茉莉酸甲酯对匍枝筋骨草细胞生长和β-蜕皮甾酮积累的影响

doi:10.11686/cyxb2017449

摘要/Abstract

摘要： 匍枝筋骨草悬浮细胞中含有较高β-蜕皮甾酮,为了进一步提高其β-蜕皮甾酮含量,通过添加茉莉酸甲酯(MeJA)进行一系列试验研究。以4~10代筋骨草悬浮细胞为试验材料,测定不同处理浓度和处理时间的MeJA对细胞生长、β-蜕皮甾酮积累的影响,细胞生长量采用称量计数,β-蜕皮甾酮含量则使用高效液相进行检测。结果表明:匍枝筋骨草悬浮细胞生长曲线及β-蜕皮甾酮积累曲线符合Logistics模型。在细胞快速生长的初始期(第4天)或中期(第7天)添加不同浓度的MeJA,对细胞生长影响相对较小,生长曲线均有小高峰,分别出现在处理后第5天和第3天,干物质积累量分别达到0.60和0.62 g。而在细胞快速生长的高峰期添加MeJA,细胞生长曲线呈下降趋势,细胞鲜重和干重显著低于CK(P<0.05)。在细胞快速生长的初始期或中期添加MeJA后细胞鲜重与β-蜕皮甾酮积累量呈显著相关。在细胞快速生长的初始期或中期添加10~50 μmol·L^-1 MeJA后,细胞鲜重与CK对比表现为显著增加,其中添加50 μmol·L^-1 MeJA后细胞鲜重最佳,最高可达到35.90 g,显著高于其他处理(P<0.05)。而同样条件下β-蜕皮甾酮表现为积累量小幅增加,最高量为0.5095 mg·g^-1。添加100~200 μmol·L^-1 MeJA均会抑制细胞的生长,其中添加200 μmol·L^-1 MeJA细胞鲜重与CK相比显著下降,抑制率最高可达到38.88%。而添加100~200 μmol·L^-1 MeJA后β-蜕皮甾酮积累量表现为大幅提升,最高可达3.5315 mg·g^-1,为同期CK的14.44倍(P<0.01)。在细胞快速生长的高峰期添加10~200 μmol·L^-1 MeJA后,细胞鲜重与CK相比都表现为下降,说明在此时添加这些浓度MeJA可抑制细胞的生长,最高抑制率可达31.01%。而在细胞快速生长的高峰期添加10~50 μmol·L^-1 MeJA后,β-蜕皮甾酮积累量可在短时间内大量激增,β-蜕皮甾酮积累量最高达到1.4136 mg·g^-1,是CK的5.06倍(P<0.01)。添加100~200 μmol·L^-1 MeJA则积累量较少。在细胞快速生长的中期添加100 μmol·L^-1 MeJA条件下对细胞的刺激较小,β-蜕皮甾酮积累量最高,达到3.5315 mg·g^-1。

