镧对人参发根中皂苷积累及合成关键酶基因表达的影响
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摘要
以不同浓度镧处理人参发根, qRT-PCR检测人参皂苷合成关键酶基因PgSS,PgDDS和PgβAS的表达水平, HPLC测定人参皂苷含量,探讨镧对人参发根生长及其人参皂苷合成影响的作用机制。镧浓度分别为5.0, 10.0和15.0 mg·L~(-1)时,促进发根伸长、根毛形成和重量增加, 20.0, 25.0和30.0 mg·L~(-1)时,抑制发根生长;PgSS和PgDDS基因表达水平对镧呈浓度依赖性变化, 20.0 mg·L~(-1)时,PgSS和PgDDS基因表达量均显著增加(P<0.05)且达到最大,高于20.0 mg·L~(-1)时,PgSS和PgDDS基因表达水平随镧浓度升高而降低;镧对PgβAS基因表达无明显影响。镧促进发根中人参皂苷Rb1, Rb2, Rc, Rd, Re, Rg1和总皂苷含量增加,人参皂苷合成积累规律与PgSS和PgDDS基因表达规律一致,当镧浓度为20.0 mg·L~(-1)时,人参皂苷产量亦达到最高。表明镧可以调节人参发根的生长,通过促进人参皂苷合成关键酶基因PgSS和PgDDS的表达增加人参皂苷的生物合成与积累。
To investigate the effect of lanthanum(La) on the ginsenosides biosynthesis and its key enzymes genes expression in hairy roots of Panax ginseng. La was added to the media of ginseng hairy roots with 0.0, 5.0, 10.0, 15.0, 20.0, 25.0 and 30.0 mg·L~(-1), respectively. The expression levels of PgSS, PgDDS and PgβAS genes were identified by qRT-PCR, and the content of ginsenoside Rb1, Rb2, Rc, Rd, Re, Rg1 and total saponins was assayed by HPLC. The correlation between expression profiles of key enzymes genes and the ginsenosides biosynthesis and accumulation was also analyzed. The results indicated that La enhances the extension, root hair formation and biomass of ginseng hairy roots at 5.0, 10.0 and 15.0 mg·L~(-1), and the growth of hairy roots is inhibited by La at 20.0, 25.0 and 30 mg·L~(-1). PgSS and PgDDS genes expression is induced by La in a concentration dependent way, and the expression level increases obviously(P<0.05) and reaches the highest level at 20.0 mg·L~(-1), and then decreases. However, La does not affect the PgβAS gene. At the meantime, biosynthesis and accumulation of ginsenoside Rb1, Rb2, Rc, Rd, Re, Rg1, total saponins increase obviously in a same way of the expression patterns of PgSS and PgDDS genes, the yeild of ginsenosides reaches to the climax at 20.0 mg·L~(-1) La. In conclusion, La can improve the expression of PgSS and PgDDS genes and its corresponding metabolites ginsenoside in hairy roots of P. ginseng. This work provides a foundation for investigation of the accumulation mechanism of ginsenosides under the control of La in P. ginseng.
To investigate the effect of lanthanum(La) on the ginsenosides biosynthesis and its key enzymes genes expression in hairy roots of Panax ginseng. La was added to the media of ginseng hairy roots with 0.0, 5.0, 10.0, 15.0, 20.0, 25.0 and 30.0 mg·L~(-1), respectively. The expression levels of PgSS, PgDDS and PgβAS genes were identified by qRT-PCR, and the content of ginsenoside Rb1, Rb2, Rc, Rd, Re, Rg1 and total saponins was assayed by HPLC. The correlation between expression profiles of key enzymes genes and the ginsenosides biosynthesis and accumulation was also analyzed. The results indicated that La enhances the extension, root hair formation and biomass of ginseng hairy roots at 5.0, 10.0 and 15.0 mg·L~(-1), and the growth of hairy roots is inhibited by La at 20.0, 25.0 and 30 mg·L~(-1). PgSS and PgDDS genes expression is induced by La in a concentration dependent way, and the expression level increases obviously(P<0.05) and reaches the highest level at 20.0 mg·L~(-1), and then decreases. However, La does not affect the PgβAS gene. At the meantime, biosynthesis and accumulation of ginsenoside Rb1, Rb2, Rc, Rd, Re, Rg1, total saponins increase obviously in a same way of the expression patterns of PgSS and PgDDS genes, the yeild of ginsenosides reaches to the climax at 20.0 mg·L~(-1) La. In conclusion, La can improve the expression of PgSS and PgDDS genes and its corresponding metabolites ginsenoside in hairy roots of P. ginseng. This work provides a foundation for investigation of the accumulation mechanism of ginsenosides under the control of La in P. ginseng.
