NO对金丝桃悬浮细胞生长及金丝桃素生物合成的促进作用研究

Enhancement of Hypericin Production and Cell Growth of Hypericum perforatum L. Suspension Cultures by Nitric Oxide

一氧化氮 (NO)是近年来发现的一种新型植物信号分子。以硝普钠 (Sodiumnitroprusside ,SNP)为一氧化氮 (NO)的供体 ,研究外源NO对金丝桃悬浮细胞生长及金丝桃素生物合成的影响。试验结果表明 ,金丝桃悬浮细胞在含 0 5和 15 0mmol LSNP的培养基中培养 2 0d后 ,细胞的干重分别为对照组的 14 0 %和 5 0 % ;细胞中金丝桃素的含量分别为对照组的 98%和2 10 %。试验结果表明 ,低浓度SNP处理有利于金丝桃悬浮细胞生长 ,而高浓度SNP可以促进金丝桃素的合成。在细胞培养初期 (0d)加入 0 5mmol LSNP并在指数生长后期 (14d)加入 15 0mmol LSNP的金丝桃悬浮细胞在培养 2 5d后 ,细胞的干重和金丝桃素的含量分别为对照组的 1 4和 1 8倍 ,金丝桃素的产量达 15 2mg L ,比对照高 3 2倍。SNP对金丝桃悬浮细胞生长及金丝桃素含量的影响可以被NO专一性淬灭剂CPITO(2 4 carboxyphenyl 4 ,4 ,5 ,5 tetramethylimidazoline 1 oxyl 3 oxide)所抑制 ,说明SNP是通过其分解产物NO影响细胞生长和金丝桃素的合成。试验结果同时表明 ,在 15 0mmol L的SNP处理下 ,金丝桃悬浮细胞中的苯丙氨酸解氨酶 (PAL)的活性显著升高 ,推测NO可能通过触发金丝桃悬浮细胞的防卫反应 ,激活了细胞中金丝桃素的生物合成途径。

Nitric oxide has emerged as a key signaling molecule in plants recently. The role of nitric oxide in elicitor induced defense responses of plants has been extensively investigated. In this work, sodium nitroprusside was utilized as the donor of nitric oxide to investigate the effects of exogenous nitric oxide on hypericin production and cell growth of suspension cell cultures of Hypericum perforatum L.. Compared with the untreated Hypericum perforatum L. suspension cells, external application of 0 5 and 15 0 mmol/L sodium nitroprusside induced 1 4 and 0 5 fold dry cell weight, and 0 9 and 2 1 fold hypericin content respectively. The results showed that low concentration of sodium nitroprusside promoted the growth of Hypericum perforatum L. suspension cells, while high concentration of sodium nitroprusside enhanced hypericin biosynthesis in Hypericum perforatum L. suspension cells. The maximum hypericin production was achieved by adding 0 5 mmol/L and 15 0 mmol/L sodium nitroprusside to the culture at day 0 and day 14 respectively, increasing the total hypericin yield by nearly 3 2 fold. The effects of sodium nitroprusside on hypericin content and growth of Hypericum perforatum L. suspension cells were abolished by nitric oxide specific scavenger 2 4 carboxyphenyl 4,4,5,5 tetramethylimidazoline 1 oxyl 3 oxide, which indicated that the effects of the application of sodium nitroprusside were caused by nitric oxide released from sodium nitroprusside rather than sodium nitroprusside itself. The results also showed that 15 0 mmol/L sodium nitroprusside stimulated the activities of phenylalanine ammonia lyase (PAL), one of the key enzymes of phenylpropanoid pathway, in suspension cells of Hypericum perforatum L., which suggested that the synthetic pathway of hypericin might be activated by NO through triggering the defense responses of Hypericum perforatum L. suspension cells.