Ce~（4+）对悬浮培养长春花细胞次生代谢产物诱导作用的研究
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摘要
为了研究稀土元素促进长春花悬浮培养细胞次生代谢产物的作用机理,以Ce~(4+)作为诱导子,建立了1mMCe~(4+)诱导长春花细胞悬浮培养体系,研究了1mMCe~(4+)诱导作用下长春花细胞的活性氧、MAPK途径、PAL、次生代谢产物以及胞内金属离子的变化,试图从信号转导角度探索Ce~(4+)诱导植物细胞次生代谢产物合成的基本规律。
在研究Ce~(4+)诱导作用下长春花细胞体系中H_2O_2的变化及其作用中发现Ce~(4+)诱导长春花细胞出现了H_2O_2迸发现象;H_2O_2清除剂CAT的加入降低了Ce~(4+)诱导的长春花细胞PAL的活化和长春质碱的合成;研究表明Ce~(4+)诱导H_2O_2的产生先于长春质碱合成的激活,由此看出Ce~(4+)诱导H_2O_2的产生处于Ce~(4+)诱导长春质碱合成的上游。外加H_2O_2也能诱导细胞PAL的激活和长春质碱的合成,证明H_2O_2是Ce~(4+)诱导长春花细胞次生代谢所必需的。研究还发现只是低浓度的Ce~(4+)(0.5mM~1mM)促进长春花细胞H_2O_2的产生和长春质碱的合成。
应用JNK、ERK和p38等途径特异性抑制剂的实验表明: JNK、ERK和p38等MAPK途径参与了Ce~(4+)诱导长春花细胞体系次生代谢产物合成的过程,而只有ERK和p38途径参与了Ce~(4+)诱导长春花细胞H_2O_2迸发的过程,但在H_2O_2迸发的下游JNK、ERK和p38等多条MAPK途径参与了将H_2O_2信号分子的传递调控次生代谢产物的合成。
应用光谱学方法研究了不同浓度Ce~(4+)对离体的过氧化氢酶(CAT)活力和构象的影响,发现0.01mM Ce~(4+)在提高CAT活力的同时使其α-螺旋含量增加,β-折叠含量减小,而0.05 mM～0.2 mM Ce~(4+)对CAT活性和二级结构的作用则相反。
运用电感耦合等离子发射光谱仪对1mMCe~(4+)诱导作用下的长春花细胞胞内的Ca~(2+)、Mg~(2+)、Zn~(2+)等金属离子进行了测定,分析了在不同的诱导时间金属离子含量的变化和吲哚生物总碱合成之间的关系。研究发现1mMCe~(4+)促进长春花细胞次生代谢产物合成的同时引起了细胞外Ca~(2+)的内流和细胞内K +的外渗。
The effect mechanism of rare earth on secondary metabolism productions in suspension cultured Catharanthus roseus was investigated by addition of 1mM Ce~(4+) to cultures. The changes of the activated oxygen species, mitogen-activated protein kinase (MAPK), PAL and secondary metabolites were studied in detail. We attempted to explain the mechanism of secondary metabolites production in Ce~(4+) induced Catharanthus roseus.
The changes and physiological role of H_2O_2 in Catharanthus roseus induced by Ce~(4+) were investigated. H_2O_2 burst after addition of Ce~(4+). PAL activation and the catharanthine production decreased significantly while CAT, a H_2O_2 scavenger, was added into cultures. Moreover, H_2O_2 generation was earlier than the catharanthine synthesis. We can conclude that H_2O_2 was the up-stream signal in catharanthine synthesis regulation. The production of catharanthine also increased after addition of H_2O_2. These results indicated that H_2O_2 play a key role in PAL activation and catharanthine synthesis. We also found that the concentration of Ce~(4+) strongly influenced H_2O_2 concentration and catharanthine production. Meanwhile, there was a saturated effect according to Ce~(4+) concentration.
Signal transduction pathway that Ce~(4+) induced secondary metabolites synthesis in Catharanthus roseus was investigated by the addition of harmacological inhibitors. JNK、ERK and p38 were all activated in secondary metabolites synthesis induced by Ce~(4+). However, only ERK and p38 were activated in H_2O_2 generation induced by Ce~(4+). H_2O_2 signal was transduced by JNK、ERK and p38 protein kinase pathway to activated the secondary metabolites synthesis.
The effects of Ce~(4+) on CAT activation and conformation were investigated by uv-visible spectra, fluorescent emission spectra, synchronous fluorescence, ultraviolet differential spectra, circular dichroism, FT-IR. The CAT conformation was related to the Ce~(4+) concentration.α-helix andβ-fold in CAT increase and decrease at lower concentration of Ce~(4+), respectively. However, the results reversed at higher concentration of Ce~(4+). These changes were related with Ce~(4+) concentration.