引文
[1]Majeed F,Malik F Z,Ahmed Z,Afreen A,Afzal M N,Khalid N.Ginseng phytochemicals as therapeutics in oncology:Recent perspectives[J].Biomed.Pharmacother.,2018,100:52.
[2]Tam D N H,Truong D H,Nguyen T T H,Quynh L N,Tran L,Nguyen H D,Shamandy B E,Le T M H,Tran D K,Sayed D,Vu V V,Mizukami S,Hirayama K,Huy N T.Ginsenoside Rh1:A systematic review of its pharmacological properties[J].Planta Med.,2018,84(3):139.
[3]Zhao C,Xu T H,Liang Y L,Zhao S J,Ren L Q,Wang Q,Dou B.Functional analysis of beta-amyrin synthase gene in ginsenoside biosynthesis by RNA interference[J].Plant Cell Rep.,2015,34(8):1307.
[4]Cao H Z,Nuruzzaman M,Xiu H,Huang J J,Wu K L,Chen X H,Li J J,Wang L,Jeong J H,Park S J,Yang F,Luo J L,Luo Z Y.Transcriptome analysis of methyl jasmonate-elicited Panax ginseng adventitious roots to discover putative ginsenoside biosynthesis and transport genes[J].Int.J.Mol.Sci.,2015,16(2):3035.
[5]Wang J,Gao W Y,Zhang J,Zuo B M,Zhang L M,Huang L Q.Advances in study of ginsenoside biosynthesis pathway in Panax ginseng C.A.Meyer[J].Acta Physiol.Plant.,2012,34(2):397.
[6]Kim T D,Han J Y,Huh G H,Choi Y E.Expression and functional characterization of three squalene synthase genes associated with saponin biosynthesis in Panax ginseng[J].Plant Cell Physiol.,2011,52(1):125.
[7]Tansakul P,Shibuya M,Kushiro T,Ebizuka Y.Dammar-enediol-II synthase,the first dedicated enzyme for ginsenoside biosynthesis,in Panax ginseng[J].FEBSLett.,2006,580(22):5143.
[8]Han J Y,Kim M J,Ban Y W,Hwang H S,Choi Y E.The involvement of beta-amyrin 28-oxidase(CYP716A52v2)in oleanane-type ginsenoside biosynthesis in Panax ginseng[J].Plant Cell Physiol.,2013,54(12):2034.
[9]袁晓凡,赵兵,王玉春.稀土元素在药用植物细胞和组织培养中的应用[J].植物学通报,2005,22(1):115.Yuan X F,Zhao B,Wang Y C.Application of rare earth elements in medicinal plant cell and tissue culture[J].Chinese Bulletin of Botany,2005,22(1):115.
[10]Wu J Y,Wang C G,Mei X G.Stimulation of taxol production and excretion in Taxus spp.cell cultures by rare earth chemical lanthanum[J].J.Biotechnol.,2001,85(1):67.
[11]元英进,胡国武,王传贵,景莹,周永洽,申泮文.镧、铈对红豆杉细胞生长及紫杉醇合成与释放的影响[J].中国稀土学报,1998,16(1):57.Yuan Y J,Hu G W,Wang C G,Jing Y,Zhou Y Q,Shen P W.Effect of rare earth compounds on the growths,taxol biosynthesis and release of Taxus cuspidata cell culture[J].Journal of the Chinese Society of Rare Earths,1998,16(1):57.
[12]元英进,胡宗定.稀土元素对长春花植物细胞培养的影响[J].稀土,1993,14(3):38.Yuan Y J,Hu Z D.Effects of rare earth elements on plant cell culture in periwinkle[J].Chinese Rare Earth,1993,14(3):38.
[13]杨世海,刘晓峰,果德安.稀土元素镧对掌叶大黄毛状根和非转化根生长及蒽醌产量的影响[J].中草药,2004,35(10):1171.Yang S H,Liu X F,Guo D A.Effect of rare-earth elements La3+on growth of hairy roots and normal roots of Rheum palmatum and their yield of anthraquinone[J].Chinese Traditional and Herbal Drugs,2004,35(10):1171.
[14]姚强,高兴喜,宫志远,任鹏飞,仇可新,刘岩.部分稀土元素对灵芝多糖和三萜类物质液体发酵的影响[J].食品科学,2011,32(5):224.Yao Q,Gao X X,Gong Z Y,Ren P F,Qiu K X,Liu Y.Effects of some rare earth elements on liquid fermentation of Ganoderma lucidum to produce polysaccharides and triterpenoids[J].Food Science,2011,32(5):224.
