人参中人参皂苷生物合成对水分调控的响应及其机制的初步研究
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摘要
探究土壤含水量对人参皂苷生物合成的影响,并从抗氧化酶系统和皂苷合成途径关键酶基因表达等角度阐释其机制。以三年生盆栽人参为试验材料,设置3个水分梯度,即饱和含水量的40%(W1),60%(W2),80%(W3),采用HPLC测定11种单体皂苷含量并以GAPDH为内参基因,利用实时荧光定量PCR分析皂苷合成途径中6个关键酶基因(HMGR,SS,β-AS,CYP716A47,CYP716A52v2,CYP716A53v2)表达量,同时对超氧化物歧化酶(SOD),过氧化物酶(POD),过氧化氢酶(CAT)活性和MDA含量进行测定。人参皂苷含量及生物量随土壤含水量的增大呈上升趋势,W3处理最适; PPD(Rb1,Rc,Rb2,Rd,Rh2,Rb3,Rg3),PPT(Rg1,Re,Rf)型人参皂苷,Ro及总皂苷均在8月30日达最大值,分别为9. 92,5. 48,0. 63 mg·g-1。整个调控期间W3抗氧化酶活性大于W1,MDA含量小于W1并且W3处理时β-AS,CYP716A47,CYP716A53v2基因表达量呈上升趋势而HMGR,SS基因在各个水分条件下表达量较稳定。经相关性分析得W3处理组HMGR,SS基因与PPD,PPT型人参皂苷及Ro呈现显著正相关,CYP716A52v2基因与Ro呈显著正相关,CYP716A47基因与PPD型人参皂苷呈极显著正相关; W1,W2处理时β-AS基因与PPD型人参皂苷呈极显著正相关。W3处理为最适水分,人参总皂苷及单体皂苷含量最高,各基因与皂苷含量相关性密切,更利于人参皂苷的积累。
The study is aimed to explore the effect of soil moisture content on ginsenoside biosynthesis and explain its mechanism from the perspectives of antioxidant enzyme system and gene expression of key enzymes in the pathway of ginsenoside synthesis. In the study,two years old Panax ginseng was used as the experimental material and three moisture gradient,40% of saturated water content( W1),60%( W2),80%( W3) were set up. The content of 11 monomeric saponins were determined by HPLC. With GAPDH as a reference gene,six key enzymes( HMGR,SS,β-AS,CYP716 A47,CYP716 A52 v2,CYP716 A53 v2) in ginseng saponin synthesis pathway expression were analyzed by fluorescent quantitative PCR and the activities of superoxide dismutase( SOD),peroxidase( POD),catalase( CAT) activity and MDA content were also determined. With the increase of soil water,the content of ginseng saponin and biomass showed an increasing trend. PPD( Rb1,Rc,Rb2,Rd,Rh2,Rb3,Rg3),PPT( Rg1,Re,Rf) ginsenoside,Ro and total ginsenoside reached the maximum value on August 30,were 9.92,5.48,0.63 mg·g-1,respectively. During the whole regulation period,the antioxidant activity of W3 was greater than that of W1,and the MDA content was less than that of W1. At W3,expression levels of β-AS,CYP716 A47 and CYP716 A53 v2 showed an increasing trend,while HMGR and SS genes showed relatively stable expression levels under various water conditions. According to the correlation analysis,HMGR and SS genes in the W3 treatment group were significantly positively correlated with PPD,PPT ginsenoside and Ro,CYP716 A52 v2 gene was significantly positively correlated with Ro,and CYP716 A47 gene was significantly positively correlated with PPD ginsenoside. There was a significant positive correlation between β-AS gene and PPD ginsenoside in W1 and W2 treatment. Therefore,W3 is the optimum moisture content,ginseng total saponins and monomer saponin content is the highest,the gene closely correlation with content of saponins and more conducive to the accumulation of ginsenosides.
