竹节参毛状根培养体系的建立及人参皂苷Re的合成

Culture of hairy roots of Panax japonicus and ginsenoside Re synthesis

目的:建立竹节参毛状根的诱导方法和培养体系,并对其生长特性和所合成的人参皂苷Re的含量进行分析。方法:用卸甲型根癌农杆菌C58C1菌株感染竹节参预培养的地上茎,诱导毛状根产生,在1/2MS培养基上扩大培养并测定其生长特性;用PCR检测竹节参转化毛状根的T-DNA及HPLC测定人参皂苷Re含量。结果:C58C1菌株能诱导竹节参地上茎产生毛状根,在浸染25min时,诱导率最高,为90%;经PCR检测,C58C1菌株Ri质粒上的rolB基因已经整合到竹节参毛状根基因组中;HPLC检测发现,竹节参毛状根均能合成人参皂苷Re,其中含量最大的是1/2MS液体培养的单克隆PJ8,其值高达60.26 mg.g-1。结论:初步建立了竹节参毛状根的诱导和培养方法,为大规模培养竹节参毛状根以生产高含量的人参皂苷Re奠定了实验基础。

Objective： To establish the induction method and culture system of hairy roots of Panax japonicus,and determine ginsenoside Re contents.Method： Hairy roots of P.japonicus was obtained through infecting pre-incubated terrestrial stem with the Agrobacterium tumefaciens strain C58C1;its enlarging culture was carried out on the 1/2MS medium,and growing characteristics were measured.The transformation of T-DNA was examined by PCR and ginsenoside Re content was determined by HPLC.Result： A.tumefaciens strain C58C1 could make terrestrial stem of P.japonicus bring about hairy roots,the max inductivity was 90% when infecting for 25 min.The PCR examination result showed that rolB genes could be inserted into the hair roots of P.japonicus.All hairy roots could synthesize ginsenoside Re,among them,the max content was PJ8 with 60.26 mg·g-1.Conclusion： It was reported for the first time that the induction method and culture system of hairy roots of P.japonicus were established successfully,which provided a foundation for producing high content ginsenoside Re through culturing the hairy roots of P.japonicus.