[15]王景余,李向高,郑友兰.稀土元素对人参皂甙含量的影响[J].中国药学杂志,1991,26(10):591.Wang J Y,Li X G,Zheng Y L.Effect of rare earth element on the content of ginsenoside in ginseng root[J].Chinese Pharmaceutical Journal,1991,26(10):591.
[16]张儒,张变玲,谢涛,李谷才,罗志勇.响应面法优化人参不定根中总皂苷的提取工艺[J].天然产物研究与开发,2015,27(4):726.Zhang R,Zhang B L,Xie T,Li G C,Luo Z Y.Optimization of extraction process of total ginsenoside from adventitious roots of Panax ginseng C.A.Meyer by response surface methodology[J].Natural Product Research and Development,2015,27(4):726.
[17]Yin J X,Zhang D H,Zhuang J J,Huang Y,Mu Y,LüS W.Study on the correlation between gene expression and enzyme activity of seven key enzymes and ginsenoside content in ginseng in over time in Ji'an,China[J].Int.J.Mol.Sci.,2017,18(12):2682.
[18]郭亚鹏,施和平.镧对美洲商陆毛状根生长、皂苷产生及抗氧化酶活性的影响[J].中国稀土学报,2017,35(5):614.Guo Y P,Shi H P.Effects of Lanthanum on growth and esculentoside production in Phytolacca americana L.hairy roots[J].Journal of the Chinese Society of Rare Earths,2017,35(5):614.
[19]Karki P,Kim C,Smith K,Son D S,Aschner M,Lee E.Transcriptional regulation of the astrocytic excitatory amino acid transporter 1(EAAT1)via NF-kappaB and Yin Yang 1(YY1)[J].J.Biol.Chem.,2015,290(39):23725.
[20]de Oliveira C,Ramos S J,Siqueira J O,Faquin V,de Castro E M,Amaral D C,Techio V H,Coelho L C,e Silva P H,Schnug E,Guilherme L R.Bioaccumulation and effects of lanthanum on growth and mitotic index in soybean plants[J].E Ecotoxicol Environ Saf,2015,122(12):136.
[21]Hu H Q,Wang L H,Li Y L,Sun J W,Zhou Q,Huang X H.Insight into mechanism of lanthanum(III)induced damage to plant photosynthesis[J].Ecotox.Environ.Safe,2016,127(5):43.
[22]崔堂兵,张长远,郑穗平,郭勇,蒋玲兰.稀土元素对银杏悬浮培养细胞生长和次级代谢产物积累的影响[J].广东农业科学,2002,(5):29.Cui T B,Zhang C Y,Zheng S P,Guo Y,Jiang L L.Effect of rare-earth elements on Ginkgo biloba cell growth and secondary products accumulation in suspension culture[J].Guangdong Agricultural Sciences,2002,(5):29.
[23]Kim Y J,Jeon J N,Jang M G,Oh J Y,Kwon W S,Jung S K,Yang D C.Ginsenoside profiles and related gene expression during foliation in Panax ginseng Meyer[J].J.Ginseng Res.,2014.38(1):66.
[24]边丽华,邹琳,周冰谦,刘伟,周洁,王晓.稀土元素镧对丹参活性物质积累及其合成途径中酶基因表达的影响[J].中国中药杂志,2016,41(23):4344.Bian L H,Zou L,Zhou B Q,Liu W,Zhou J,Wang X.Effect of Lanthanum on accumulation of activeconstituent and key enzymes expression of Salvia miltiorrhiza hairy root[J].China Journal of Chinese Materia Medica,2016,41(23):4344.
[25]边丽华,邹琳,周冰谦,刘伟,周洁,王晓.稀土元素镧与丹参毛状根中活性成分及其生物合成中关键酶的量效关系[J].中国现代中药,2017,19(3):387.Bian L H,Zou L,Zhou B Q,Liu W,Zhou J,Wang X.The dose-effect relationship between La and active constituent and key enzymes expression of Salvia miltiorrhiza hairy root[J].Modern Chinese Medicine,2017,19(3):387.
[26]罗建平,曹磊,潘利华,查学强.稀土元素对怀槐悬浮培养细胞异黄酮合成及氧化还原态的影响[J].应用与环境生物学报,2008,14(3):362.Luo J P,Cao L,Pan L H,Zha X Q.Effects of rare earth elements on isoflavone biosynthesis and redox status of Maackia amurensis cells in suspension cultures[J].Chinese Journal of Applied and Environmental Biology,2008,14(3):362.