The study is aimed to explore the effect of soil moisture content on ginsenoside biosynthesis and explain its mechanism from the perspectives of antioxidant enzyme system and gene expression of key enzymes in the pathway of ginsenoside synthesis. In the study,two years old Panax ginseng was used as the experimental material and three moisture gradient,40% of saturated water content( W1),60%( W2),80%( W3) were set up. The content of 11 monomeric saponins were determined by HPLC. With GAPDH as a reference gene,six key enzymes( HMGR,SS,β-AS,CYP716 A47,CYP716 A52 v2,CYP716 A53 v2) in ginseng saponin synthesis pathway expression were analyzed by fluorescent quantitative PCR and the activities of superoxide dismutase( SOD),peroxidase( POD),catalase( CAT) activity and MDA content were also determined. With the increase of soil water,the content of ginseng saponin and biomass showed an increasing trend. PPD( Rb1,Rc,Rb2,Rd,Rh2,Rb3,Rg3),PPT( Rg1,Re,Rf) ginsenoside,Ro and total ginsenoside reached the maximum value on August 30,were 9.92,5.48,0.63 mg·g-1,respectively. During the whole regulation period,the antioxidant activity of W3 was greater than that of W1,and the MDA content was less than that of W1. At W3,expression levels of β-AS,CYP716 A47 and CYP716 A53 v2 showed an increasing trend,while HMGR and SS genes showed relatively stable expression levels under various water conditions. According to the correlation analysis,HMGR and SS genes in the W3 treatment group were significantly positively correlated with PPD,PPT ginsenoside and Ro,CYP716 A52 v2 gene was significantly positively correlated with Ro,and CYP716 A47 gene was significantly positively correlated with PPD ginsenoside. There was a significant positive correlation between β-AS gene and PPD ginsenoside in W1 and W2 treatment. Therefore,W3 is the optimum moisture content,ginseng total saponins and monomer saponin content is the highest,the gene closely correlation with content of saponins and more conducive to the accumulation of ginsenosides.
引文
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[14] Caelles C,Ferrer A,Balcells L,et al. Isolation and structural characterization of a c DNA encoding Arabidopsis thaliana 3-hydroxy-3-methylglutaryl coenzyme A reductase[J]. Plant Mol Biol,1989,13(6):627.
[15]张风侠,梁新华,王俊.植物三萜皂苷生物合成及关键酶鲨烯合酶的研究进展[J].农业科学研究,2009,30(3):64.
[16] Braga M V,Urbina J A,Souza W. Effects of squalene synthase inhibitors on the growth and ultrastructure of Trypanosoma cruzi[J]. Int J Antimicrob Agents,2004,24(1):72.
[17]杨林林,张涛,杨利民,等.人参叶片中人参皂苷含量、关键酶基因表达和生态因子的相关性分析[J].华南农业大学学报,2018,39(03):39.
[18]杨利民.中药资源生态学及其科学问题[J].吉林农业大学学报,2008,30(4):506.
[19]焦晓林,高微微.环境因子对药用植物三萜皂苷合成影响的研究进展[J].中草药,2011,42(2):398.
[20]胡国强,陈顺钦,袁媛,等.外源生长素对黄芩悬浮细胞有效成分和内源激素含量的影响[J].中国实验方剂学杂志,2011,17(17):127.
[21]胡国强,张学文,李旻辉,等.植物生长调节剂缩节胺对黄芩活性成分含量的影响[J].中国中药杂志,2012,37(21):3215.
[22]苏文华,张光飞,李秀华,等.植物药材次生代谢产物的积累与环境的关系[J].中草药,2005(9):139.
[23]张志良.植物生理学实验指导[M].北京:高等教育出版社,1990.
[24]李合生.植物生理生化实验原理和技术[M].北京:高等教育出版社,2000.
[25]杨萍,王红兰,张燕,等.土壤水分含量对羌活植株生长及有效成分积累的影响[J].中国中药杂志,2018,43(24):4824.
[26]黄璐琦,郭兰萍.中药资源生态学[M].上海:上海科学技术出版社,2009:44.
[27]杨利民,张永刚,林红梅,等.中药材质量形成理论与控制技术研究进展[J].吉林农业大学学报,2012,34(2):1199.
