| 题名 | 牛角瓜及其发根中碳氢化合物与强心苷的代谢调控 |
| 作者 | 孙健 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-04-04 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 赵兵 |
| 关键词 | 牛角瓜 发根 碳氢化合物 代谢调控 强心苷 |
| 其他题名 | Regulation of Hydrocarbon and Cardenolides Metabolism in Calotropis gigantea and Its Hairy Root Cultures |
| 学位专业 | 生物化工 |
| 中文摘要 | 作为一种传统药用植物，牛角瓜（Calotropis gigantea）主要活成分为强心苷类物质等。近年来的研究也表明牛角瓜中含有一定量的碳氢化合物，可以作为能源植物加以利用。但野生牛角瓜中的强心苷及碳氢化合物的含量较低，无法满足商业化利用的要求。提高牛角瓜药用成份和能源成份的含量才能打破其商业化利用的瓶颈。转基因育种具有周期短、目标明确等特点，因此本研究首先分析了牛角瓜作为能源植物的潜力，利用发根农杆菌的介导将烷烃合成基因转入了牛角瓜植株，并对获得的牛角瓜发根及转基因植株的碳氢化合物及强心苷进行了代谢调控，提高了强心苷和碳氢化合物的合成能力。本文主要研究结果如下：首先对牛角瓜的再生途径进行了研究，结果表明牛角瓜很难通过愈伤途径进行再生，而发根途径对于牛角瓜再生来说是一种较合适的方式。发根农杆菌LBA9402对牛角瓜叶片外植体的侵染效率较高，当菌液的OD600为0.5，稀释15倍牛角瓜叶片30min，侵染后的叶片在Cef浓度为300mg/l下进行除菌，发根诱导效果最好。获得的发根在MS+NAA 0.5 mg/l+6-BA 4.0 mg/l固体培养基上可以诱导产生丛生芽，诱导率为10%。丛生芽在MS固体培养基上很容易生根，生根率为100%。对培养牛角瓜发根的无机营养进行了优化，当牛角瓜发根在MMS-CG培养基中添加3%的蔗糖时其碳氢化合物的含量最高，为304.81 mg/l；通过一个2升的鼓泡塔生物反应培养对其最佳培养条件进行了分析，发现在光照强度为34.17 μmol/m2/s、气体流量为0.40 vvm 、接种量为56.00 g/l、培养30天时其碳氢化合物的产量可以达到356.14 mg/l。另外，研究还表明加入100mg/l可溶性壳聚糖，培养25天后其碳氢化合物的产量明显提高，可以达到614.45 mg/l。通过发根农杆菌的ATCC15834的介导将烷烃合成相关的基因WAX2转入牛角瓜，并产生转基因发根。对得到的转WAX2的发根的碳氢化合物及烷烃的含量进行分析发现其较野生牛角瓜植株均有较大提高。通过对转基因发根的诱导已经获得了转WAX2基因的牛角瓜植株，其碳氢化合物含量较野生植株提高了3.39%，烷烃含量提高了23.91%。本研究还建立了牛角瓜发根中强心苷的提取及检测方法，在该方法的基础上对牛角瓜发根中强心苷的最优生产条件进行了探索，发现牛角瓜受到损伤后10天左右其强心苷含量达到最高值（39.31±0.24 mg/g DW）。另外，研究了不同诱导子对牛角瓜发根中强心苷产量的影响，结果表明50 mg/l壳聚糖处理20天后牛角瓜发根中强心苷产量可以达到1054.47±55.02 mg/l，较对照提高了2.71倍。 |
| 英文摘要 | Calotropis gigantea is a tropical plant which possesses the potential of serving as both a medicinal and energy resource. However, its production of medicinal ingredients and energy substances under natural conditions is too low to meet the requirement of industrial scaling up. Due to the significant advantages of molecular breeding, such as short cycle and definite goal, it was used for the production of high hydrocarbon yielding transgenetic C. gigantea in this study. Main results of this research are as follows: 1. The regeneration of C. gigantea from the callus was studied and the result showed that the callus of C. gigantea was less sensitive to plant growth regulators and difficult to obtain regenerated shoots through tissue culture. As an alternative way for plant regeneration, the regeneration from hairy root was also studied. Optimal induction rate was obtained when Agrobactorium rhizogenes (LBA9402) suspension (OD600=0.5) was diluted 15 times before infection, the C. gigantea leaves were infected with LBA9402 for 30min and then sterilized under 300mg / l Cef . Adventitious shoots were obtained from the hairy roots of C. gigantea on MS+NAA 0.5 mg/l+6-BA 4.0 mg/l solid medium, and the regenerated shoot induction frequency was 10%. Regenerated shoots rooted easily on MS medium and the regenerated root induction frequency was 100%. 2. To improve the production of hydrocarbons of C. gigantea hairy roots, the hairy root culture medium were optimized. The highest yield of 304.81 mg/l was achieved when the hairy roots were cultured in MMS-CG medium with 3% sucrose. A 2 liters bubble column bioreactor was also used for the production of hydrocarbons. Under the optimal culture condition (light intensity of 34.17 μmol/m2 .s, air flow of 0.40 vvm and inoculum density of 56.00 g/l), the hydrocarbon yield was 356.14 mg/l. In addition, the adding of 100mg / l chitosan significantly improved the hydrocarbon yield of C. gigantea hairy roots and it can achieve 614.45 mg/l at 25 day。 3. In order to further enhance the production of hydrocarbons in the hairy root system of C. gigantea, ATCC15834 genes of Agrobacterium rhizogenes was selected to mediate two alkanes related genes (WAX2 and CER1) into the hair root. Hair root which was transferred with WAX2 produced higher amount of hydrocarbon compared with the wild ones. The WAX2 transgenic line was also obtained and its hydrocarbon and alkane content increased 3.39% and 23.91% than wild plants, respectively. 4. The extraction and detection methods for cardenolides in the hairy roots of C. gigantea were established, based upon which the optimal extraction conditions for cardenolides were explored. The result revealed that the content of cardenolides reached the highest value 10 days after infection (39.31±0.24 mg/g DW). In addition, the effects of elicitors on the enhancement of cardenolides were studied. The result showed that the addition of chitosan (50 mg/l) for 20 days arose a hightest yield of cardenolides (1054.47 mg/l), which was 2.71-fold than the control. |
| 语种 | 中文 |
| 公开日期 | 2013-09-25 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1875]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 孙健. 牛角瓜及其发根中碳氢化合物与强心苷的代谢调控[D]. 中国科学院研究生院. 2012. |
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