摘要
目的:建立钩藤发根的高效诱导体系,筛选最适于钩藤发根生长和活性成分积累的培养基。方法:用发根农杆菌C58C1(pRiA4)浸染钩藤无菌的茎、嫩叶和老叶,探究C58C1对不同外植体的发根诱导率;用6种植物培养基(1/2MS、MS、N6、White、B5、WPM)培养钩藤发根,HPLC测定钩藤碱和异钩藤的含量。结果:农杆菌C58C1对3种外植体的诱导率都超过了80%,嫩叶的最高达到了91.8%;PCR检测表明农杆菌中rolB和rolC基因均已整合入钩藤发根的基因组;钩藤发根能合成钩藤碱和异钩藤碱2种活性成分,且钩藤碱占优势;B5培养基最适于钩藤发根的生长和2种生物碱的积累,钩藤碱与异钩藤碱产量最高,100 mL培养基分别可积累0.506 mg和0.160 mg;1/2MS是次优培养基。结论:该研究建立了钩藤发根的高效诱导体系和最适培养体系,为钩藤活性成分的大量生产和农杆菌介导的发根遗传转化体系研究奠定了技术基础。
Objective:To establish highly efficient induction system of Uncaria rhynchophylla hairy root and screen for optimum medium for hairy root growth and active constituent accumulation.Methods:Sterile Uncaria rhynchophylla stem,young leaf and old leaf were used as explants and infected by Agrobacterium rhizogenes C58 C1(pRiA4)solution,the induction rate of the C58 C1 to different explants was investigated,and then six plant culture media(1/2 MS,MS,N6,White,B5,WPM)were adopted for cultivation,rhynchophylline and isorhynchophylline contents were determined by HPLC method.Results:The hairy root induction rates of three types of explants infected by Agrobacterium rhizogenes C58 C1 all exceeded 80%,the highest induction rate reached 91.8%when young leaves were as explants.PCR detection showed that two genes of rolB and rolC in Agrobacterium rhizogenes C58 C1 had integrated into the genome of Uncaria rhynchophylla hairy root.Hairy root could synthesize rhynchophylline and isorhynchophylline,and rhynchophylline was allways the predominant alkaloid.B5 medium was most suitable for Uncaria rhynchophylla hairy root growth and two alkaloids accumulation.Yields of rhynchophylline and isorhynchophylline were the highest in B5 medium and could reach 0.506 mg and 0.160 mg in 100 mL medium,respectively.1/2 MS was a suboptimal medium.Conclusion:The efficient induction system and optimum culture system of Uncaria rhynchophylla hairy root are established in this study,and it lays the technical foundation for mass production of Uncaria rhynchophylla active constituents and Agrobacterium rhizogenes-mediated genetic transformation system of hairy root.
作者
姜育松
强玮
马东迪
左婷婷
苏畅凤
代玉玉
路星星
JIANG Yu-song;QIANG Wei;MA Dong-di;ZUO Ting-ting;SU Chang-feng;DAI Yu-yu;LU Xing-xing(College of Life Sciences,Guizhou University/Institute of Agro-bioengineering/Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region,Ministry of Education/Collaborative Innovation Center for Mountain Ecology&Agro-Bioengineering,Guiyang 550025,China)
出处
《中药材》
CAS
北大核心
2021年第11期2516-2520,共5页
Journal of Chinese Medicinal Materials
基金
贵州大学“SRT计划”项目资助(贵大SRT字[2018]190号)
贵州大学引进人才科研项目(贵大人基合字[2017]58)
贵州省生物学一流学科建设项目(GNYL[2017]009)
国家重点研发计划课题(2016YFC0502604)
贵州省科技计划重大专项课题(黔科合平台人才[2017]5411-06)
关键词
钩藤
发根农杆菌
发根
钩藤碱
培养基优化
Uncaria rhynchophylla(Miq.)Miq.ex Havil.
Agrobacterium rhizogenes
Hairy root
Rhynchophyll
Medium optimization