关键词: 茉莉酸甲酯, 筋骨草, β, -蜕皮甾酮, 细胞生长, 次生代谢产物

Abstract: High β-ecdysterone (β-EC) levels were contained in a cell suspension of Ajuga lobata. In order to further improve β-EC content, jasmonic acid methylester was added. The effects of adding jasmonic acid methylester was based on 4-10 generations of cell suspension of A. lobata and the influence of different MeJA concentrations and adding times on cell growth and β-EC accumulation was assessed. For β-EC content, high performance liquid chromatography (HPLC) was utilised. Results showed that the growth curve of the cell suspension and accumulative line of β-EC followed the Logistics model. Adding different concentrations of MeJA in the early (the 4th day of culture) or middle stages (the 7th day of culture) of the rapid cell growth period did not greatly affect cell growth; the growth curves were similar and each produced a peak. Adding MeJA on the 4th day and 7th day of culture resulted in peaks mostly occurring on the 5th and 3rd day after MeJA addition respectively. Dry weights of the cell suspension reached 0.60 g and 0.62 g respectively. Adding different concentrations of MeJA at the peak (the 10th day of culture) of rapid cell growth, resulted in a decline in growth in all cell cultures. The growth of suspended cells was significantly reduced by adding MeJA. Fresh and dry weights were usually significantly lower than that in the control group (P<0.05). The cell fresh weights were significantly correlated with β-EC contents after adding MeJA into the culture system during the early or middle stages of rapid cell growth. After adding 10-50 μmol·L^-1 MeJA cell fresh weights increased significantly compared with that of the control. Adding 50 μmol·L^-1 MeJA achieved the highest cell fresh weight (35.90 g), significantly higher than other treatments (P<0.05). Similarly β-EC content (0.5095 mg·g^-1) was also highest with 50 μmol·L^-1 MeJA. Adding 200 μmol·L^-1 MeJA, significantly reduced cell fresh weights compared with the control (38.88% reduction). After adding 100-200 μmol·L^-1 MeJA β-EC content was greatly increased, reaching 3.5315 mg·g^-1, 14.44 times the control treatment(P<0.01). At the peak of rapid cell growth (10th day of culture), adding 10-200 μmol·L^-1MeJA, reduced cell fresh weights compared with that of the control. The results illustrated that adding MeJA during cell growth could inhibit cell growth. Adding 10-50 μmol·L^-1 MeJA at the peak of rapid cell growth resulted in a massive surge in β-EC content shortly after adding, the highest level being 1.4136 mg·g^-1, 5.06 times the control (P<0.01). When the MeJA concentration reached 100-200 μmol·L^-1, the accumulation of β-EC was significantly reduced. We concluded that cell growth was mildly stimulated by adding 100 μmol·L^-1 MeJA treatment in the middle stage of cell rapid growth period with β-EC content reaching 3.5315 mg·g^-1.

Key words: jasmonic acid methylester (MeJA), Ajuga lobata, beta-ecdysterone (β-EC), cell growth, secondary metabolic products

王晓梅, 迟德富, 宇佳. 茉莉酸甲酯对匍枝筋骨草细胞生长和β-蜕皮甾酮积累的影响[J]. 草业学报, 2018, 27(9): 95-109.

WANG Xiao-mei, CHI De-fu, YU Jia. The effect of jasmonic acid methylester on cell growth and β-ecdysterone accumulation in Ajuga lobata[J]. Acta Prataculturae Sinica, 2018, 27(9): 95-109.