[27]Yang J L,Hu Z F,Zhang T T,Gu A D,Gong T,Zhu P.Progress on the studies of the key enzymes of ginsenosidebiosynthesis[J].Molecules,2018,23(3):589.
[2]Tam D N H,Truong D H,Nguyen T T H,Quynh L N,Tran L,Nguyen H D,Shamandy B E,Le T M H,Tran D K,Sayed D,Vu V V,Mizukami S,Hirayama K,Huy N T.Ginsenoside Rh1:A systematic review of its pharmacological properties[J].Planta Med.,2018,84(3):139.
[3]Zhao C,Xu T H,Liang Y L,Zhao S J,Ren L Q,Wang Q,Dou B.Functional analysis of beta-amyrin synthase gene in ginsenoside biosynthesis by RNA interference[J].Plant Cell Rep.,2015,34(8):1307.
[4]Cao H Z,Nuruzzaman M,Xiu H,Huang J J,Wu K L,Chen X H,Li J J,Wang L,Jeong J H,Park S J,Yang F,Luo J L,Luo Z Y.Transcriptome analysis of methyl jasmonate-elicited Panax ginseng adventitious roots to discover putative ginsenoside biosynthesis and transport genes[J].Int.J.Mol.Sci.,2015,16(2):3035.
[5]Wang J,Gao W Y,Zhang J,Zuo B M,Zhang L M,Huang L Q.Advances in study of ginsenoside biosynthesis pathway in Panax ginseng C.A.Meyer[J].Acta Physiol.Plant.,2012,34(2):397.
[6]Kim T D,Han J Y,Huh G H,Choi Y E.Expression and functional characterization of three squalene synthase genes associated with saponin biosynthesis in Panax ginseng[J].Plant Cell Physiol.,2011,52(1):125.
[7]Tansakul P,Shibuya M,Kushiro T,Ebizuka Y.Dammar-enediol-II synthase,the first dedicated enzyme for ginsenoside biosynthesis,in Panax ginseng[J].FEBSLett.,2006,580(22):5143.
[8]Han J Y,Kim M J,Ban Y W,Hwang H S,Choi Y E.The involvement of beta-amyrin 28-oxidase(CYP716A52v2)in oleanane-type ginsenoside biosynthesis in Panax ginseng[J].Plant Cell Physiol.,2013,54(12):2034.
[9]袁晓凡,赵兵,王玉春.稀土元素在药用植物细胞和组织培养中的应用[J].植物学通报,2005,22(1):115.Yuan X F,Zhao B,Wang Y C.Application of rare earth elements in medicinal plant cell and tissue culture[J].Chinese Bulletin of Botany,2005,22(1):115.
[10]Wu J Y,Wang C G,Mei X G.Stimulation of taxol production and excretion in Taxus spp.cell cultures by rare earth chemical lanthanum[J].J.Biotechnol.,2001,85(1):67.
[11]元英进,胡国武,王传贵,景莹,周永洽,申泮文.镧、铈对红豆杉细胞生长及紫杉醇合成与释放的影响[J].中国稀土学报,1998,16(1):57.Yuan Y J,Hu G W,Wang C G,Jing Y,Zhou Y Q,Shen P W.Effect of rare earth compounds on the growths,taxol biosynthesis and release of Taxus cuspidata cell culture[J].Journal of the Chinese Society of Rare Earths,1998,16(1):57.
[12]元英进,胡宗定.稀土元素对长春花植物细胞培养的影响[J].稀土,1993,14(3):38.Yuan Y J,Hu Z D.Effects of rare earth elements on plant cell culture in periwinkle[J].Chinese Rare Earth,1993,14(3):38.
[13]杨世海,刘晓峰,果德安.稀土元素镧对掌叶大黄毛状根和非转化根生长及蒽醌产量的影响[J].中草药,2004,35(10):1171.Yang S H,Liu X F,Guo D A.Effect of rare-earth elements La3+on growth of hairy roots and normal roots of Rheum palmatum and their yield of anthraquinone[J].Chinese Traditional and Herbal Drugs,2004,35(10):1171.
[14]姚强,高兴喜,宫志远,任鹏飞,仇可新,刘岩.部分稀土元素对灵芝多糖和三萜类物质液体发酵的影响[J].食品科学,2011,32(5):224.Yao Q,Gao X X,Gong Z Y,Ren P F,Qiu K X,Liu Y.Effects of some rare earth elements on liquid fermentation of Ganoderma lucidum to produce polysaccharides and triterpenoids[J].Food Science,2011,32(5):224.
