稀土元素对怀槐培养细胞异黄酮合成及氧化还原态的影响
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摘要
怀槐(Maackia amurensis Rupr．et Maxim)又名朝鲜槐、高丽槐，是我国一种珍贵的野生药用植物，其乙醇提取物中的总异黄酮成分已被证明具有保肝等作用。目前的研究表明适量稀土元素能在短时间内促进植物细胞生长和异黄酮等植物次生代谢产物的合成，稀土元素还能通过改变超氧化物歧化酶(SOD)、过氧化氢酶(CAT)等氧化酶有效调节植物细胞的氧化还原状态。
通过改变细胞的氧化态如谷胱甘肽(GSH)刺激也能调节次生代谢产物的合成。为了探究植物细胞次生代谢产物合成与氧化还原态变化之间的关系，本论文选择稀土元素作为诱导子，用合成异黄酮的怀槐培养细胞考察稀土元素刺激异黄酮合成和细胞氧化还原态变化的关系。
通过实验发现20μmol/L硝酸铈(Ce(NO_3)_3)和10μmol/L硝酸镧(La(NO_3)_3)是怀槐培养细胞异黄酮合成的有效诱导子。动态分析表明Ce(NO_3)_3处理组的异黄酮含量在诱导第4d达到最大值，比同期对照提高了49％；La(NO_3)_3处理组在诱导的第3d达到最大值，比同期对照提高了77％。苯丙氨酸解氨酶(PAL)和异黄酮合酶(IFS)活性动态分析发现，20μmol/L Ce(NO_3)_3和10μmol/L La(NO_3)_3能够激活PAL和IFS活性，表明适量稀土元素促进异黄酮合成可能和稀土元素调节细胞中次生代谢途径中关键酶活性有关。
GSH库和ASC库水平的活性动态变化分析表明，适量稀土元素在显著提高GSH、ASC含量和GSH/GSSG、ASC/DHA比率的同时，又明显降低了细胞中GSSG、DHA含量。通过分析比较异黄酮、PAL酶活性、IFS酶活性、GSH(或ASC)含量、GSH/GSSG(或ASC/DHA)比率等峰值出现时间的差异，可以推测20μmol/L Ce(NO_3)_3和10μmol/L La(NO_3)_3首先通过改变怀槐培养细胞中的氧化还原态，再影响次生代谢途径中PAL酶和IFS酶的表达，最终调节了异黄酮的合成。
Macackia amurensis is a precious wild medicinal tree in China, also named Amur Maackia. The isoflavones isolated from the EtOH extracts of heatwood of Macackia amurensis have protective effects on the liver. It is known that Rare Earth Elements (REEs) could not only stimulate plant cell growth and biosynthesis of secondary metabolites such as isoflavones in the short time, but also regulate the redox status of plant cell through changing the content of oxidase including SOD, CAT, etc. It is reported that the change of redox status in the plant cell can stimulate the biosynthesis of secondary metabolite. To determine the relationship between the biosynthesis of secondary metabolite and the change of redox status in the plant cell, the REEs here were utilized as elicitors to investigate their effect on the production of isoflavones and changes in the redox status in Maackia amurensis cell suspension cultures.
By selection experiments, 20μmol/L Ce(NO_3)_3 and 10μmol/L La(NO_3)_3 were chosen as effective concentrations of REEs elicitors to promote isoflavone synthesis in cell cultures of M.amurensis. It was revealed that the content of isoflavone came to maximum in the fourth and third day by the elicitation of Ce(NO_3)_3 and La(NO_3)_3, which were enhanced by 49% and 77% in contrast to the control, respectively. This study also indicated that the activities of PAL and IFS could be increased by the elicitation of Ce(NO_3)_3 and La(NO_3)_3. The obtained results suggested an increase in isoflavone biosynthesis by the REEs could be related to the change of the key enzyme of secondary metabolism.