参考文献

[1] Xu X Y, Zheng Y M, Wang L.The research progress of plant ecdysone. Journal of Biology, 2005, 22(2): 12-14.
胥秀英, 郑一敏, 王龙. 植物蜕皮激素的研究进展. 生物学杂志, 2005, 22(2): 12-14.
[2] Zhu W M, Yang X S, He H P, et al. Phytoecdysones from Porana discifera. Acta Botanica Yunnanica, 2000, 22(3): 351-357.
朱伟明, 杨小生, 何红平, 等. 搭棚藤植物中的蜕皮激素. 云南植物研究, 2000, 22(3): 351-357.
[3] Zhou J, Li J, Weng Q, et al. The regulation of ecdysteroid on insect growth and productive processes. Chinese Journal of Applied Entomology, 2013, 50(5): 1413-1418.
周娇, 李娟, 翁强, 等. 蜕皮激素对昆虫生长及生殖过程的调控. 应用昆虫学报, 2013, 50(5): 1413-1418.
[4] Zhao Y L, Xing Z Y, Chen J L, et al. Research advances in pharmacological effects of phytoecdysones. Anti-Infection Pharmacy, 2010, 7(3): 147-152.
赵一璐, 邢朝云, 陈晶磊, 等. 植物蜕皮激素药理活性研究进展. 抗感染药学, 2010, 7(3): 147-152.
[5] Qiu M H.The diversity of plant secondary metabolites from the plant ecdysone//Chen Y Y. Biodiversity and human future——The 2nd national conference on biodiversity conservation and sustainable use. Beijing: China Forestry Press, 1998: 54-62.
邱明华. 从植物蜕皮激素看植物次生代谢产物的多样性//陈宜瑜. 生物多样性与人类未来——第二届全国生物多样性保护与持续利用研讨会论文集. 北京: 中国林业出版社, 1998: 54-62.
[6] Coll J, Tandron Y A, Zeng X N.New phytoecdysteroids from cultured plants of Ajuga nipponensis Makino. Steroids, 2007, 72(3): 270-277.
[7] Ramazonov N S, Mamadalieva N Z, Bobaev I D.Phytoecdysteroids from five species of the genus Silene. Chemistry of Natural Compounds, 2007, 43(1): 117-118.
[8] Qiu M H, Nie R L.Development and utilization of plant ecdysone. Natural Resources, 1989, (3): 42.
邱明华, 聂瑞麟. 植物蜕皮激素的开发和利用. 自然资源, 1989, (3): 42.
[9] Zhu X D, An Y L.A study on the technology for extracting pure β- ecdysone from Cyanotis arachnoidea. Journal of Southwest Forestry College, 2003, 23(2): 23-25.
朱向东, 安银岭. 从露水草中提取纯化β-蜕皮激素工艺研究. 西南林学院学报, 2003, 23(2): 23-25.
[10] Guo X D, Huang Z S, Bao Y D, et al. Chemical constituents of Ajuga decumbens. Chinese Traditional and Herbal Drugs, 2005, 36(5): 646-648.
郭新东, 黄志纾, 鲍雅丹, 等. 筋骨草的化学成分研究. 中草药, 2005, 36(5): 646-648.
[11] Qiu Z G, Chi D F.Phytoecdysones in Ajuga multiflora and A. linearifolia. Journal of Northeast Forestry University, 1997, 25(5): 85-86.
邱志国, 迟德富. 两种筋骨草中蜕皮激素类物质的分析. 东北林业大学学报, 1997, 25(5): 85-86.
[12] Chen X X, Lu W.The determination of phytoecdysones content in three Ajuga plants of Fujian province. Strait Pharmaceutical Journal, 2007, 19(12): 64-65.
陈小霞, 卢伟. 福建省三种常用筋骨草属植物中蜕皮甾酮的含量测定. 海峡药学, 2007, 19(12): 64-65.
[13] Li X, Qian J J, Li X C, et al. A study on edysterone production of Ajuga lobata D.Don on cell lines in suspension culture. Chinese Agricultural Science Bulletin, 2013, 29(34): 127-133.
黎昕, 钱晶晶, 李晓灿, 等. 匍枝筋骨草悬浮培养生产蜕皮甾酮的研究. 中国农学通报, 2013, 29(34): 127-133.
[14] Qian J J, Li X, Yang Y Y, et al. β-ecdysterone accumulation and regulation in Ajuga lobata suspension culture. Journal of Beijing Forestry University, 2015, 37(9): 91-100.
钱晶晶, 黎昕, 杨月月, 等. 匍枝筋骨草悬浮培养中β-蜕皮甾酮的积累与调控. 北京林业大学学报, 2015, 37(9): 91-100.
[15] Xue R H, Jin S A.Methyl jasmonate: A vital signaling molecule. Letters In Biotechnology, 2006, 17(6): 985-988.
薛仁镐, 金圣爱. 茉莉酸甲酯:一种重要的植物信号转导分子. 生物技术通讯, 2006, 17(6): 985-988.
