【作者】 沈国栋；

【导师】 罗建平；

【作者基本信息】 合肥工业大学 ， 农产品加工及贮藏工程， 2004， 硕士

【摘要】 怀槐是我国珍贵的野生药用植物，资源面临枯竭，其药用成分异黄酮具有显著的保肝、抗肿瘤、抗氧化及防治骨质疏松等药理活性。本论文旨在利用植物细胞培养技术考察水杨酸调节怀槐细胞次生代谢合成异黄酮与碳氮代谢之间的关系，为进一步开展保肝药用植物怀槐异黄酮生产的次生代谢细胞工程，保护和可持续利用怀槐药用资源提供基础。 论文采用薄层层析（TLC）和高效液相色谱（HPLC）技术证明怀槐培养细胞具有合成异黄酮的能力，细胞中异黄酮的积累与细胞生长之间呈正相关性，其中活性最强的染料木素含量明显高于植株幼苗和种子。以CCl₄急性肝损伤小鼠为模型证实了培养细胞异黄酮提取物能有效降低血清谷草转氨酶（GOT，EC2.6.1.1）和谷丙转氨酶（GPT，EC 2.6.1.2）活性、升高血清白蛋白和总蛋白水平，具有显著的保肝作用。 通过实验发现水杨酸（SA）是怀槐培养细胞异黄酮合成的有效诱导子，其最佳浓度为100 μmol／L，它显著促进怀槐大豆苷元、染料木素及芒柄花黄素合成，培养12d时细胞中染料木素的积累水平达到对照的2.6倍。苯丙氨酸（Phe）合成关键酶3-脱氧-α-5可拉伯庚酮糖酸-7-磷酸合酶（DAHPS，EC 4.1.2.15）和Phe进入异黄酮合成途径的关键酶苯丙氨酸氨基裂解酶（PAL，EC 4.3.1.5）活性动态分析发现，SA能够激活DAHPS和PAL，表明SA促进异黄酮合成可能和SA调节细胞Phe合成有关。 SA促进异黄酮合成与其能够有效调节细胞的碳氮代谢相关。细胞内外碳源和氮源水平及相关代谢酶活性变化分析表明，SA一方面可显著提高细胞壁结合蔗糖酶（WBI，EC 3.2.1.26）活性，加速蔗糖分解，提供给细胞更多的碳源和能量，同时诱导葡萄糖-6-磷酸脱氢酶（G6PD，EC 1.1.1.49）活性升高，促进细胞中葡萄糖进入磷酸戊糖途径，为经过3-脱氧-α-阿拉伯庚酮糖酸-7-磷酸（DAHP）合成异黄酮合成前体Phe提供碳架和还原力：另一方面SA通过激发硝酸还原酶（NR，EC 1.6.6.1）和谷氨酸合酶（GOGAT，EC 1.4.1.14）活性增强导致细胞快速将NO₃^-转化为NH₄⁺，并经过GS-GOGAT循环有效固定NH₄⁺，为经过DAHP合成异黄酮合成前体Phe提供氨基。 通过透射电镜观察发现，SA处理显著影响细胞超微结构，胞内嗜锇小体（EDB）增加很多，分析表明细胞内EDB的出现及数量和异黄酮积累表现出正相关性。从细胞超微结构上证实了SA对怀槐细胞异黄酮合成的诱导作用。更多还原

【Abstract】 Maackia amurensis is a precious wild medicinal tree in China, but its resource is becoming exhausted. The isoflavones isolated from this plant have marked pharmacological effects, such as hepatoprotection, antitumor, antioxidation and osteoporosis inhibition. The objective of this thesis was to investigate the relationship between isoflavone synthesis and carbon- and nitrogen-metabolism regulated by salicylic acid in cultured cells of M. amurensis by means of plant biotechnology, in order to provide a foundation for isoflavone production in the cell cultures by cell secondary metabolism engineering, protecting and sustaining to utilize the medicinal metabolite resource of M. amurensis.By the technologies of TLC and HPLC, the ability of the obtained cell cultures to produce isoflavones was found, and there was a positive relationship between cell growth and isoflavone accumulation. Genistein, which has a potent bioactivity among isoflavones, was obtained more in the cell cultures than in the plants and seeds of M. amurensis. Extraction of isoflavones from the cell cultures could effectively attenuate the activities of serum aminotransferases (GOT, EC 2.6.1.1 and GPT, EC 2.6.1.2) and elevate the content of serum albumin against acute CCl4-hepatitis in mice, thus its remarkable hepatoprotective effect was showed.By the experiments in this paper, SA was chosen as an effective elicitor of isoflavones synthesis in cell cultures of M. amurensis. 100 mol/l which had the best effect among the different concentrations of SA, markedly promoted daidzein, genistein and formononetin synthesis, especially the highest genistein accumulation to 2.6-fold of the control in the cells elicited for 12d. By the analysis on changes in the activities of key enzymes, 3-deoxy-a-arabino-heptulosonate-7-phosphate synthase (DAHPS, EC 4.1.2.15) and phenylalanine-ammonia lyase (PAL, EC 4.3.1.5) could be elicited by SA, and the corelationship between the improved isoflavone accumulation and promoted Phe content by SA was showed.In this paper it was revealed that the elicited isoflavone synthesis was associated with regulated carbon- and nitrogen-metabolism by SA. After the levels of carbon- and nitrogen-source in liquid media and cultured cells and the changes in the activities of the involved key enzymes were studied, SA was found to improve the activity in cell wall-bound invertase (WBI, EC 3.2.1.26) greatly, thus more sucrose degraded and more carbon-source and energy supplied for the cell metabolism, at the same time to strenthen glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), so more glucose utilised in pentose phosphate pathway and more carbon-skeleton and reducing power given for Phe and isoflavone synthesis. In the other hand, nitrate reductase (NR, EC 1.6.6.1) and glutamate synthase (GOGAT, EC 1.4.1.14) were elicited by SA, thus NO 3- was transformed to NH 4+ rapidly, and NH 4+ was fixed via the recycle of GOGAT - GS to provide for Phe and isoflavone synthesis. By means of transmission electron microscopy, the occurring and quantity of electron-dense body (EDB) grew much more in the treated cells by SA than in the control, and the positive relationship between the occurring of EDB and isoflavone accumulation in the treated cells was showed by statistics analysis. The changes in the treated cell ulfrastructure strenthened the explanation about the elicitation of isoflavone accumulation in the cell cultures of M. amurensis by SA.更多还原