| 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. |
