南方红豆杉毛状根诱导体系的建立及毛状根中 紫杉醇的分离纯化研究

目的:研究发根农杆菌对南方红豆杉转化的转化体系和毛状根中紫杉醇的提取纯化工艺;方法:采用发根农杆菌对南方红豆杉不同外植体进行诱导,并对影响毛状根转化的因素进行分析。采用超临界CO2流体萃取法、硅胶柱色谱法以及高效液相法对毛状根中的紫杉醇进行分离提纯;结果:成功构建了南方红豆杉毛状根诱导体系。其中农杆菌的种类、外植体类型、预培养和共培养时间、培养基中激素浓度均影响毛状根的转化率。毛状根经高压纸电泳检测显示表达冠瘿碱。毛状根中紫杉醇经超临界CO2法提取及硅胶柱色谱法分离提纯,采用高效液相法分析显示其纯度可达到98%。结论:发根农杆菌诱导南方红豆杉产生的毛状根中可产生紫杉醇,可作为紫杉醇的主要来源。

Induction of Taxus chinensis var. mairei Hairy Roots and Isolation and Purification of Paclitaxel in Hairy Roots

Objective: To explore Induction system of Taxus chinensis var.mairei hairy roots and isolation and purification of paclitaxel in hairy roots.Methods: Genetic transformation of Taxus chinensis var.mairei by three different Agrobacterium rhizogenes,the effect of some factors on transformation efficiency were analyzed.Paclitaxel in hairy roots was isolated and purified by supercritical CO2 fluid extraction,silica gel column chromatography and high performance liquid chromatography(HPLC).Results: The induction system of Taxus chinensis var.mairei hairy roots was established.The factors influenced transformation efficiency contain Agrobacterium species of Agrobacterium rhizogenes,types of explants,pro-culture,co-culture time and hormone concentrations in the medium.High voltage paper electrophoresis(HVPE) indicated that crown gall were expressed in Taxus chinensis var.mairei hairy roots.Paclitaxel in hairy roots was successfully separated and purified by supercritical CO2 fluid extraction and silica gel column chromatography.The purity of paclitaxel was 98% by HPLC assay.Conclusion: Taxus chinensis var.mairei hairy roots induced by Agrobacterium rhizogenes can produce paclitaxel,as source of paclitaxel.