Ri质粒诱导喜树毛状根培养基中喜树碱的研究

Study on hairy root cultures of Camptotheca acuminata (Nyssaceae) and the camptothecin (CPT) from medium

喜树(Camptotheca acuminate)可产生具有抗癌作用的喜树碱,有许多用细胞培养方法制备喜树碱的尝试,但都因极低的产率而无从深入。植物毛状根培养的应用将成为一种生物碱生产的替代方法。以发根农杆菌(Agrobacterium rhizogenes)A41.2556感染喜树诱导产生毛状根,建立喜树碱毛状根产生体系。甘露碱检测及PCR结果证实发根农杆菌Ri质粒的rolB基因已在喜树毛状根基因组中整合并得到表达。确定了用RP-HPLC测定发根培养基中喜树碱的的方法,比较了不同的吸附树脂对培养基中喜树碱的富集能力,从培养30d的喜树培养基中获得喜树碱干质量分数为0.14mg·g-1。

Camptothecin （CPT） is an anticancer and antiviral alkaloid produced by the Camptotheca acuminata （Nyssaceae）. Several attempts have been made to produce CPT from cell suspensions. However,the low yields obtained limit this approach. The ability of plant hairy root cultures may be an alternative source of alkaloid production. Hairy root cultures of C. acuminata were established from tissue transformed with Agrobacterium rhizogenes strains As1.2556. Integration of the genes responsible for the hairy-root phenotype （rolB genes） into the plant genome was verified by DNA PCR analysis. As the method for the determination of Camptothecin （CPT） from the medium by RP-HPLC was established,the method for the extracting of Camptothecin （CPT） from the medium with polyacrylamid was established. The cultures were able to synthesize the alkaloids at levels equal to 0.14 mg/g dry weight for CPT from the hairy root.