[28]张永刚,韩梅,姜雪,等.黄芩对干旱复水的生理生态响应[J].中国中药杂志,2013,38(22):3845.
[29] Henry M,Rahier A,And Taton M. Effect of gypsogenin 3-o-glucuronide pretreatment of Gypsohila paniculata and Saponaria officinalis cell suspension cultures on the activities of microsomal 2,3-oxidosqualene cycloartenol and amyrin cyclases[J]. Phytochemistry,1992,31:3855.
[30] Liu P,Guo W S,Pu H C,et all.Effects of high temperature on antioxidant enzymes and lipid peroxidation in flag leaves of wheat during grain filling period[J].Agr Sci China,2006,5(6):425.
[31] Ekmekc I Y,Tanyolac D,Ayhan B.Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars[J].J Plant Physiol,2008,165(6):600.
[32] Lee D H,Lee C B.Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber:in gel enzyme activity assays[J].Plant Sci,2000,159(1):75.
[33]李璇,岳红,黄璐琦,等.影响植物抗氧化酶活性的因素及其研究热点和现状[J].中国中药杂志,2013,38(7):973.
[34]张宇,周自云,夏鹏国,等.干旱胁迫对柴胡中皂苷合成关键酶基因表达及皂苷含量的影响[J].中国中药杂志,2016,41(4):643.
[35]黄璐琦,郭兰萍.环境胁迫下次生代谢产物的积累及道地药材的形成[J].中国中药杂志,2007,32(4):277.
[36]王铁生,王化民,赵亚惠,等.不同供水条件对人参生育和生理特性的影响[J].中药通报,1987(7):17.
[37]尤伟,孟庆文,黄泽成.人参不同生育期需水量的探讨[J].特产研究,1994(3):24.
[2]黎阳,张铁军,刘素香,等.人参化学成分和药理研究进展[J].中草药,2009,40(1):164.
[3]徐俞悦,尹超,陈波,等.人参超微粉对正常和免疫力低下小鼠免疫功能的影响[J].中国现代应用药学,2018,35(11):1665.
[4] Zhang Z,Jiang B,Zheng X,et al. Immune enhancing effects of Rg3 on peripheral lymphocytes in vitro in cancer patients treated with radiotherapy[J]. Chin Pharm J,2004,39(4):261.
[5] Sui D Y,Yu X F,Qu S C,et al. Effects of Panax quinquefolium20S-2protopanaxdiol saponins on experimental ventricular remodeling in rat[J]. Chin Pharm J,2007,42(2):108.
[6] Lee Y,Jin Y,Lim W,et al. A ginsenoside-Rh1,a component of ginseng saponin,activates estrogen receptor in human breast carcinoma MCF-7 cells[J]. J Steroid Biochem,2003,84(4):463.
[7] William C S,Chung W S,Sally K W,et al. Ginsenoside Re of Panax ginseng possesses significant antioxidant and antihyperlipidemic efficacies in streptozotocininduced diabetic rats[J]. Eur J Pharmacol,2006,550(1/3):173.
[8] Kim S E,Lee Y H,Park H J,et al. Ginsenoside-Rs4,a new type of ginseng saponin concurrently induces apoptosis and selectively elevates protein levels of p53 and p21 WAF1 in human hepatoma SK-HEP-1 cells European[J]. Eur J Cancer,1999,35(3):507.
[9]林彦萍,张美萍,王康宇,等.人参皂苷生物合成研究进展[J].中国中药杂志,2016,41(23):4292.
[10] Kim D S,Chang Y J,Zedk P,et al. Dammarane saponins from Panax ginseng[J]. Phytochemistry,1995,40(5):1493.
[11] Tung N H,Song G Y,Nhiem N X,et al. Dammarane-type saponins from the flower buds of Panax ginseng and their intracellular radical scavenging capacity[J]. J Agric Food Chem,2010,58(2):868.
[12] Liu G Y,Li X W,Wang N B,et al. Three new dammarane-type triterpene saponins from the leaves of Panax ginseng C.A. Meyer[J]. J Asian Nat Prod Res,2010,12(10):865.
