水杨酸对丹参培养细胞中迷迭香酸生物合成及其相关酶的影响

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

摘要:

迷迭香酸 (RA) 是丹参中一种重要的酚酸类次生代谢物。为探讨水杨酸 (SA) 诱导子对丹参悬浮培养细胞中RA的生物合成及其相关酶的影响，考察了SA诱导子和酪氨酸氨基转移酶 (TAT) 的竞争性抑制剂 (AOPP) 对RA合成积累量、苯丙氨酸解氨酶 (PAL) 和TAT活性的影响。发现在培养的第6天用浓度为6.25 mg/L的SA处理后，PAL活性在诱导后4 h出现高峰，为对照组水平的124％；RA的积累量在诱导后8 h出现峰值 (5.914±0.296) mg/g。用浓度为0.1 μmol/L的AOPP处理，6 h 后AOPP对TAT活性影响较小 (与对照组无显著差异)，但明显抑制了PAL活性 (为对照组水平的44％)，且在PAL活性明显降低的同时RA的积累量显著减少 (4.709±0.204) mg/g。进一步用0.1 μmol/L AOPP和6.25 mg/L SA共处理，AOPP对PAL的抑制作用可得到一定程度的缓解，且RA的积累量较AOPP单独处理的高。表明SA可以诱导丹参悬浮培养细胞中RA积累量的增加，且在RA合成过程中PAL的限速作用比TAT明显。

Abstract:

Rosmarinic acid (RA), a phenolic acid, is one of the important secondary metabolites produced in Salvia miltiorrhiza. To observe the influence of salicylic acid (SA), an elicitor, on the synthesis of RA and related enzymes, we treated the cell suspension cultures of S. miltiorrhiza with SA and L-α-aminooxy-β-phenylpropionic acid (AOPP), a competitive inhibitor of tyrosine aminotransferase (TAT). Under this condition, the activities of related enzymes, such as phenylalanine ammonia-lyase and TAT were traced and assayed; the accumulative amount of RA was measured. The results showed that the PAL activity reached the peak at 4 h, 124% higher than that of the control, and the content of RA reached its maximum ((5.914±0.296) mg/g dry weight) at 8 h, after treated by 6.25 mg/L SA on day 6 of the suspension culture. The results of treatment with 0.1 μmol/L AOPP showed that AOPP affected little on the TAT activity, while the PAL activity was significantly influenced, with 44% lower than that of the control at 6 h. Meanwhile, the reduced accumulation of RA ((4.709±0.204) mg/g dry weight) paralleled with the decrease in PAL activity. The co-treatment by 0.1 μmol/L AOPP and 6.25 mg/L SA relieved the restriction imposed by AOPP on PAL, and made the cell cultures accumulate more RA than sole treatment with AOPP, indicated that SA induced the accumulation of RA in suspension cell culture of S. miltiorrhiza, and the rate-limiting effect of PAL was stronger than TAT.