在线富集生物反应器培育特异性花生毛状根合成白藜芦醇

This research we concentrated on two tasks: in first we establishes the system of the hairy roots culture of a new peanut 仲恺花21# that optimizated with the distant hybridization technique. And screening the varieties of peanut with several factors such as the resveratrol content in high, suitable for hairy root culture and easy to accept the Ri plasmid. Then the culture system will be made become a efficient biosynthesis system for resveratrol . Secondly, the molecular imprinted polymer(MIP)will be prepared ,which is designed to be with high selectivity and high adsorption of resveratrol, and the main part of bioreactor is made of this material, which will promote the secretion of resveratrol into media by the peanut hairy roots and the performance of the bioreactor .The resveratrol in peanut hairy roots culture media will be in situ enrichment with MIP on-line. Resveratrol is a polyphenol with health care function. It is advance for selection the new peanut varieties with high resveratrol content to establish the system of the peanut hairy roots culture, we propose to improve the performance of the bioreactor by applicated the molecular imprinting technique.It also promotes the secretion of resveratrol from beanut hairy root .This provides the necessary fundamental study to chieve for scale in production secondary metabolite of plants by use of hairy roots culture technique.In this research we focus on two innovative items. 1. we propose to establishe a hairy roots culture system efficient synthesizing resveratrol with the specific peanut varieties. 2. Application of the molecular imprinting technique, the bioreactor will be made with the function of in situ enrichment of resveratrol on-line in peanut hairy roots culture medium .

1、基于分子印迹聚合物独特的选择性和亲和力,制备对白藜芦醇高选择性、高吸附性的分子印迹吸附材料，并用此材料构建生物反应器的核心部件，促进花生毛状根白藜芦醇的分泌，优化生物反应器的性能；同时利用分子印迹吸附剂直接从发根培养基中在线原位抓捕富集白藜芦醇。2、应用远缘杂交技术优化的仲恺花21号系列花生品种培养发根，筛选白藜芦醇含量高、根系适合诱导毛状根的特异性花生品种，高效生物合成白藜芦醇。白藜芦醇是有特殊医疗保健功能的多酚物质，利用花生毛状根合成白藜芦醇，便于选择高产白藜芦醇的花生株系；用分子印迹技术改造生物反应器性能，促进花生毛状根白藜芦醇分泌，并进行原位富集，为实现毛状根培养生物合成植物次生代谢物大规模生产提供必要的基础研究。 本项目着重进行如下的创新性探索：1、以分子印迹技术创建在线富集生物反应器。2、构建特异性的花生毛状根培育体系高效合成白藜芦醇。

1、对秋季播种成熟期20个花生品种，春季播种花针期95个品种，成熟期130个品种的根、茎进行了白藜芦醇含量对比筛选，结果表明：春、秋季播种成熟期根、茎白藜芦醇含量均明显高于其他品种；当植株到达成熟期后，根白藜芦醇含量呈升高趋势，含量高于茎部。若根、茎整株质量比为1：15计算，秋季成熟期ZK08单株白藜芦醇提取量达2.842 mg；春季播种成熟期S21单株白藜芦醇提取量达到6.974 mg；对高含量白藜芦醇花生品种亲源信息进行了对比：70%为珍珠豆型植株，亲源信息方面， 40%植株由粤油系列或湛油系列杂交而成，25%植株为广西种质资源，湛油、粤油杂交系列与广西种质系列可能在白藜芦醇含量方面更具优势。.2、本研究以优质无菌苗品种为YH02、AS09和C1花生的叶子、下胚轴及子叶作为实验材料，成功获取花生的毛状根及发根组织的基因组DNA，利用HPLC检测，花生YH02自然生长的根部、无菌苗的根部、过发根农杆菌R1000介导过根的白藜芦醇含量分别为486.0µg/g、 1.381µg/g、22.93µg/g 。.3、探究沉淀聚合法、悬浮聚合法制备白藜芦醇分子印迹聚合物（MIPs），结果表明，沉淀聚合法最佳条件为：白藜芦醇、 MAA和EGDMA的摩尔比为1:4:25， AIBN为0.1.0 g，甲醇20.0 mL，微球吸附容量为72.70 μmol/g；悬浮聚合法最佳条件为：白藜芦醇、MAA和EGDMA摩尔比为1:12:50，氯仿60.0mL，合成转速195r/min，分散剂4.0g，微球对白藜芦醇吸附量为16.97μmol/g，对白藜芦醇苷吸附量为6.642μmol/g。.4、生物反应器构建及运行，本课题构建的生物反应器装置优化培养条件为：CO2浓度为750 ppm、温度为25℃、湿度为85%。萌芽期每隔3小时雾化喷淋一次，幼苗期每隔4小时雾化喷淋一次。萌芽期3天，9-10天可长成约10cm左右的花生苗，且根系较发达。在花生培养的同时将分子印迹聚合物置于装置中，利用分子印迹聚合物对花生的分泌物中的白藜芦醇进行特异性吸附，再将白藜芦醇从分子印迹中解吸附。对生物反应器的传质动力特性及持续运行调控进行了研究。