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[摘要]
目的 分析外源一氧化碳(CO)对白桦悬浮细胞生长和三萜累积的影响。方法 在白桦悬浮细胞培养的第8天添加5、10、20、30 μmol/L的CO供体高铁血红素处理0.25~8 d,采用高效液相色谱法、比色法和实时荧光定量PCR方法分析白桦三萜含量及其合成关键酶基因的表达。结果 除20和30 μmol/L CO处理2 d后显著降低白桦悬浮细胞干质量外,CO处理未对白桦悬浮细胞干质量产生显著影响。除30 μmol/L CO处理提高pH值和丙二醛含量外,CO处理未对pH值和丙二醛含量产生显著影响。CO处理不同程度地增加了三萜、白桦酯醇和齐墩果酸含量,其中,30 μmol/L CO处理1 d时三萜积累量达最大值,为同期对照的1.3倍;10 μmol/L CO处理4 d时白桦酯醇含量达最高值,为对照的1.5倍;10 μmol/L CO处理2 d时齐墩果酸含量达最高值,为对照的4.2倍。三萜合成关键酶FPPS、LUS、CAS1、CAS2和β-AS基因的实时荧光定量PCR检测结果进一步证实了CO对白桦三萜累积的促进作用。结论 CO处理促进了白桦悬浮细胞中三萜的合成。
[Key word]
[Abstract]
Objective To analyze the effect of carbon monoxide (CO) on the growth of Betula platyphylla suspension cells and triterpenoid production. Methods CO donor hematin of 5, 10, 20, and 30 μmol/L were added to eight-day-old B. platyphylla suspension cells, and the change of triterpenoid content and gene expression of FPPS, LUS, CAS1, CAS2, and β-AS related to the triterpenoid synthesis were analyzed by chemical colorimetry, high performance liquid chromatography and real-time PCR, respectively. Results CO treatment did not have a significant effect on dry weight of B. platyphylla suspension cells except for 2-8 d at 20 μmol/L and 30 μmol/L CO treatment. CO treatment also did not have a significant effect on pH value and malondialdehyde content except for 30 μmol/L CO treatment. CO treatment significantly increased the content of triterpenoid, betulin and oleanolic acid. the content of triterpenoid, betulin and oleanolic acid were the highest after 1 d at 30 μmol/L CO treatment, 4 d at 10 μmol/L CO treatment and 2 d at 10 μmol/L CO treatment, which was 1.3, 1.5, and 4.2 times of the control, respectively. Triterpenoid production in B. platyphylla suspension cells induced by CO was further confirmed by key enzyme genes of FPPS, LUS, CAS1, CAS2, and β-AS related to the triterpenoid synthesis by RT-PCR. Conclusion CO treatment could effectively enhance the triterpenoid production in B. platyphylla suspension cell culture.
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[基金项目]
中央高效基本科研业务专项资金项目(2572017EA05);中央高效基本科研业务专项资金项目(2572018CJ02);黑龙江自然科学基金资助项目(C2016005)