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草业学报 ›› 2018, Vol. 27 ›› Issue (9): 95-109.DOI: 10.11686/cyxb2017449

• 研究论文 • 上一篇    下一篇

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

王晓梅, 迟德富*, 宇佳   

  1. 东北林业大学林学院, 黑龙江 哈尔滨 150040
  • 收稿日期:2017-10-23 修回日期:2017-11-23 出版日期:2018-09-20 发布日期:2018-09-20
  • 通讯作者: E-mail: chidefu@126.com
  • 作者简介:王晓梅(1993-),女,山东烟台人,在读博士。E-mail: 2550691573@qq.com
  • 基金资助:
    国家自然科学基金项目(31370649)资助

The effect of jasmonic acid methylester on cell growth and β-ecdysterone accumulation in Ajuga lobata

WANG Xiao-mei, CHI De-fu*, YU Jia   

  1. College of Forestry, Northeast Forestry University, Harbin 150040, China
  • Received:2017-10-23 Revised:2017-11-23 Online:2018-09-20 Published:2018-09-20

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