摇瓶培养条件下雷公藤不定根生长及次生代谢产物含量的研究
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摘要
以雷公藤(Tripterygium wilfordii Hook. f.)不定根为材料,研究摇瓶悬浮培养条件下接种密度、装液比例、逐级放大、消泡剂、大孔吸附树脂种类及浓度对雷公藤不定根增长量、不定根及培养基中雷公藤内酯醇、雷公藤吉碱、雷公藤次碱含量的影响。结果显示,接种密度在15 g/L (FW)时较适合不定根的继代培养和次生代谢产物的积累。250 m L摇瓶中装入100 m L培养基,即装液量为2/5时,培养基利用率最高。随着摇瓶体积的逐渐放大,不定根增长量和3种次生代谢产物含量略有下降,5 L摇瓶中不定根增长量为对照的91.6%,内酯醇、吉碱和次碱的含量分别为对照的91.8%、91.7%和96.9%。6种大孔吸附树脂中,XAD-7处理对不定根的生长有明显促进作用,培养结束时,3种次生代谢产物产量显著提高,当XAD-7浓度为0.5 g/瓶时不定根增长量为对照的1.2倍,内酯醇、吉碱和次碱产量最高,分别为对照的2.9、2.4和2.2倍。培养基中添加消泡剂后不定根增长量、3种次生代谢产物总产量均不同程度下降,其中,LX-603处理后,虽然不定根增长量为对照的85%,内酯醇、吉碱和次碱产量分别为对照的78%、64%和87%,但明显抑制了培养过程中泡沫的产生。研究结果表明筛选的摇瓶逐级放大培养雷公藤不定根的方法效果较好,可为雷公藤不定根生物反应器放大培养奠定基础。
Tripterygium wilfordii Hook.f.,adventitious roots were used in vitro to study the effects of inoculation density,liquid medium volume per flask,step-by-step amplification,defoaming agent,macroporous adsorption(MA) resin type,and concentrations of triptolide,wilforgine,and wilforine in roots and medium on adventitious root growth after shake-flask culture.Results showed that an inoculation density of 15 g/L was suitable for the subculture of adventitious roots and accumulation of secondary metabolite.The medium utilization rate was the highest in a 250-m L flask with 100 m L of liquid medium.Adventitious root growth declined slightly withthe increase in flask volume.In a 5-L bottle,the adventitious root growth and triptolide,wilforgine,and wilforine concentrations were 91.6%,91.8%,91.7%,and 96.9%,respectively,compared to the control,XAD-7 resin markedly enhanced adventitious root growth and secondary metabolite content among the six kinds of MA resin.The adventitious root growth and triptolide,wilforgine,and wilforine content increased 1.2-,2.9-,2.4-,and2.2-fold,respectively,compared to the control when XAD-7 resin was at 0.5 g/bottle.Adventitious root growth and secondary metabolite content all decreased at differing degrees when the defoaming agent was added to the medium.Adventitious root growth and triptolide,wilforgine,and wilforine contents were 85%,78%,64%,and 87%,respectively,compared to the control when LX-603 was added to the medium;however,foam was markedly inhibited during the culturing process.This study demonstrated that step-by-step amplification was a feasible approach for adventitious root culture.Furthermore,this research provides the basis for the scale-up culture of T.wilfordii adventitious roots using a bioreactor.