[16] Wang X W.The research progress of Jasmonic acid involved in plant adversity stress. Anhui Agricultural Science Bulletin, 2008, 14(6): 24-29.
汪新文. 茉莉酸参与植物逆境胁迫的研究进展. 安徽农学通报, 2008, 14(6): 24-29.
[17] Cai K Z, Dong T X, Xu T.The physiological roles and resistance control in stress environment of jasmonates. Ecology and Environment, 2006, 15(2): 397-404.
蔡昆争, 董桃杏, 徐涛. 茉莉酸类物质(JAs)的生理特性及其在逆境胁迫中的抗性作用. 生态环境, 2006, 15(2): 397-404.
[18] Zhang X B.Effects of methyl jasmonate in plant growth and defense to stress. Biology Teaching, 2006, 31(12): 2-4.
张小冰. 茉莉酸类物质在植物生长发育和胁迫防御中的作用. 生物学教学, 2006, 31(12): 2-4.
[19] Zhao X J, Chi D F, Li X C.Study on the regeneration system of Ajuga lobata. Chinese Agricultural Science Bulletin, 2011, 27(4): 120-124.
赵晓杰, 迟德富, 李晓灿. 匍枝筋骨草再生体系构建. 中国农学通报, 2011, 27(4): 120-124.
[20] Wang W X, An Q, Wang Y, et al. A study on cell suspension culture and flavonoids accumulation of Stellera chamaejasme. Acta Prataculturae Sinica, 2010, 19(6): 132-139.
王文星, 安琪, 汪莹, 等. 瑞香狼毒细胞悬浮培养及黄酮积累的研究. 草业学报, 2010, 19(6): 132-139.
[21] Lin L D, Zhang D X, Chen D.Establishment and analysis of variation curve of biomass accumulation in single-cell cloned Scutellaria callus. Journal of Anhui Agricultural Sciences, 2010, 38(19): 10072-10074.
林立东, 张东向, 陈妲. 黄芩单细胞克隆愈伤组织生物量积累变化曲线的建立与分析. 安徽农业科学, 2010, 38(19): 10072-10074.
[22] Wang T, Li L, Lin L B, et al. Establishment of cell suspension culture system and growth model Salvia miltiorrhiza from Shandong. Journal of Sichuan Agricultural University, 2015, 33(1): 33-38.
王涛, 李乐, 林良斌, 等. 山东丹参细胞悬浮培养体系及生长模型的建立. 四川农业大学学报, 2015, 33(1): 33-38.
[23] Gui L Y, Liu S S, Chen Z M.Plant resistance to insects induced by application of exogenous jasmonic acid and methyl jasmonate. Acta Entomologica Sinica, 2004, 47(4): 507-514.
桂连友, 刘树生, 陈宗懋. 外源茉莉酸和茉莉酸甲酯诱导植物抗虫作用及其机理. 昆虫学报, 2004, 47(4): 507-514.
[24] Beltrano J, Ronco M G, Montaldi E R, et al. Senescence of flag leaves ears of wheat hastened by methyl jasmonate. Journal of Plant Growth Regulation, 1998, 17(1): 53-57.
[25] Shunsuke I, Kasuhito H, Makiko N, et al. Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in tobacco cell cultures. Plant Molecular Biology, 1998, 38(6): 1101-1111.
[26] Grsic S, Kirchheim B, Pieper K, et al. Induction of auxin biosynthetic enzymes by jasmonic acid and in club root diseased Chinese cabbage plants. Physiologia Plantarum, 1999, 105(3): 521-531.
[27] Jan T, Anders F, Lars R.A wound and methyl jasmonate-inducible transcript coding for a myrosinase associated protein with similarities to an early nodulin. Plant Physiology, 1996, 110(2): 483-491.
[28] Wang B J, Zhang X Q, Sun L W, et al. Effect of methyl jasmonate on suspension cells growth and hyperforin production of Hypericum perforatum L. Plant Physiology Communications, 2008, 44(4): 669-672.
王保军, 张秀清, 孙立伟, 等. 茉莉酸甲酯(Me JA)对贯叶连翘悬浮细胞生长和贯叶金丝桃素产量的影响. 植物生理学通讯, 2008, 44(4): 669-672.
[29] Sirvent T, Gibson D.Induction of hypericins and hyperforin in Hypericum peforatum L. in response to biotic and chemical elicitors. Physiological and Molecular Plant Pathology, 2002, 60: 311-320.
[30] Xu Q Y, Yu Y H, Bin J H.Effects of MeJA and ABA on the growth of mungbean hypocotyls. Journal of Guangxi Agriculture and Biology Science, 2003, 22(2): 114-118.
