Ca^(2+)对丹参培养细胞中迷迭香酸合成及其相关酶活性的影响

Effects of calcium on synthesis of rosmarinic acid and related enzymes in suspension cultures of Salvia miltiorrhiza

探究了外界Ca2+(0~50 mmol/L)对丹参培养细胞迷迭香酸合成及其相关酶活性的影响,并利用细胞膜钙离子通道抑制剂异搏定(Verpamil,VP)及钙离子载体A23187初步探讨了外界Ca2+浓度变化影响丹参培养细胞次生代谢的机制。结果显示:培养6 d时的丹参细胞中迷迭香酸积累量与外界Ca2+浓度显著相关,其中10 mmol/L Ca2+最有利于迷迭香酸的合成,迷迭香酸最大积累量达20.149 mg/g DW,比1 mmol/L和3 mmol/LCa2+处理分别高37.3%和20.4%。分析迷迭香酸合成的两条支路上的关键酶PAL和TAT活性变化发现,两种酶活性亦受外界Ca2+浓度影响,且活性变化先于迷迭香酸的积累,说明这两种酶均参与迷迭香酸的生物合成,但PAL比TAT促进作用更明显。进一步用VP和A23187处理发现,外界Ca2+影响迷迭香酸的合成是通过影响胞内Ca2+浓度实现的,胞外Ca2+内流可能参与了这一过程。

We studied the influence of the concentration of Ca2＋ （0-50 mmol/L） in culture medium on the synthesis of rosmarinic acid （RA） and related enzymes in Salvia mi#iorrhiza suspension cultures. Using verpamil （VP, a calcium channel antagonist） and ionophore A23187, we studied the mechanism of secondary metabolites of Salvia miltiorrhiza suspension cultures influenced by the concentration of Ca2＋ in the culture medium. The synthesis of intraceUular RA in 6-day incubation was significantly dependent on the medium Ca2＋ concentration. At the optimal Ca2＋ concentration of 10 mmol/L, a maximal RA content of 20.149 mg/g biomass dry weight was reached, which was about 37.3% and 20.4% higher than that at Ca2＋concentrations of 1 and 3 mmol/L, respectively. The variation of the activity of PAL and TAT, two key enzymes of the two branches of RA, could be affected by the concentration of Ca2＋ in culture medium. The change of their activity occurred prior to the accumulation of RA, which suggested both of the key enzymes be involved in the synthesis of RA. Meanwhile, the enzymatic action of PAL was more distinct than TAT. The treatment of VP and A23187, respectively, indicated that the influence of RA affected by the concentration of Ca2＋ in the culture medium was accomplished by the intracellular Ca2＋, and the flow of Ca2＋ from the extracellular to the intracellular environment could also participate in this process.