Tripterygium wilfordii Hook.f.,adventitious roots were used in vitro to study the effects of inoculation density,liquid medium volume per flask,step-by-step amplification,defoaming agent,macroporous adsorption(MA) resin type,and concentrations of triptolide,wilforgine,and wilforine in roots and medium on adventitious root growth after shake-flask culture.Results showed that an inoculation density of 15 g/L was suitable for the subculture of adventitious roots and accumulation of secondary metabolite.The medium utilization rate was the highest in a 250-m L flask with 100 m L of liquid medium.Adventitious root growth declined slightly withthe increase in flask volume.In a 5-L bottle,the adventitious root growth and triptolide,wilforgine,and wilforine concentrations were 91.6%,91.8%,91.7%,and 96.9%,respectively,compared to the control,XAD-7 resin markedly enhanced adventitious root growth and secondary metabolite content among the six kinds of MA resin.The adventitious root growth and triptolide,wilforgine,and wilforine content increased 1.2-,2.9-,2.4-,and2.2-fold,respectively,compared to the control when XAD-7 resin was at 0.5 g/bottle.Adventitious root growth and secondary metabolite content all decreased at differing degrees when the defoaming agent was added to the medium.Adventitious root growth and triptolide,wilforgine,and wilforine contents were 85%,78%,64%,and 87%,respectively,compared to the control when LX-603 was added to the medium;however,foam was markedly inhibited during the culturing process.This study demonstrated that step-by-step amplification was a feasible approach for adventitious root culture.Furthermore,this research provides the basis for the scale-up culture of T.wilfordii adventitious roots using a bioreactor.
引文
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[2]Swingle WT,Haller HL,Siegler EH,Swingle MC.A Chinese insecticidal plant,Tripterygium wilfordii,introduced into the United States[J].Science,1941,93(2403):60-61.
[3]Tao X,Cai JJ,Lipsky P.The identity of immunosuppressive components of the ethyl acetate extract and chloroform methanol extract(T2)of Tripterygium wilfordii Hook.F.[J].Journal of Pharmacology and Experimental Therapeutics,1995,272(3):1305-1316.
[4]岗艳云,张正行.雷公藤及其单体的药理作用研究进展[J].中国药科大学学报,1995,26(4):252-256.
[5]Liu QY.Triptolide and its expanding multiple pharmacological functions[J].International immunopharmacology,2011,11(3):377-383.
[6]Lu Y,Zhang Y,Li L,Feng XJ,Ding S,Zheng W,Li JX,Shen PP.TAB1:A target of triptolide in macrophages[J].Chemistry&Biology,2014,21:246-256.
[7]周琳,马志卿,冯俊涛,李修伟,张兴.雷公藤生物碱制品对小菜蛾和菜青虫的控制效果[J].西北农林科技大学学报:自然科学版,2006,34(12):169-173.Zhou L,Ma ZQ,Feng JT,Li XW,Zhang X.Control efficacy of alkaloid products from Tripterygium wilfordii Hook against Plutella xylostella and Pieris rape[J].Journal of Northwest Sci-Tech University of Agriculture and Forestry:Natural Science Edition,2006,34(12):169-173.
[8]周琳,李俊领,刘向阳,马志卿,冯俊涛,张兴.雷公藤总生物碱对粘虫幼虫神经系统酶活性及神经递质含量的影响[J].昆虫学报,2015,58(8):856-863.Zhou L,Li JL,Liu XY,Ma ZQ,Feng JT,Zhang X.Effects of total alkaloids from Tripterygium wilfordii on the activities of neural enzymes and contents of neurotransmitters in larval Mythimna separata[J].Acta Entomologica Sinica,2015,58(8):856-863.
[9]Luo DQ,Zhang X,Tian X,Liu JK.Insecticidal compounds from Tripterygium wilfordii active against Mythimna separata[J].Zeitschrift fur Naturforschung.C,A Journal of Biosciences,2004,59(5-6):421-426.
[10]黄文华,张蕊,郭宝林,斯金平.雷公藤药材总生物碱含量及影响因素的研究[J].中国中药杂志,2008,33(1):15-18.Huang WH,Zhang R,Guo BL,Si JP.Study on total alkaloids content and its influential factors in medicinal materials of Tripterygium[J].China Journal of Chinese Materia Medica,2008,33(1):15-18.
[11]张璟,陈蒙蒙,蒲时,祝传书,张兴.雷公藤中雷公藤甲素、雷公藤吉碱和次碱的高效液相色谱-电喷雾串联质谱分析方法[J].农药学学报,2018,20(2):197-203.Zhang J,Chen MM,Pu S,Zhu CS,Zhang X.Determination of triptolide,wilforgine and wilforine in Tripterygium wilfordii by high performance liquid chromatography-electrospray ionization tandem mass spectrometry[J].Chinese Journal of Pesticide Science,2018,20(2):197-203.
