[关键词]
[摘要]
目的 提高药用植物颠茄Atropa belladonna有效成分的产量,为颠茄在实际生产中提供经济高效的处理方法,为相关药用植物次生代谢的机制研究提供基础性参考。方法 通过向培养基中添加4种不同诱导子,包括茉莉酸甲酯(MJ)、硝酸银(AgNO3)、水杨酸(SA)、酵母提取物(YE),研究诱导子对颠茄毛状根有效成分的积累及代谢途径中的关键酶基因1,4-丁二氨-氮-甲基转移酶(putrescine N-methyl transferase,pmt)基因、托品酮还原酶-I(tropinone reductase-I,trI)基因和莨菪碱6-β-羟化酶(hyoscyamine-6-β-hydroxylase,h6h)基因表达的影响。实验将0.5 g新鲜颠茄毛状根接种于盛有B5液体培养基的三角瓶中,暗培养12 d后,将培养基更换为含有诱导子的4种新培养基,相同条件下再培养2 d后采集毛状根样品,测定每瓶毛状根的鲜质量、干质量、部分生理指标、托品烷类生物碱的量及3种关键酶基因的表达量。结果 MJ抑制了颠茄毛状根的生长及托品烷类生物碱的产生,pmt、trI基因的表达量与对照组相比也有所下降;AgNO3虽然抑制了毛状根的生长,但却显著提高了托品烷类生物碱的含量,同时pmt、trI、h6h基因的表达量均升高;SA对颠茄毛状根的生长影响不显著,但使东莨菪碱的含量提高;YE对颠茄毛状根的生长及托品烷类生物碱的含量均有促进作用,pmt、trI、h6h基因的表达量也相应升高。结论 AgNO3是提高颠茄毛状根中托品烷类生物碱含量的最佳诱导子,YE在促进颠茄毛状根生长的同时也可促进其次生代谢。推断诱导子通过调控某种或某几种关键酶基因的表达来影响生物碱的代谢合成,为生产中提高颠茄毛状根药用成分含量提供了可参考的方法。
[Key word]
[Abstract]
Objective To improve the yield of the effective medicinal ingredients of Atropa belladonna, to provide economic and efficient method for the actual production of A. belladonna, therefore to provide a basic reference for the related research on the mechanism of secondary metabolism of medicinal plants. Methods In this study, the influences of four kinds of elicitors, including methyl jasmonate (MJ), AgNO3, salicylic acid (SA), yeast extract (YE), on the accumulation of active components and the expression level of key metabolic enzyme genes, including putrescine N-methyl transferase (pmt), tropinone reductase-I (trI), and hyoscyamine-6-β-hydroxylase (h6h), were studied in hairy roots of A. belladonna. 0.5 g fresh hairy roots of A. belladonna were cultivated in B5 liquid medium. 12 d later, these mediums were replaced with the new medium containing one kind of four elicitors. Hairy roots were taken samples after 2 d to mensurate the fresh weight, dry weight, the content of tropane alkaloids, some physiological indexes, and three key genes expression level. Results MJ inhibited the growth and tropane alkaloids accumulation of hairy roots. The gene expression level of pmt and trI also decreased compared with control group (CK). The contents of tropane alkaloids and the expression level of pmt, trI and h6h were all increased compared with CK by the treatment of AgNO3, while the growth of hairy rootswas inhibited; SA contributed to the increased content of hyoscyamine, but with no obvious influence on growth of hairy roots. As to YE, the content of tropane alkaloids and the expression level of pmt, trI, h6h were all increased correspondingly. Further more, YE was benefit for the growth of hairy roots. Conclusion Elicitors had selective influence on growth and tropane alkaloids accumulation in hairy roots of A. belladonna. The best elicitor on accumulation of tropane alkaloids was AgNO3. YE could effectively improve of the growth of hairy and contents of tropane alkaloids. This study concluded that these elicitors influence the secondary metabolism of A. belladonna by regulating and controlling the expression level of some genes of key metabolic Enzyme, which could provide an effective method to enhance the medicinal composition in the culture of hairy roots of A. belladonna.
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[基金项目]
国家自然科学基金资助项目(30500041);重庆市科技攻关项目(cstc2012gg-yyjs80013)