Intracellular contents of Ca~(2+), Mg~(2+), Zn~(2+), Mn~(2+), Cu~(2+), Fe~(2+) and K+ were analyzed by an inductively coupled plasma-optical emission spectrometry and the relationships between ions contents and total alkaloid in cells at different induced time were investigated. The results showed that the secondary metabolism productions synthesis induced by 1mM Ce~(4+) was companying the Ca~(2+) influx and K+ efflux.
在研究Ce~(4+)诱导作用下长春花细胞体系中H_2O_2的变化及其作用中发现Ce~(4+)诱导长春花细胞出现了H_2O_2迸发现象;H_2O_2清除剂CAT的加入降低了Ce~(4+)诱导的长春花细胞PAL的活化和长春质碱的合成;研究表明Ce~(4+)诱导H_2O_2的产生先于长春质碱合成的激活,由此看出Ce~(4+)诱导H_2O_2的产生处于Ce~(4+)诱导长春质碱合成的上游。外加H_2O_2也能诱导细胞PAL的激活和长春质碱的合成,证明H_2O_2是Ce~(4+)诱导长春花细胞次生代谢所必需的。研究还发现只是低浓度的Ce~(4+)(0.5mM~1mM)促进长春花细胞H_2O_2的产生和长春质碱的合成。
应用JNK、ERK和p38等途径特异性抑制剂的实验表明: JNK、ERK和p38等MAPK途径参与了Ce~(4+)诱导长春花细胞体系次生代谢产物合成的过程,而只有ERK和p38途径参与了Ce~(4+)诱导长春花细胞H_2O_2迸发的过程,但在H_2O_2迸发的下游JNK、ERK和p38等多条MAPK途径参与了将H_2O_2信号分子的传递调控次生代谢产物的合成。
应用光谱学方法研究了不同浓度Ce~(4+)对离体的过氧化氢酶(CAT)活力和构象的影响,发现0.01mM Ce~(4+)在提高CAT活力的同时使其α-螺旋含量增加,β-折叠含量减小,而0.05 mM～0.2 mM Ce~(4+)对CAT活性和二级结构的作用则相反。
运用电感耦合等离子发射光谱仪对1mMCe~(4+)诱导作用下的长春花细胞胞内的Ca~(2+)、Mg~(2+)、Zn~(2+)等金属离子进行了测定,分析了在不同的诱导时间金属离子含量的变化和吲哚生物总碱合成之间的关系。研究发现1mMCe~(4+)促进长春花细胞次生代谢产物合成的同时引起了细胞外Ca~(2+)的内流和细胞内K +的外渗。
The effect mechanism of rare earth on secondary metabolism productions in suspension cultured Catharanthus roseus was investigated by addition of 1mM Ce~(4+) to cultures. The changes of the activated oxygen species, mitogen-activated protein kinase (MAPK), PAL and secondary metabolites were studied in detail. We attempted to explain the mechanism of secondary metabolites production in Ce~(4+) induced Catharanthus roseus.
The changes and physiological role of H_2O_2 in Catharanthus roseus induced by Ce~(4+) were investigated. H_2O_2 burst after addition of Ce~(4+). PAL activation and the catharanthine production decreased significantly while CAT, a H_2O_2 scavenger, was added into cultures. Moreover, H_2O_2 generation was earlier than the catharanthine synthesis. We can conclude that H_2O_2 was the up-stream signal in catharanthine synthesis regulation. The production of catharanthine also increased after addition of H_2O_2. These results indicated that H_2O_2 play a key role in PAL activation and catharanthine synthesis. We also found that the concentration of Ce~(4+) strongly influenced H_2O_2 concentration and catharanthine production. Meanwhile, there was a saturated effect according to Ce~(4+) concentration.
Signal transduction pathway that Ce~(4+) induced secondary metabolites synthesis in Catharanthus roseus was investigated by the addition of harmacological inhibitors. JNK、ERK and p38 were all activated in secondary metabolites synthesis induced by Ce~(4+). However, only ERK and p38 were activated in H_2O_2 generation induced by Ce~(4+). H_2O_2 signal was transduced by JNK、ERK and p38 protein kinase pathway to activated the secondary metabolites synthesis.
The effects of Ce~(4+) on CAT activation and conformation were investigated by uv-visible spectra, fluorescent emission spectra, synchronous fluorescence, ultraviolet differential spectra, circular dichroism, FT-IR. The CAT conformation was related to the Ce~(4+) concentration.α-helix andβ-fold in CAT increase and decrease at lower concentration of Ce~(4+), respectively. However, the results reversed at higher concentration of Ce~(4+). These changes were related with Ce~(4+) concentration.
Intracellular contents of Ca~(2+), Mg~(2+), Zn~(2+), Mn~(2+), Cu~(2+), Fe~(2+) and K+ were analyzed by an inductively coupled plasma-optical emission spectrometry and the relationships between ions contents and total alkaloid in cells at different induced time were investigated. The results showed that the secondary metabolism productions synthesis induced by 1mM Ce~(4+) was companying the Ca~(2+) influx and K+ efflux.
引文
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