SmGGPPS2对丹参酮合成的影响
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摘要
香叶基香叶基焦磷酸合酶(GGPPS)是植物二萜类次生代谢物合成过程中的重要调控位点。在药用模式植物丹参(Salvia miltiorrhiza)中,GGPPS基因家族成员SmGGPPS2的生物学功能及其在丹参酮有效成分合成过程中的作用尚不明确。分别在丹参植株中过表达和RNA干涉SmGGPPS2基因,并对转基因丹参中丹参酮含量和丹参酮合成相关基因表达量以及转基因植物生理指标进行检测,结果表明,过表达SmGGPPS2株系中的丹参酮IIA和铁锈醇等脂溶性成分含量高于野生型; RNA干涉SmGGPPS2株系中的丹参酮IIA和铁锈醇等脂溶性成分含量均低于野生型。调节表达SmGGPPS2后,丹参株系中SmHMGR1和SmCPS1等多个关键酶基因的表达都呈现明显的变化。此外,调节表达SmGGPPS2还影响丹参植株抗性。以上结果表明, SmGGPPS2在丹参酮等萜类物质的合成中起重要的调控作用。
Geranylgerany pyrophosphate synthase(GGPPS) is an important regulator in the plant diterpenoid biosynthesis pathway. The GGPPS gene family plays a critical role in the development of the medicinal model plant Salvia miltiorrhiza. However, the biological function of SmGGPPS2, especially in the biosynthesis of tanshinone or other active ingredients, is still unclear. For functional investigation, SmGGPPS2 expression was up-or down-regulated in S. miltiorrhiza plants via overexpression or RNA interference, respectively. Then we detected the content of tanshinones, the expression of genes related to tanshinone biosynthesis, and the physiological indexes of transgenic S. miltiorrhiza plants. The content of fat-soluble components, such as tanshinone IIA and ferruginol, was increased significantly in SmGGPPS2-overexpressed lines compared with the wild type, and the content of fat soluble-components was lower in SmGGPPS2-RNAi lines than those in the wild type lines. With the regulation of SmGGPPS2, the expression of key enzyme genes related to tanshinone biosynthesis in S. miltiorrhiza, such as Sm HMGR1 and SmCPS1, was changed. In addition, the regulation of SmGGPPS2 expression also affected the resistance of S. miltiorrhiza. Our results indicate that SmGGPPS2 plays an important regulatory role in tanshinone biosynthesis.
Geranylgerany pyrophosphate synthase(GGPPS) is an important regulator in the plant diterpenoid biosynthesis pathway. The GGPPS gene family plays a critical role in the development of the medicinal model plant Salvia miltiorrhiza. However, the biological function of SmGGPPS2, especially in the biosynthesis of tanshinone or other active ingredients, is still unclear. For functional investigation, SmGGPPS2 expression was up-or down-regulated in S. miltiorrhiza plants via overexpression or RNA interference, respectively. Then we detected the content of tanshinones, the expression of genes related to tanshinone biosynthesis, and the physiological indexes of transgenic S. miltiorrhiza plants. The content of fat-soluble components, such as tanshinone IIA and ferruginol, was increased significantly in SmGGPPS2-overexpressed lines compared with the wild type, and the content of fat soluble-components was lower in SmGGPPS2-RNAi lines than those in the wild type lines. With the regulation of SmGGPPS2, the expression of key enzyme genes related to tanshinone biosynthesis in S. miltiorrhiza, such as Sm HMGR1 and SmCPS1, was changed. In addition, the regulation of SmGGPPS2 expression also affected the resistance of S. miltiorrhiza. Our results indicate that SmGGPPS2 plays an important regulatory role in tanshinone biosynthesis.
引文
化文平,刘文超,王喆之,李翠芹(2016).干涉丹参SmORA1对植物抗病和丹参酮类次生代谢的影响.中国农业科学49,491-502.
化文平,宋双红,智媛,王喆之(2014).丹参SmGGPPS3基因的克隆及表达分析.植物科学学报32,50-57.
王海燕,李玉琴,王广旭(2011).白花丹参脂溶性成分超临界二氧化碳流体萃取与气相色谱-质谱分析.医药导报30,978-981.
张蕾(2009).丹参牻牛儿基牻牛儿基焦磷酸合酶基因的克隆与功能研究.博士论文.北京:中国人民解放军军事医学科学院.pp.27-40.
Aharoni A,Giri AP,Deuerlein S,Griepink F,de Kogel WJ,Verstappen FWA,Verhoeven HA,Jongsma MA,Schwab W,Bouwmeester HJ(2003).Terpenoid metabolism in wild-type and transgenic Arabidopsis plants.Plant Cell 15,2866-2884.
Chang TH,Guo RT,Ko TP,Wang AHJ,Liang PH(2006).Crystal structure of type-III geranylgeranyl pyrophosphate synthase from Saccharomyces cerevisiae and the mechanism of product chain length determination.J Biol Chem281,14991-15000.
