牛角瓜5β黄体酮还原酶基因(Cgp5Br)克隆与序列分析
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摘要
5β黄体酮还原酶是强心苷合成途径中的第一个关键酶,在强心苷合成中起着重要的调控作用。为了研究牛角瓜中强心苷生物合成途径的关键酶基因,本研究依据牛角瓜转录组数据设计特异性引物,采用RT-PCR方法从牛角瓜中克隆得到关键酶基因p5Br(5β黄体酮还原酶基因)的2个开放阅读框(ORF),分别命名为Cgp5Br1(GeneBank登录号MH094753)和Cgp5Br2(GeneBank登录号MH094754)。序列分析结果表明,Cgp5Br1的序列长1 164bp,编码387个氨基酸,Cgp5Br2的序列长1 158bp,编码385个氨基酸。进化树分析发现,Cgp5Br1和Cgp5Br2在进化上属于旁系同源。qRT-PCR分析表明,Cgp5Br1和Cgp5Br2在牛角瓜的根、茎、叶、花和种子中均有表达。Cgp5Br1在根中相对表达量最高,而Cgp5Br2在根、茎、叶和花中的表达量都相对较高,种子中最低。本研究为今后利用代谢工程提高强心苷含量提供了重要的基因资源。
Progesterone 5β-reductase is the first key enzyme in the synthesis of cardiac glycosides,which plays an important regulatory role in the synthesis of cardiac glycosides.In order to study the key enzyme genes in the cardiac glycoside biosynthesis pathway,in this work,according to the transcriptome data,specific primers were designed and two open reading frames(ORFs)of the key enzyme gene p5Br were cloned from C.gigantea by RT-PCR,which were named as Cgp5 Br1(MH094753)and Cgp5 Br2(MH094754)respectively.Sequence analysis showed that the sequence lengths of Cgp5 Br1 and Cgp5 Br2 were 1 164 bp and 1 158 bp,encoding 387 and 385 amino acids respectively.Phylogenetic analysis demonstrated that Cgp5 Br1 and Cgp5 Br2 belong to paralogs in evolution.qRT-PCR analysis indicated that Cgp5 Br1 and Cgp5 Br2 were expressed in all the tested tissues of C.gigantea, including root,stem,leaf,flower and seed.Cgp5 Br1 showed the highest expression in the root,while the expression levels of Cgp5 Br2 were relatively high in root,stem,leaf and flower and was the lowest in seed.This study provided important genetic resources for metabolic engineering to improve the content of cardiac glycosides in the future.
Progesterone 5β-reductase is the first key enzyme in the synthesis of cardiac glycosides,which plays an important regulatory role in the synthesis of cardiac glycosides.In order to study the key enzyme genes in the cardiac glycoside biosynthesis pathway,in this work,according to the transcriptome data,specific primers were designed and two open reading frames(ORFs)of the key enzyme gene p5Br were cloned from C.gigantea by RT-PCR,which were named as Cgp5 Br1(MH094753)and Cgp5 Br2(MH094754)respectively.Sequence analysis showed that the sequence lengths of Cgp5 Br1 and Cgp5 Br2 were 1 164 bp and 1 158 bp,encoding 387 and 385 amino acids respectively.Phylogenetic analysis demonstrated that Cgp5 Br1 and Cgp5 Br2 belong to paralogs in evolution.qRT-PCR analysis indicated that Cgp5 Br1 and Cgp5 Br2 were expressed in all the tested tissues of C.gigantea, including root,stem,leaf,flower and seed.Cgp5 Br1 showed the highest expression in the root,while the expression levels of Cgp5 Br2 were relatively high in root,stem,leaf and flower and was the lowest in seed.This study provided important genetic resources for metabolic engineering to improve the content of cardiac glycosides in the future.
引文
[1]中国科学院植物志编辑委员会.中国植物志[M].北京:科学出版社,1977.
[2]Kadiyala M,Ponnusankar S,Elango K.Calotropis gigantiea(L.)R.Br(Apocynaceae):A phytochemical and pharmacological review [J].Journal of Ethnopharmacology,2013,150(1):32-50.
[3]中国科学院昆明植物研究所.云南植物志[M].北京:科学出版社,1983.
[4]Deshmukh P T,Fernandes J,Atul A,et al.Wound healing activity of Calotropis gigantea root bark in rats [J].Journal of Ethnopharmacology,2009,125(1):178-181.
[5]Subramanian S P,Saratha V.Evaluation of antibacterial activity of Calotropis gigantea latex extract on selected pathogenic bacteria[J].Journal of Pharmacy Research,2010(3):517-521.
[6]Mbhele N,Balogun F O,Kazeem M I,et al.In vitro studies on the antimicrobial,antioxidant and antidiabetic potential of cephalaria gigantea [J].Bangladesh Journal of Pharmacology,2015,10(1):214-221.
[7]Habib M R,Karim M R.Antimicrobial and cytotoxic cctivity of Di-(2-ethylhexyl)phthalate and anhydrosophoradiol-3-acetate isolated from Calotropis gigantea(Linn.)flower [J].Mycobiology,2009,37(1):31-36.