[15]王景余,李向高,郑友兰.稀土元素对人参皂甙含量的影响[J].中国药学杂志,1991,26(10):591.Wang J Y,Li X G,Zheng Y L.Effect of rare earth element on the content of ginsenoside in ginseng root[J].Chinese Pharmaceutical Journal,1991,26(10):591.
[16]张儒,张变玲,谢涛,李谷才,罗志勇.响应面法优化人参不定根中总皂苷的提取工艺[J].天然产物研究与开发,2015,27(4):726.Zhang R,Zhang B L,Xie T,Li G C,Luo Z Y.Optimization of extraction process of total ginsenoside from adventitious roots of Panax ginseng C.A.Meyer by response surface methodology[J].Natural Product Research and Development,2015,27(4):726.
[17]Yin J X,Zhang D H,Zhuang J J,Huang Y,Mu Y,LüS W.Study on the correlation between gene expression and enzyme activity of seven key enzymes and ginsenoside content in ginseng in over time in Ji'an,China[J].Int.J.Mol.Sci.,2017,18(12):2682.
[18]郭亚鹏,施和平.镧对美洲商陆毛状根生长、皂苷产生及抗氧化酶活性的影响[J].中国稀土学报,2017,35(5):614.Guo Y P,Shi H P.Effects of Lanthanum on growth and esculentoside production in Phytolacca americana L.hairy roots[J].Journal of the Chinese Society of Rare Earths,2017,35(5):614.
[19]Karki P,Kim C,Smith K,Son D S,Aschner M,Lee E.Transcriptional regulation of the astrocytic excitatory amino acid transporter 1(EAAT1)via NF-kappaB and Yin Yang 1(YY1)[J].J.Biol.Chem.,2015,290(39):23725.
[20]de Oliveira C,Ramos S J,Siqueira J O,Faquin V,de Castro E M,Amaral D C,Techio V H,Coelho L C,e Silva P H,Schnug E,Guilherme L R.Bioaccumulation and effects of lanthanum on growth and mitotic index in soybean plants[J].E Ecotoxicol Environ Saf,2015,122(12):136.
[21]Hu H Q,Wang L H,Li Y L,Sun J W,Zhou Q,Huang X H.Insight into mechanism of lanthanum(III)induced damage to plant photosynthesis[J].Ecotox.Environ.Safe,2016,127(5):43.
[22]崔堂兵,张长远,郑穗平,郭勇,蒋玲兰.稀土元素对银杏悬浮培养细胞生长和次级代谢产物积累的影响[J].广东农业科学,2002,(5):29.Cui T B,Zhang C Y,Zheng S P,Guo Y,Jiang L L.Effect of rare-earth elements on Ginkgo biloba cell growth and secondary products accumulation in suspension culture[J].Guangdong Agricultural Sciences,2002,(5):29.
[23]Kim Y J,Jeon J N,Jang M G,Oh J Y,Kwon W S,Jung S K,Yang D C.Ginsenoside profiles and related gene expression during foliation in Panax ginseng Meyer[J].J.Ginseng Res.,2014.38(1):66.
[24]边丽华,邹琳,周冰谦,刘伟,周洁,王晓.稀土元素镧对丹参活性物质积累及其合成途径中酶基因表达的影响[J].中国中药杂志,2016,41(23):4344.Bian L H,Zou L,Zhou B Q,Liu W,Zhou J,Wang X.Effect of Lanthanum on accumulation of activeconstituent and key enzymes expression of Salvia miltiorrhiza hairy root[J].China Journal of Chinese Materia Medica,2016,41(23):4344.
[25]边丽华,邹琳,周冰谦,刘伟,周洁,王晓.稀土元素镧与丹参毛状根中活性成分及其生物合成中关键酶的量效关系[J].中国现代中药,2017,19(3):387.Bian L H,Zou L,Zhou B Q,Liu W,Zhou J,Wang X.The dose-effect relationship between La and active constituent and key enzymes expression of Salvia miltiorrhiza hairy root[J].Modern Chinese Medicine,2017,19(3):387.
[26]罗建平,曹磊,潘利华,查学强.稀土元素对怀槐悬浮培养细胞异黄酮合成及氧化还原态的影响[J].应用与环境生物学报,2008,14(3):362.Luo J P,Cao L,Pan L H,Zha X Q.Effects of rare earth elements on isoflavone biosynthesis and redox status of Maackia amurensis cells in suspension cultures[J].Chinese Journal of Applied and Environmental Biology,2008,14(3):362.
[27]Yang J L,Hu Z F,Zhang T T,Gu A D,Gong T,Zhu P.Progress on the studies of the key enzymes of ginsenosidebiosynthesis[J].Molecules,2018,23(3):589.
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