徐曲毅, 禹艳红, 宾金华. 茉莉酸甲酯和脱落酸对绿豆下胚轴生长的影响. 广西农业生物科技, 2003, 22(2): 114-118.
[31] Zhang J J, Xu M J.Effects of nitric oixde and mehtyl jasmonate on the biaealin porduction and cell growth in suspension cultures of Scuetllaria baicalensis. Chinese Bulletin of Botany, 2006, 23(4): 374-379.
张进杰, 徐茂军. NO和茉莉酸甲醋对黄芩悬浮细胞生长及黄芩苷合成的影响. 植物学通报, 2006, 23(4): 374-379.
[32] Lu M B, Su X N, Mei X G.The influence of fungi inducer on cell in Taxus chinensis. Journal Huazhong University of Science Technology, 1998, 26(7): 107-109.
鲁明波, 苏湘那, 梅兴国. 真菌诱导物对红豆杉细胞的影响. 华中理工大学学报, 1998, 26(7): 107-109.
[33] Li H L, Sun Z Y, Zhao L J, et al. Effects of methyl jasmonate on growth and development of Oriental Lily. Journal of China Agricultural University, 2010, 15(1): 25-30.
李红利, 孙振元, 赵梁军, 等. 茉莉酸甲酯对东方百合生长发育的影响. 中国农业大学学报, 2010, 15(1): 25-30.
[34] Song P, Xia K, Wu C W, et al. Differential response of floret opening in male sterile and male fertile rices to methyl jasmonate. Acta Botanica Sinica, 2001, 43(5): 480-485.
宋平, 夏凯, 吴传万, 等. 雄性不育和可育水稻开颖对茉莉酸甲酯响应的差异. 植物学报, 2001, 43(5): 480-485.
[35] Zeng X C, Zhou X, Zhang W, et al. Opening of rice floret in rapid response to methyl jasmonate. Plant Growth Regulation, 1999, 18: 153-158.
[36] Boland W, Hopke J, Donath J, et al. Jasmonic acid and coronatin induce odor production in plants. Angewandte Chemie (International Edition in English), 1995, 34(15): 1600-1602.
[37] Dicke M, Gols R, Ludeking D, et al. Jasmonic acid and herbivory differentially induce carnivore attracting plant volatiles in lima bean plants. Journal of Chemical Ecology, 1999, 25(8): 1907-1922.
[38] Farmer E E.Surface-to-air signals. Nature, 2001, 411(6839): 854-856.
[39] Xu Y J, He Z P, Lin W.Effects of methyl jasmonate on the growth and accumulation of picfeltrarraenin glycosides of Picria fel-terrae Lour. Crops, 2013, 12(2): 80-84.
谢阳姣, 何志鹏, 林伟. 茉莉酸甲酯对苦玄参生长及苦玄参苷积累的影响研究. 作物杂志, 2013, 12(2): 80-84.
[40] Aftab T, Khan M M A, Idrees M, et al. Methyl jasmonate counteracts boron toxicity by preventing oxidative stress and regulating antioxidant enzyme activities and artemisinin biosynthesis in Artemisia annua L. Protoplasma, 2011, 248(3): 601-612.
[41] Miao X Y, Zhu W H, Zhang X M, et al. Effects of salicylic acid, methyl jasmonate and ultrasound on the production of cumarins and growth of Radix Glehniae Callus. Food Science, 2016, 37(9): 181-185.
苗晓燕, 朱维红, 张筱梅, 等. 水杨酸、茉莉酸甲酯及超声波对北沙参愈伤组织生长及产香豆素的影响. 食品科学, 2016, 37(9): 181-185.
[42] Zhu H T, Li J, Li Y, et al. Effects of methy jasmonate on the content of total saponin in tissue culture seedlings of Panax notoginseng. Journal of West China Forestry Science, 2014, 43(2): 72-78.
朱宏涛, 李江, 李元, 等. 茉莉酸甲酯对三七组培苗中总皂苷含量的影响. 西部林业科学, 2014, 43(2): 72-78.
[43] Cai J, Ma Y, Chen J W, et al. Effects of methyl jasmonate on accumulation of furanocoumarins in Changium smyrnioides and its suspension cells. Chinese Journal of Traditional Chinese Medicine, 2017, 42(1): 76-82.
蔡静, 马赟, 陈建伟, 等. 茉莉酸甲酯对明党参植株和悬浮细胞中呋喃香豆素积累的影响. 中国中药杂志, 2017, 42(1): 76-82.
[44] Wang H Y, Luo H, Sun M.Application of elicitor to cell culture of medicinal plants. Chinese Traditional and Herbal Drugs, 2004, 35(8): 3-7.
王和勇, 罗恒, 孙敏. 诱导子在药用植物细胞培养中的应用. 中草药, 2004, 35(8): 3-7.