[13]杨鹤,宋述尧,许永华,等.人参三萜皂苷的研究进展及其生态学作用[J].中草药,2017,48(8):1692.
[14] Caelles C,Ferrer A,Balcells L,et al. Isolation and structural characterization of a c DNA encoding Arabidopsis thaliana 3-hydroxy-3-methylglutaryl coenzyme A reductase[J]. Plant Mol Biol,1989,13(6):627.
[15]张风侠,梁新华,王俊.植物三萜皂苷生物合成及关键酶鲨烯合酶的研究进展[J].农业科学研究,2009,30(3):64.
[16] Braga M V,Urbina J A,Souza W. Effects of squalene synthase inhibitors on the growth and ultrastructure of Trypanosoma cruzi[J]. Int J Antimicrob Agents,2004,24(1):72.
[17]杨林林,张涛,杨利民,等.人参叶片中人参皂苷含量、关键酶基因表达和生态因子的相关性分析[J].华南农业大学学报,2018,39(03):39.
[18]杨利民.中药资源生态学及其科学问题[J].吉林农业大学学报,2008,30(4):506.
[19]焦晓林,高微微.环境因子对药用植物三萜皂苷合成影响的研究进展[J].中草药,2011,42(2):398.
[20]胡国强,陈顺钦,袁媛,等.外源生长素对黄芩悬浮细胞有效成分和内源激素含量的影响[J].中国实验方剂学杂志,2011,17(17):127.
[21]胡国强,张学文,李旻辉,等.植物生长调节剂缩节胺对黄芩活性成分含量的影响[J].中国中药杂志,2012,37(21):3215.
[22]苏文华,张光飞,李秀华,等.植物药材次生代谢产物的积累与环境的关系[J].中草药,2005(9):139.
[23]张志良.植物生理学实验指导[M].北京:高等教育出版社,1990.
[24]李合生.植物生理生化实验原理和技术[M].北京:高等教育出版社,2000.
[25]杨萍,王红兰,张燕,等.土壤水分含量对羌活植株生长及有效成分积累的影响[J].中国中药杂志,2018,43(24):4824.
[26]黄璐琦,郭兰萍.中药资源生态学[M].上海:上海科学技术出版社,2009:44.
[27]杨利民,张永刚,林红梅,等.中药材质量形成理论与控制技术研究进展[J].吉林农业大学学报,2012,34(2):1199.
[28]张永刚,韩梅,姜雪,等.黄芩对干旱复水的生理生态响应[J].中国中药杂志,2013,38(22):3845.
[29] Henry M,Rahier A,And Taton M. Effect of gypsogenin 3-o-glucuronide pretreatment of Gypsohila paniculata and Saponaria officinalis cell suspension cultures on the activities of microsomal 2,3-oxidosqualene cycloartenol and amyrin cyclases[J]. Phytochemistry,1992,31:3855.
[30] Liu P,Guo W S,Pu H C,et all.Effects of high temperature on antioxidant enzymes and lipid peroxidation in flag leaves of wheat during grain filling period[J].Agr Sci China,2006,5(6):425.
[31] Ekmekc I Y,Tanyolac D,Ayhan B.Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars[J].J Plant Physiol,2008,165(6):600.
[32] Lee D H,Lee C B.Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber:in gel enzyme activity assays[J].Plant Sci,2000,159(1):75.
[33]李璇,岳红,黄璐琦,等.影响植物抗氧化酶活性的因素及其研究热点和现状[J].中国中药杂志,2013,38(7):973.
[34]张宇,周自云,夏鹏国,等.干旱胁迫对柴胡中皂苷合成关键酶基因表达及皂苷含量的影响[J].中国中药杂志,2016,41(4):643.
[35]黄璐琦,郭兰萍.环境胁迫下次生代谢产物的积累及道地药材的形成[J].中国中药杂志,2007,32(4):277.
[36]王铁生,王化民,赵亚惠,等.不同供水条件对人参生育和生理特性的影响[J].中药通报,1987(7):17.
[37]尤伟,孟庆文,黄泽成.人参不同生育期需水量的探讨[J].特产研究,1994(3):24.