茉莉酸甲酯诱导刺人参不定根三萜皂苷合成及其关键酶基因表达研究  

Biosynthesis of Triterpenoid Saponins and Key Enzymes Gene Expression from Adventitious Root of Oplopanax elatus Induced by Methyl Jasmonate

作　　者：向文珍 张贺 王鑫[1] 康孟莹 王宇[1] 李玉花[1] 吴昊[1] XIANG Wen-zhen;ZHANG He;WANG Xin;KANG Meng-ying;WANG Yu;LI Yu-hua;WU Hao(Heilongjiang Key Laboratory of Plant Bioactive Substance Biosynthesis and Utilization/Key Laboratory of Saline-alkali Vegetation Ecology Restoration,Ministry of Education,Northeast Forestry University,Harbin 150040,China)

机构地区：[1]黑龙江省植物天然活性物质的生物合成与利用重点实验室/东北林业大学东北盐碱植被恢复与重建教育部重点实验室,黑龙江哈尔滨150040

出　　处：《中药材》2024年第9期2146-2153,共8页Journal of Chinese Medicinal Materials

基　　金：国家自然科学基金项目(U21A20243,32271823);黑龙江省自然基金项目(TD2022C001,YQ2021C003)。

摘　　要：目的:建立生物反应器液体发酵培养刺人参组培不定根的繁殖体系,研究MeJA诱导下刺人参三萜总皂苷的积累量及其生物合成关键酶基因的表达量。方法:以刺人参根为外植体,筛选出诱导不定根的最适培养基激素配比;利用液体培养基进行悬浮培养,在10 L气升式生物反应器中扩大培养刺人参不定根;用0、50、100和200μmol/L茉莉酸甲酯(MeJA)诱导刺人参不定根,采用香草醛-冰醋酸法测定不定根三萜总皂苷含量;利用qPCR测定MeJA诱导0、6及12 h后刺人参不定根中三萜皂苷合成途径关键酶基因SQS,SQE,BAS,P450及UGT的表达量差异。结果:在固体MS培养基中添加3 mg/L IBA可诱导刺人参不定根,在液体MS培养基中添加3 mg/L IBA可实现刺人参不定根悬浮培养增殖,利用10 L气升式生物反应器培养约30 d不定根生物量增加约40倍;在100μmol/L MeJA诱导下,刺人参三萜总皂苷的含量达对照组的2.3倍,刺人参三萜皂苷合成途径关键酶基因SQS,SQE,BAS,P450及UGT的表达量升高至对照的1.3~58.6倍。结论:成功建立刺人参不定根液体发酵培养繁殖体系,外源添加MeJA可以诱导刺人参三萜皂苷合成途径关键酶基因表达的上调,从而提升了三萜总皂苷的积累量。本研究为阐明刺人参中三萜皂苷生物合成机制提供了依据。Objective:To establish a propagation system for the tissue culture of Oplopanax elatus adventitious roots using liquid fermentation in a bioreactor,to investigate the effects of methyl jasmonate(MeJA)on the accumulation of triterpenoid total saponins and the expression of key enzyme gene involved in biosynthesis.Methods:The optimum medium hormone ratio for inducing adventitious root was selected by taking Oplopanax elatus adventitious roots as explants.The adventitious root was expanded in 10 L airlift bioreactor by suspension culture of liquid medium.The Oplopanax elatus adventitious roots was induced by 0,50,100 and 200μmol/L methyl jas-monate(MeJA),the content of total saponins in adventitious roots was determined by vanillin and glacial acetic acid method.The ex-pression levels of key enzyme genes SQS,SQE,BAS,P450 and UGT in the triterpene saponin synthesis pathway of Oplopanax elatus adven-titious roots were determined by qPCR after 0,6 and 12 hours induction by MeJA.Results:Adding 3 mg/L IBA in the solid MS medium could induce Oplopanax elatus adventitious roots,adding 3 mg/L IBA in the liquid MS medium could achieve the suspension culture pro-liferation of Oplopanax elatus adventitious roots.The biomass increased by about 40 times after 30 days of adventitious roots cultivation with 10 L airlift bioreactor.Under the induction of 100μmol/L MeJA,the content of triterpenoid saponins of Oplopanax elatus adventitious roots was 2.3 times that of the control group.The expression levels of SQS,SQE,BAS,P450 and UGT,which were the key genes of triter-pene saponin synthesis pathway,increased to 1.3~58.6 times of the control.Conclusion:The liquid fermentation culture and propagation system of Oplopanax elatus adventitious roots is successfully established.Exogenous addition of MeJA can induce the up-regulation of key enzyme gene expression in the pathway of triterpene saponins synthesis,thereby increasing the accumulation of triterpene saponins.This study provides a basis for elucidating the biosynthesis mechanism o

关 键 词：刺人参 不定根 茉莉酸甲酯(MeJA) 三萜皂苷 气升式生物反应器 

分 类 号：R282.2[医药卫生—中药学]