Chen C,Zhang Y,Qiakefu K,Zhang X,Han LM,Hua WP,Yan YP,Wang ZZ(2016).Overexpression of tomato Prosystemin(LePS)enhances pest resistance and the production of tanshinones in Salvia miltiorrhiza Bunge.JAgric Food Chem 64,7760-7769.
Chen W,He SZ,Liu DG,Patil GB,Zhai H,Wang FB,Stephenson TJ,Wang YN,Wang B,Valliyodan B,Nguyen HT,Liu QC(2015).A sweetpotato geranylgeranyl pyrophosphate synthase gene,Ib GGPS,increases carotenoid content and enhances osmotic stress tolerance in Arabidopsis thaliana.PLo S One 10,e0137623.
Cheng QQ,Su P,Hu YT,He YF,Gao W,Huang LQ(2014).RNA interference-mediated repression of SmCPS(Copalyldiphosphate synthase)expression in hairy roots of Salvia miltiorrhiza causes a decrease of tanshinones and sheds light on the functional role of SmCPS.Biotechnol Lett 36,363-369.
Cui GH,Duan LX,Jin BL,Qian J,Xue ZY,Shen GA,Snyder JH,Song JY,Chen SL,Huang LQ,Peters RJ,Qi XQ(2015).Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza.Plant Physiol169,1607-1618.
Han JL,Liu BY,Ye HC,Wang H,Li ZQ,Li GF(2006).Effects of overexpression of the endogenous farnesyl diphosphate synthase on the artemisinin content in Artemisia annua L.J Integr Plant Biol 48,482-487.
Hua WP,Song J,Li CQ,Wang ZZ(2012).Molecular cloning and characterization of the promoter of SmGGPPs and its expression pattern in Salvia miltiorrhiza.Mol Biol Rep 39,5775-5783.
Hua WP,Zhang Y,Song J,Zhao LJ,Wang ZZ(2011).De novo transcriptome sequencing in Salvia miltiorrhiza to identify genes involved in the biosynthesis of active ingredients.Genomics 98,272-279.
Kai GY,Liao P,Zhang T,Zhou W,Wang J,Xu H,Liu YY,Zhang L(2010).Characterization,expression profiling,and functional identification of a gene encoding geranylgeranyl diphosphate synthase from Salvia miltiorrhiza.Biotechnol Bioproc Eng 15,236-245.
Kai GY,Xu H,Zhou CC,Liao P,Xiao JB,Luo XQ,You LJ,Zhang L(2011).Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.Metab Eng 13,319-327.
Lin TH,Hsieh CL(2010).Pharmacological effects of Salvia miltiorrhiza(Danshen)on cerebral infarction.Chin Med 5,22.
Ma XH,Ma Y,Tang JF,He YL,Liu YC,Ma XJ,Shen Y,Cui GH,Lin HX,Rong QX,Guo J,Huang LQ(2015).The biosynthetic pathways of tanshinones and phenolic acids in Salvia miltiorrhiza.Molecules 20,16235-16254.
Ma YM,Yuan LC,Wu B,Li XE,Chen SL,Lu SF(2012).Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza.J Exp Bot 63,2809-2823.
Shi M,Luo XQ,Ju GH,Li LL,Huang SX,Zhang T,Wang HZ,Kai GY(2016).Enhanced diterpene tanshinone accumulation and bioactivity of transgenic Salvia miltiorrhiza hairy roots by pathway engineering.J Agric Food Chem64,2523-2530.
Shi M,Luo XQ,Ju GH,Yu XH,Hao XL,Huang Q,Xiao JB,Cui LJ,Kai GY(2014).Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl Co A reductase and 1-deoxy-Dxylulose 5-phosphate reductoisomerase.Funct Integr Genom 14,603-615.
Tata SK,Jung J,Kim YH,Choi JY,Jung JY,Lee IJ,Shin JS,Ryu SB(2016).Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth,early flowering and increased seed yield.Plant Biotechnol J 14,29-39.
Vandesompele J,De Preter K,Pattyn F,Poppe B,Van Roy N,De Paepe A,Speleman F(2002).Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol 3,research0034.
Yan YP,Wang ZZ(2007).Genetic transformation of the medicinal plant Salvia miltiorrhiza by Agrobacterium tumefaciens-mediated method.Plant Cell Tiss Org 88,175-184.
Young AJ,Lowe GM(2001).Antioxidant and prooxidant properties of carotenoids.Arch Biochem Biophys 385,20-27.
化文平,宋双红,智媛,王喆之(2014).丹参SmGGPPS3基因的克隆及表达分析.植物科学学报32,50-57.
王海燕,李玉琴,王广旭(2011).白花丹参脂溶性成分超临界二氧化碳流体萃取与气相色谱-质谱分析.医药导报30,978-981.
张蕾(2009).丹参牻牛儿基牻牛儿基焦磷酸合酶基因的克隆与功能研究.博士论文.北京:中国人民解放军军事医学科学院.pp.27-40.
