讯作者: 魏建和 Tel/Fax:86-10-57833359,E-mail:wjianh@263.net
Email: wjianh@263.net
相关功能
PDF(7176KB) Free
打印本文
0
作者相关文章
刘娟  在本刊中的所有文章
韩晓敏  在本刊中的所有文章
梁良  在本刊中的所有文章
刘庆昌  在本刊中的所有文章
徐艳红  在本刊中的所有文章
杨成民  在本刊中的所有文章
张争  在本刊中的所有文章
孙晶  在本刊中的所有文章
魏建和  在本刊中的所有文章

参考文献:
[1] China Pharmacopoeia Committee. Pharmacopoeia of People's Republic of China (中华人民共和国药典: 一部) [S]. Beijing: China Medical Science Press, 2010: 172.
[2] Du Q, Wang ZH, Liang HZ, et al. Study on tissue culture of Aquilaria sinensis [J]. China J Chin Mater Med (中国中药杂志), 2001, 26: 679-680.
[3] Liu Y, Chen H, Yang Y, et al. Whole-tree agarwood-induced technique: an efficient novel technique for producing high-quality agarwood in cultivated Aquilaria sinensis trees [J]. Molecules, 2013, 18: 3086-3106.
[4] CITES. Amendments to Appendix Ⅰand Ⅱof CITES [C]//Proceedings of Thirteenth Meeting of the Conference of the Parties. Bangkok, Thailand, 2004: 2-4.
[5] Xu Y, Yang X, Zhang Z, et al. Cloning and expression analysis of HMG-CoA reductase from Aquilaria sinensis (Lour.) Gilg [J]. Acta Pharm Sin (药学学报), 2013, 48: 953-959.
[6] Zhang Z, Yang Y, Wei J, et al. Advances in studies on mechanism of agarwood formation in Aquilaria sinensis and its hypothesis of agarwood formation induced by defense response [J]. Chin Tradit Herb Drugs (中草药), 2010, 41: 156-159.
[7] Dai J, Zhu W, Wu Y, et al. Effects of precursors and fungal elicitors on GKB production in suspension cultured cells of Ginkgo biloba L [J]. Acta Pharm Sin (药学学报), 2000, 35: 151-155.
[8] Sang Y, Sun M, Zhang Q. Study on cell suspension culture of Opisthopappus taihangensis [J]. J Henan Agric Univ (河南农业大学学报), 2011, 45: 177-182.
[9] Bai MZ, Liu X, Zhang X, et al. Study on the cell suspension culture of Aquilaria sinensis [J]. J Anhui Agric Sci (安徽农业科学), 2009, 37: 15150-15152.
[10] Kumeta Y, Ito M. Characterization of δ-guaiene synthases from cultured cells of Aquilaria, responsible for the formation of the sesquiterpenes in agarwood [J]. Plant Physiol, 2010, 154: 1998-2007.
[11] Ito M, Okimoto K, Yagura T, et al. Induction of sesquiterpenoid production by methyl jasmonate in Aquilaria sinensis cell suspension culture [J]. J Essent Oil Res, 2005, 17: 175-180.
[12] Okudera Y, Ito M. Production of agarwood fragrant constituents in Aquilaria calli and cell suspension cultures [J]. Plant Biotechnol, 2007, 26: 307.
[13] He ML, Qi SY, Hu LJ. Inducible formation of 2-(2-phenylethyl) chromones in cell suspension culture of Aquilaria sinensis [J]. Guihaia, 2007, 27: 627-632.
[14] Xu YH, Zhang Z, Wang MX, et al. Identification of genes related to agarwood formation: transcriptome analysis of healthy and wounded tissues Aquilaria sinensis [J]. BMC Genomics, 2013, 14: 227.
[15] Chen HQ, Yang Y, Xue J, et al. Comparison of composi-tions and antimicrobial activities of essential oils from chemically stimulated agarwood, wild agarwood and healthy trees of Aquilaria sinensis (Lour.) Gilg [J]. Molecules, 2011, 16: 1.
[16] Wei JH, Zhang Z, Yang Y, et al. Study on synthesis mechanism and application of induced medicinal secondary metabolites [C]//The Ninth National Medicinal Plants and Herbal Medicine Symposium Sets (第九届全国药用植物及植物药学术研讨会论文集), 2010: 19.