[8]Adak M,Gupta J K.Evaluation of anti-inflammatory activity of Calotropis gigantea(AKANDA)in various biological system [J].Nepal Medical College Journal,2006,8(3):156-161.
[9]Kovendan K,Murugan K,Prasanna Kumar K,et al.Mosquitocidal properties of Calotropis gigantea(Family:Asclepiadaceae)leaf extract and bacterial insecticide,Bacillus thuringiensis,against the mosquito vectors [J].Parasitology Research,2012,111(2):531-544.
[10]Pathak A K,Argal A.Analgesic activity of Calotropis gigantea flower [J].Fitoterapia,2007,78(1):40-42.
[11]Parhira S,Zhu G Y,Chen M,et al.Cardenolides from Calotropis gigantea as potent inhibitors of hypoxia-inducible factor-1 transcriptional activity[J].Journal of Ethnopharmacology,2016,194:930-936.
[12]Nguyen K D H,Dang P H,Nguyen H X,et al.Phytochemical and cytotoxic studies on the leaves of,Calotropis gigantea[J].Bioorganic and Medicinal Chemistry Letters,2017,27(13):2902-2906.
[13] Habib M R,Karim M R.Evaluation of antitumour activity of Calotropis gigantea L.root bark against ehrlich ascites carcinoma in Swiss albino mice[J].Asian Pacific Journal of Tropical Medicine,2011,4(10):786-790.
[14]Park H Y,Toume K,Arai M A,et al.Calotropin:a cardenolide from calotropis gigantea that inhibits Wnt signaling by increasing casein kinase 1 alpha in colon cancer cells [J].Chembiochem,2014,15(6):872-878.
[15]Saratha V,Subramanian S P.Lupeol,a triterpenoid isolated fromCalotropis gigantealatex ameliorates the primary and secondary complications of FCA induced adjuvant disease in experimental rats[J].Inflammopharmacology,2012,20(1):27-37.
[16]Chitme H,Chandra R,Kaushik S.Studies on anti-diarrhoeal activity of Calotropis gigantea R.Br.in experimental animals [J].Journal of Pharmacy and Pharmaceutical Sciences,2004,7(1):70-75.
[17]Rathod N R,Chitme H R,Irchhaiya R,et al.Hypoglycemic effect of Calotropis gigantea Linn.leaves and flowers in Streptozotocin-induced diabetic rats [J].Oman Medical Journal,2011,26(2):104-108.
[18]Chan E W C,Sweidan N I,Wong S K,et al.Cytotoxic cardenolides from calotropis species:ashort review [J].Records of Natural Products,2017,11(4):334-344.
[19]Prassas I,Diamandis E P.Novel therapeutic applications of cardiac glycosides[J].Nature Reviews Drug Discovery,2008,7(11):926-935.
[20]Tripathi P K,Awasthi S,Kanojiya S,et al.Callus culture and in vitro biosynthesis of cardiac glycosides from Calotropis gigantea(L.)Ait[J].In Vitro Cellular and Developmental Biology - Plant,2013,49(4):455-460.
[21]Pérez-Bermúdez P,Moya García A A,Tunón I,et al.Digitalis purpurea P5 beta R2,encoding steroid 5 beta-reductase,is a novel defense-related gene involved in cardenolide biosynthesis [J].New Phytologist,2010,185(3):687-700.
[22]Thitima L,Somyote S.19-Nor- and 18,20-epoxy-cardenolides from the leaves of Calotropis gigantea[J].Journal of Natural Products,2006,69(8):1249-1251.
[23] Hagimori M,Matsumoto T,Obi Y.effects of mineral salts,initial pH and precursors on digitoxin formation by shoot-forming cultures of Digitalis purpurea L.grown in liquid media[J].Journal of the Agricultural Chemical Society of Japan,1983,47(3):565-571.
[24]Gartner D E,Ulrich Seitz H.Enzyme activities in Cardenolide-accumulating,mixotrophic shoot cultures of Digitalis purpurea L[J].Journal of Plant Physiology,1993,141(3):269-275.
[25]Grtner D E,Keilholz W,Seitz H U.Purification,characterization and partial peptide microsequencing of progesterone 5 beta-reductase from shoot cultures of Digitalis purpurea[J].Febs Journal,2010,225(3):1125-1132.
[26]Herl V,Fischer G,Mulleruri F,et al.Molecular cloning and heterologous expression of progesterone 5beta-reductase from Digitalis lanata Ehrh[J].Phytochemistry,2006,67(3):225-231.
[27]Herl V,Albach D C,MüllerUriF,et al.Using progesterone 5β-reductase,a gene encoding a key enzyme in the cardenolide biosynthesis,to infer the phylogeny of the genus Digitalis[J].Plant Systematics and Evolution,2008,271(1/2):65-78.
[28]Bauer P,Munkert J,Brydziun M,et al.Highly conserved progesterone 5 beta-reductase genes(P5 beta R)from 5 beta-cardenolide-free and 5 beta-cardenolide-producing angiosperms [J].Phytochemistry,2010,71(13):1495-1505.
