摘要
唐古特大黄(Rheum tanguticum)是《中国药典》收载的三种“正品大黄”之一。本研究采用四因素三水平正交试验方法,探讨菌液浓度、外植体类型、乙酰丁香酮浓度和共培养时间四个关键因素对唐古特大黄毛状根诱导的影响,并针对5种不同的发根农杆菌菌株和侵染时间进行试验。结果表明,菌液浓度(OD_(600))为0.4、外植体为胚轴、乙酰丁香酮浓度为100 mg·L^(-1)、共培养时间为4 d时毛状根诱导率最高。PCR技术检测诱导出的毛状根,初步证实了发根农杆菌Ri质粒中的rol C基因已整合到唐古特大黄毛状根中。此外,我们构建过表达载体进行毛状根遗传转化,经PCR扩增初步确认目的基因已整合到唐古特大黄毛状根中。本研究建立了发根农杆菌介导的唐古特大黄遗传转化体系,为唐古特大黄基因功能研究和毛状根次生代谢产物的产业化提供技术参考。
Rheum tanguticum is one of the three"official rhubarb"species recorded in the Chinese Pharmacopoeia.In this study,an orthogonal design L_(9)(3^(4))was used to investigate the effects of four key factors(bacterial concentration,explant type,acetosyringone concentration and co-culture time)on hairy root induction in R.tanguticum.Additionally,five different strains of Rhizobium rhizogenes and various infection times were tested.The results showed that the highest hairy root induction rate was achieved under the following conditions:a bacterial concentration(OD6oo)of 0.4,hypocotyl explant,an acetosyringone concentration of 100 mg·L^(-1),and a co-culture time of 4 d.PCR analysis of the induced hairy roots preliminarily confirmed the integration of the rolC gene from the Ri plasmid of R.rhizogenes into the hairy roots of R.tanguticum.Furthermore,genetic transformation of hairy roots was performed using constructed overexpression vectors,and PCR amplification indicated the integration of the target gene into the R.tanguticum hairy roots.Therefore,a R.rhizogenes-mediated genetic transformation system of R.tanguticum was successfully established.This will provide technical support for further research on gene function in R.tanguticum and the industrial production of the secondary metabolites derived from its hairy roots.
作者
孙晴
张会轩
李欣
李毅
刘宝龙
胡延萍
SUN Qing;ZHANG Huixuan;LI Xin;LI Yi;LIU Baolong;HU Yanping(Qinghai Provincial Key Laboratory of Qinghai-Xizang Plateau Biological Rescources,Northwest Institute of Plateau Biology,Chinese Academy of Sciences,Xining 810008,China;University of Chinese Academy of Sciences,Bejing 100049,China)
出处
《植物生理学报》
北大核心
2025年第8期1171-1179,共9页
Plant Physiology Journal
基金
青海省自然科学基金面上项目(2024-ZJ-915)
2020年中国科学院“西部之光”人才培养计划“西部青年学者”一般项目。
关键词
唐古特大黄
发根农杆菌
遗传转化
毛状根
诱导率
Rheum tanguticum
Rhizobium rhizogenes
genetic transformation
hairy root
induction rate