The dynamic changes of GSH sink and ASC sink showed that REEs could increase the GSH and ASC contents, the ratio of GSH/GSSG and ASC/DHA. Also REEs reduced the GSSG and DHA contents. Through analyzing time course of peak value of PAL activity, IFS activity and GSH (or ASC) content as well as GSH/GSSG (ASC/DHA) ratio changes, it can be concluded that 20μmol/L Ce(NO_3)_3 and 10μmol/L La(NO_3)_3 changed redox status of M.amurensis firstly, then influenced PAL and IFS activity, finally regulated isoflavone biosynthesis.
通过改变细胞的氧化态如谷胱甘肽(GSH)刺激也能调节次生代谢产物的合成。为了探究植物细胞次生代谢产物合成与氧化还原态变化之间的关系，本论文选择稀土元素作为诱导子，用合成异黄酮的怀槐培养细胞考察稀土元素刺激异黄酮合成和细胞氧化还原态变化的关系。
通过实验发现20μmol/L硝酸铈(Ce(NO_3)_3)和10μmol/L硝酸镧(La(NO_3)_3)是怀槐培养细胞异黄酮合成的有效诱导子。动态分析表明Ce(NO_3)_3处理组的异黄酮含量在诱导第4d达到最大值，比同期对照提高了49％；La(NO_3)_3处理组在诱导的第3d达到最大值，比同期对照提高了77％。苯丙氨酸解氨酶(PAL)和异黄酮合酶(IFS)活性动态分析发现，20μmol/L Ce(NO_3)_3和10μmol/L La(NO_3)_3能够激活PAL和IFS活性，表明适量稀土元素促进异黄酮合成可能和稀土元素调节细胞中次生代谢途径中关键酶活性有关。
GSH库和ASC库水平的活性动态变化分析表明，适量稀土元素在显著提高GSH、ASC含量和GSH/GSSG、ASC/DHA比率的同时，又明显降低了细胞中GSSG、DHA含量。通过分析比较异黄酮、PAL酶活性、IFS酶活性、GSH(或ASC)含量、GSH/GSSG(或ASC/DHA)比率等峰值出现时间的差异，可以推测20μmol/L Ce(NO_3)_3和10μmol/L La(NO_3)_3首先通过改变怀槐培养细胞中的氧化还原态，再影响次生代谢途径中PAL酶和IFS酶的表达，最终调节了异黄酮的合成。
Macackia amurensis is a precious wild medicinal tree in China, also named Amur Maackia. The isoflavones isolated from the EtOH extracts of heatwood of Macackia amurensis have protective effects on the liver. It is known that Rare Earth Elements (REEs) could not only stimulate plant cell growth and biosynthesis of secondary metabolites such as isoflavones in the short time, but also regulate the redox status of plant cell through changing the content of oxidase including SOD, CAT, etc. It is reported that the change of redox status in the plant cell can stimulate the biosynthesis of secondary metabolite. To determine the relationship between the biosynthesis of secondary metabolite and the change of redox status in the plant cell, the REEs here were utilized as elicitors to investigate their effect on the production of isoflavones and changes in the redox status in Maackia amurensis cell suspension cultures.
By selection experiments, 20μmol/L Ce(NO_3)_3 and 10μmol/L La(NO_3)_3 were chosen as effective concentrations of REEs elicitors to promote isoflavone synthesis in cell cultures of M.amurensis. It was revealed that the content of isoflavone came to maximum in the fourth and third day by the elicitation of Ce(NO_3)_3 and La(NO_3)_3, which were enhanced by 49% and 77% in contrast to the control, respectively. This study also indicated that the activities of PAL and IFS could be increased by the elicitation of Ce(NO_3)_3 and La(NO_3)_3. The obtained results suggested an increase in isoflavone biosynthesis by the REEs could be related to the change of the key enzyme of secondary metabolism.
The dynamic changes of GSH sink and ASC sink showed that REEs could increase the GSH and ASC contents, the ratio of GSH/GSSG and ASC/DHA. Also REEs reduced the GSSG and DHA contents. Through analyzing time course of peak value of PAL activity, IFS activity and GSH (or ASC) content as well as GSH/GSSG (ASC/DHA) ratio changes, it can be concluded that 20μmol/L Ce(NO_3)_3 and 10μmol/L La(NO_3)_3 changed redox status of M.amurensis firstly, then influenced PAL and IFS activity, finally regulated isoflavone biosynthesis.
引文
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