Aharoni A,Giri AP,Deuerlein S,Griepink F,de Kogel WJ,Verstappen FWA,Verhoeven HA,Jongsma MA,Schwab W,Bouwmeester HJ(2003).Terpenoid metabolism in wild-type and transgenic Arabidopsis plants.Plant Cell 15,2866-2884.
Chang TH,Guo RT,Ko TP,Wang AHJ,Liang PH(2006).Crystal structure of type-III geranylgeranyl pyrophosphate synthase from Saccharomyces cerevisiae and the mechanism of product chain length determination.J Biol Chem281,14991-15000.
Chen C,Zhang Y,Qiakefu K,Zhang X,Han LM,Hua WP,Yan YP,Wang ZZ(2016).Overexpression of tomato Prosystemin(LePS)enhances pest resistance and the production of tanshinones in Salvia miltiorrhiza Bunge.JAgric Food Chem 64,7760-7769.
Chen W,He SZ,Liu DG,Patil GB,Zhai H,Wang FB,Stephenson TJ,Wang YN,Wang B,Valliyodan B,Nguyen HT,Liu QC(2015).A sweetpotato geranylgeranyl pyrophosphate synthase gene,Ib GGPS,increases carotenoid content and enhances osmotic stress tolerance in Arabidopsis thaliana.PLo S One 10,e0137623.
Cheng QQ,Su P,Hu YT,He YF,Gao W,Huang LQ(2014).RNA interference-mediated repression of SmCPS(Copalyldiphosphate synthase)expression in hairy roots of Salvia miltiorrhiza causes a decrease of tanshinones and sheds light on the functional role of SmCPS.Biotechnol Lett 36,363-369.
Cui GH,Duan LX,Jin BL,Qian J,Xue ZY,Shen GA,Snyder JH,Song JY,Chen SL,Huang LQ,Peters RJ,Qi XQ(2015).Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza.Plant Physiol169,1607-1618.
Han JL,Liu BY,Ye HC,Wang H,Li ZQ,Li GF(2006).Effects of overexpression of the endogenous farnesyl diphosphate synthase on the artemisinin content in Artemisia annua L.J Integr Plant Biol 48,482-487.
Hua WP,Song J,Li CQ,Wang ZZ(2012).Molecular cloning and characterization of the promoter of SmGGPPs and its expression pattern in Salvia miltiorrhiza.Mol Biol Rep 39,5775-5783.
Hua WP,Zhang Y,Song J,Zhao LJ,Wang ZZ(2011).De novo transcriptome sequencing in Salvia miltiorrhiza to identify genes involved in the biosynthesis of active ingredients.Genomics 98,272-279.
Kai GY,Liao P,Zhang T,Zhou W,Wang J,Xu H,Liu YY,Zhang L(2010).Characterization,expression profiling,and functional identification of a gene encoding geranylgeranyl diphosphate synthase from Salvia miltiorrhiza.Biotechnol Bioproc Eng 15,236-245.
Kai GY,Xu H,Zhou CC,Liao P,Xiao JB,Luo XQ,You LJ,Zhang L(2011).Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.Metab Eng 13,319-327.
Lin TH,Hsieh CL(2010).Pharmacological effects of Salvia miltiorrhiza(Danshen)on cerebral infarction.Chin Med 5,22.
Ma XH,Ma Y,Tang JF,He YL,Liu YC,Ma XJ,Shen Y,Cui GH,Lin HX,Rong QX,Guo J,Huang LQ(2015).The biosynthetic pathways of tanshinones and phenolic acids in Salvia miltiorrhiza.Molecules 20,16235-16254.
Ma YM,Yuan LC,Wu B,Li XE,Chen SL,Lu SF(2012).Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza.J Exp Bot 63,2809-2823.
Shi M,Luo XQ,Ju GH,Li LL,Huang SX,Zhang T,Wang HZ,Kai GY(2016).Enhanced diterpene tanshinone accumulation and bioactivity of transgenic Salvia miltiorrhiza hairy roots by pathway engineering.J Agric Food Chem64,2523-2530.
Shi M,Luo XQ,Ju GH,Yu XH,Hao XL,Huang Q,Xiao JB,Cui LJ,Kai GY(2014).Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl Co A reductase and 1-deoxy-Dxylulose 5-phosphate reductoisomerase.Funct Integr Genom 14,603-615.
Tata SK,Jung J,Kim YH,Choi JY,Jung JY,Lee IJ,Shin JS,Ryu SB(2016).Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth,early flowering and increased seed yield.Plant Biotechnol J 14,29-39.
Vandesompele J,De Preter K,Pattyn F,Poppe B,Van Roy N,De Paepe A,Speleman F(2002).Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol 3,research0034.
Yan YP,Wang ZZ(2007).Genetic transformation of the medicinal plant Salvia miltiorrhiza by Agrobacterium tumefaciens-mediated method.Plant Cell Tiss Org 88,175-184.
Young AJ,Lowe GM(2001).Antioxidant and prooxidant properties of carotenoids.Arch Biochem Biophys 385,20-27.