腐胺与过氧化氢交互调控白桦三萜合成的初步研究

Interaction between putrescine and hydrogen peroxide on regulating triterpenoid production in Betula platyphylla

目的分析腐胺和过氧化氢调控白桦三萜合成中是否存在交互作用。方法在白桦悬浮细胞的生长末期添加1 mmol/L腐胺(Put)、0.1 mmol/L过氧化氢(H2O2)和40μg/mL真菌诱导子,采用比色法、高效液相色谱法和荧光显微镜分析白桦悬浮细胞中三萜量、多胺量和H2O2荧光强度变化。结果 1 mmol/LPut增加了细胞中H2O2的荧光强度和三萜量。0.1 mmol/LH2O2促进了三萜合成,却抑制了多胺中亚精胺(Spd)、精胺(Spm)的合成,而对Put无影响。Put与H2O2同时处理组,细胞中H2O2的荧光强度和多胺的量均介于Put与H2O2单独处理组之间,而三萜量却显著高于其单独处理组;进一步药理学实验分析发现,真菌诱导子与H2O2清除剂过氧化氢酶(CAT)处理后,细胞中的Put和Spd量比真菌诱导后增加了0.62%和16.05%,而对Spm无显著影响。真菌诱导子与多胺(Pas)合成抑制剂D-精氨酸(D-arg)处理组,细胞中的H2O2荧光强度比真菌诱导子处理组有所降低了;在真菌诱导子、CAT和D-arg同时处理下,细胞中的H2O2荧光强度、PAs量和三萜量均低于真菌诱导子与CAT或D-arg处理,但三萜量仍高于对照组。结论 Put与H2O2在促进白桦三萜合成中存在交互作用。

Objective To analyze the interaction between putrescine （Put） and hydrogen peroxide （H2O2） on the regulation of triterpenoid production in Betula platyphylla suspension cell culture. Methods Put （1 mmol/L）, H2O2 （0.1 mmol/L）, and fungal elicitors （40μg/mL） were added into the eight-day-old B. platyphylla suspension cell culture, and the changes of triterpenoid content, polyamine content, and H2O2 fluorescence intensity were analyzed by chemical colorimetry, HPLC, and fluorescence microscopy, respectively. Results Put （1 mmol/L） increased the H2O2 fluorescence intensity and triterpenoid content in B. platyphylla suspension cell culture. H2O2 （0.1 mmol/L） promoted the triterpenoid production, inhibited the production of spermidine （Spd） and spermine （Spm）, and had no effectt on Put content. Under the treatment of 1 mmol/L Put and 0.1 mmol/L H2O2, H2O2 fluorescence intensity and polyamine （PAs） content were between its separated treatment, and triterpenoid content was significantly higher than its separated treatment. Comparing with the fungal elicitor, fungal elicitor and H2O2 scavenger catalase （CAT） induced an increase of 0.62% and 16.05% in Put and Spd content, respectively, while no effect on Spm content. Fungal elicitor and PAs synthesis inhibitor D-arginine （D-arg） decreased the H2O2 fluorescence intensity. Under the treatment of fungal elicitor, the fluorescence intensity of CAT and D-arg, H2O2 and the contents of PAs and triterpenoid were lower than those of fimgal elicitor and CAT or D-arg, but triterpenoid content was still higher than that of the control. Conclusion This can be inferred that the interaction between Put and H2O2 regulates the triterpenoid production in birch suspension cell culture.