【作者】 何梦玲；

【导师】 周吉源；

【作者基本信息】 华中师范大学 ， 植物学， 2001， 硕士

【摘要】 喜树碱是一种色氨酸-萜烯类生物碱，功能抗癌、清热、杀虫，是一种极有开发前景的植物药。由于资源有限，且化学合成困难、成本高，通过高等植物细胞培养生产次生代谢物质——喜树碱将是一个很好的选择。本文以喜树（Camptotheca acuminata Decsne.）幼嫩叶片为材料，诱导和筛选了愈伤组织，初步研究了固体培养条件下光照、激素及一些理化因子对喜树细胞生长、生理生化特性的影响，使喜树培养细胞生长速度有明显提高，为进一步研究积累了资料。 具体实验结果如下： 不同种类、不同浓度的激素及其组合对细胞生长的效应均不相同，本文按正交设计法设计实验，得出喜树愈伤组织诱导及增殖的最佳培养基为：B₅+2，4-D₀₅+NAA₁₀+KT₀₅。 以黑暗为对照，在绿、白、蓝、黄、红五种不同光质条件下，培养细胞生长周期均为27天左右，细胞增殖曲线为“S”型，可大致分为延迟期、指数生长期和静止期三个时期。绿光对喜树愈伤组织细胞生长有促进作用，而蓝光则有抑制作用。在光照和黑暗两种条件下，喜树固体培养细胞的可溶性蛋白质含量变化曲线相似，均出现两个峰值，分别在第15和21天；过氧化物酶、超氧化物歧化酶和酯酶的活性均出现两个高峰，但出现的时间不同。前者的第一个峰值出现在第3天，光照下的第二个峰值在第15天，黑暗下在第18天出现；后者的第二个峰值出现时间相同，在第24天，光照条件下第一个峰值在第15天出现，黑暗下在第18天；酯酶活性变化的第一个高峰在第3天，光照下第二峰值在第15天，黑暗下在第18天，黑暗条件下的酶活性峰值均晚于光照条件下的，三种酶活性的变化均与光照条件下喜树细胞生长速率较黑暗条件下快相一致。蛋白质组分及过氧化物酶、超氧化物歧化酶和酯酶同工酶电泳分析结果表明，在愈伤组织培养周期中，它们都发生了变化。在细胞快速生长时期，出现了新的蛋白带；同样的，细胞生长速度较快时，酶活性较高，表现在酶带染色较深，且同工酶酶带数在数量上有增加，与ribeat，IL6Ai／％；bi丫’．不、y＼1＼引ERS 丁旺SIS 细胞生长速度慢时有明显区别，显示了细胞生长和生理生化特性之间的 相关性。 在喜树细胞固体培养中，适宜的接种量为3g鲜重／50ml培养基；蔗 糖的最佳添加量为309几；作为碳源，葡萄糖的效果优于蔗糖，随着葡 萄糖的浓度升高，细胞生长速率呈上升趋势，且在葡萄糖浓度为309几 时最有利于细胞生长；添加 0＊％的琼脂糖能促进喜树的细胞主长；而水 解乳蛋白却对细胞生长有抑制作用。更多还原

【Abstract】 Camptothecin is a kind of anticancer medicine having good prospect of exploitation. But lacking of resources, production of camptothecin by cell culture may be a promising choice. In this paper, based on the young leaves of Camptotheca acuminata Decsne., callus were induced and screened, and the influences of light, hormones and several factors on the growth, physiological, biochemical characters of solid culture of Camptotheca acuminata Decsne. were primarily studied, the results were as follows: The optimal medium of callus induction and growth of Camptotheca acuminata Decsne. was B~ with the addition of 0.Smg/L 2.4-D. 1 .Omg/LNAA and 0.5 mg/LKT. In different light qualities, the cell growth circles were about 27 days. and the cell growth curve was like 搒? The green light accelerated callus抯 growth. while the blue light did the opposite effect. When Camptotheca acuminata Decsne. was cultured in different illuminative conditions, the contents of soluble protein, activities of peroxidase(POD). superoxide dismutase (SOD) and esterase all showed two peaks which were on the different days; The varieties of three enzymes?activity were all consistent with which C.acuminata抯 cell trowth rate in illumination was quicker than that of in dark. The electrophoresis analysis of protein抯 composition and isozymes showed: they all had changed in callus culture cycle and revealed the correlations between the callus growth and physiological and biochemical characters. In the solid culture, the inoculum quantity of 3g.FW/50m1 medium was better; the optimal concentration of Sucrose was 30g/L ; as the sources of Carbon. the effect of Glucose was better than that of Sucrose, and the medium with 30g/L Glucose was good to cell growth; the medium with addition of 0.1% Agarose increased the quantity of cell growth, but LH inhibited it.更多还原