[12]Brinker AM,Raskin I.Determination of triptolide in root extracts of Tripterygium wilfordii by solid-phase extraction and reverse-phase high-performance liquid chromatography[J].J Chromatogr A,2005,1070(1-2):65-70.
[13]Ni L,Ma J,Li CJ,Li L,Guo JM,Yuan SP,Hou Q,Guo Y,Zhang DM.Novel rearranged and highly oxygenated abietane diterpenoids from the leaves of Tripterygium wilfordii[J].Tetrahedron Lett,2015,56(10):1239-1243.
[14]洪伟,李键,吴承祯,洪滔,范海兰,唐佳栋,陈灿.雷公藤栽培及利用研究综述[J].福建林学院学报,2007,27(1):92-96.Hong W,Li J,Wu CZ,Hong T,Fan HL,Tang JD,Chen C.Research summary of cultivation and utilization of Tripterygium wilfordii[J].Journal of Fujian College of Forestry,2007,27(1):92-96.
[15]李琰,冯俊涛,史晓燕,吴勇延,张兴.雷公藤组培产物中雷公藤甲素和总生物碱含量的测定[J].农药学学报,2009,11(3):367-372.Li Y,Feng JT,Shi XY,Wu YY,Zhang X.Study on the determination of triptolide and total alkaloids in Tripterygium wilfordii Hook.f.cultures[J].Chinese Journal of Pesticide Science,2009,11(3):367-372.
[16]张兴,李琰,冯俊涛,王智辉,王永宏.一种由雷公藤悬浮细胞诱导不定根的方法:ZL200810017697[P].2008-08-27[2018-07-09].
[17]刘跃,张红蕊,赵林,刘新利.消泡剂在发酵工业中的应用[J].齐鲁工业大学学报:自然科学版,2014,28(2):37-39.Liu Y,Zhang HR,Zhao L,Liu XL.Application of antifoams in fermentation processes[J].Journal of Qilu University of Technology:Natural Science Edition,2014,28(2):37-39.
[18]梁小玲,黄伟敏,黄区,李俏,周建湘.泡沫对食品发酵的影响及控制[J].现代食品,2016(14):22-23.Liang XL,Huang WM,Huang Q,Li Q,Zhou JX.The influence and control of foam in food fermentation[J].Modern Food,2016(14):22-23.
[19]Miao GP,Zhu CS,Yang YQ,Feng MX,Ma ZQ,Feng JT,Zhang X.Elicitation and in situ adsorption enhanced secondary metabolites production of Tripterygium wilfordii Hook.f.adventitious root fragment liquid cultures in shake flask and a modified bubble column bioreactor[J].Bioproc Biosyst Eng,2014,37(4):641-650.
[20]Yan Q,Hu ZD,Tan RX,Wu JY.Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption,elicitation and semi-continuous operation[J].J Biotechnol,2005,119(4):416-424.
[21]Miao GP,Zhu CS,Feng JT,Han J,Song XW,Zhang X.Aggregate cell suspension cultures of Tripterygium wilfordii Hook.f.for triptolide,wilforgine,and wilforine production[J].Plant Cell Tiss Org,2013,112(1):109-116.
[22]祝传书,刘艳,陈蒙蒙,蒲时,崔蕾,李琰,张兴.雷公藤转录因子Tw WRKY1基因的克隆与表达分析[J].农业生物技术学报,2018,26(4):595-605.Zhu CS,Liu Y,Chen MM,Pu S,Cui L,Li Y,Zhang X.Cloning and expression analysis of transcription factor TwWRKY1 gene in Tripterygium wilfordii[J].Journal of Agricultural Biotechnology,2018,26(4):595-605.
[23]徐步青,李振中,张俊,王芬,刘幸佳,崔永一.不同培养条件对铁皮石斛类原球茎生物反应器培养的影响[J].中草药,2014,45(5):709-713.Xu BQ,Li ZZ,Zhang J,Wang F,Liu XJ,Cui YY.Effect of different culture conditions on protocorm-like bodies of Dendrobium candidum in bioreactor culture[J].Chinese Traditional and Herbal Drugs,2014,45(5):709-713.
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