[29]Newman R A,Yang P,Pawlus A D,et al.Cardiac glycosides as novel cancer therapeutic agents[J].Molecular Interventions,2008,8(1):36-49.
[30]Sun Y,Huang Y H,Huang F Y,et al.3’-epi-12 beta-hydroxyfroside,a new cardenolide,induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells [J].Theranostics,2018,8(7):2044-2060.
[31]Gavidia I,Tarr O R,Rodr G F,et al.Plant progesterone 5β-reductase is not homologous to the animal enzyme.Molecular evolutionary characterization of P5βR from Digitalis purpurea [J].Phytochemistry,2007,68(6):853-864.
[32]Muriira N G,Xu W,Muchugi A,et al.De novo sequencing and assembly analysis of transcriptome in the Sodom apple(Calotropis gigantea)[J].Bmc Genomics,2015,16(1):723.
[33]Munkert J,Pollier J,Miettinen K,et al.Iridoid synthase activity is common among the plant progesterone 5 beta-reductase family[J].Molecular Plant,2015,8(1):136-152.
[2]Kadiyala M,Ponnusankar S,Elango K.Calotropis gigantiea(L.)R.Br(Apocynaceae):A phytochemical and pharmacological review [J].Journal of Ethnopharmacology,2013,150(1):32-50.
[3]中国科学院昆明植物研究所.云南植物志[M].北京:科学出版社,1983.
[4]Deshmukh P T,Fernandes J,Atul A,et al.Wound healing activity of Calotropis gigantea root bark in rats [J].Journal of Ethnopharmacology,2009,125(1):178-181.
[5]Subramanian S P,Saratha V.Evaluation of antibacterial activity of Calotropis gigantea latex extract on selected pathogenic bacteria[J].Journal of Pharmacy Research,2010(3):517-521.
[6]Mbhele N,Balogun F O,Kazeem M I,et al.In vitro studies on the antimicrobial,antioxidant and antidiabetic potential of cephalaria gigantea [J].Bangladesh Journal of Pharmacology,2015,10(1):214-221.
[7]Habib M R,Karim M R.Antimicrobial and cytotoxic cctivity of Di-(2-ethylhexyl)phthalate and anhydrosophoradiol-3-acetate isolated from Calotropis gigantea(Linn.)flower [J].Mycobiology,2009,37(1):31-36.
[8]Adak M,Gupta J K.Evaluation of anti-inflammatory activity of Calotropis gigantea(AKANDA)in various biological system [J].Nepal Medical College Journal,2006,8(3):156-161.
[9]Kovendan K,Murugan K,Prasanna Kumar K,et al.Mosquitocidal properties of Calotropis gigantea(Family:Asclepiadaceae)leaf extract and bacterial insecticide,Bacillus thuringiensis,against the mosquito vectors [J].Parasitology Research,2012,111(2):531-544.
[10]Pathak A K,Argal A.Analgesic activity of Calotropis gigantea flower [J].Fitoterapia,2007,78(1):40-42.
[11]Parhira S,Zhu G Y,Chen M,et al.Cardenolides from Calotropis gigantea as potent inhibitors of hypoxia-inducible factor-1 transcriptional activity[J].Journal of Ethnopharmacology,2016,194:930-936.
[12]Nguyen K D H,Dang P H,Nguyen H X,et al.Phytochemical and cytotoxic studies on the leaves of,Calotropis gigantea[J].Bioorganic and Medicinal Chemistry Letters,2017,27(13):2902-2906.
[13] Habib M R,Karim M R.Evaluation of antitumour activity of Calotropis gigantea L.root bark against ehrlich ascites carcinoma in Swiss albino mice[J].Asian Pacific Journal of Tropical Medicine,2011,4(10):786-790.
[14]Park H Y,Toume K,Arai M A,et al.Calotropin:a cardenolide from calotropis gigantea that inhibits Wnt signaling by increasing casein kinase 1 alpha in colon cancer cells [J].Chembiochem,2014,15(6):872-878.
[15]Saratha V,Subramanian S P.Lupeol,a triterpenoid isolated fromCalotropis gigantealatex ameliorates the primary and secondary complications of FCA induced adjuvant disease in experimental rats[J].Inflammopharmacology,2012,20(1):27-37.
[16]Chitme H,Chandra R,Kaushik S.Studies on anti-diarrhoeal activity of Calotropis gigantea R.Br.in experimental animals [J].Journal of Pharmacy and Pharmaceutical Sciences,2004,7(1):70-75.
[17]Rathod N R,Chitme H R,Irchhaiya R,et al.Hypoglycemic effect of Calotropis gigantea Linn.leaves and flowers in Streptozotocin-induced diabetic rats [J].Oman Medical Journal,2011,26(2):104-108.
[18]Chan E W C,Sweidan N I,Wong S K,et al.Cytotoxic cardenolides from calotropis species:ashort review [J].Records of Natural Products,2017,11(4):334-344.
[19]Prassas I,Diamandis E P.Novel therapeutic applications of cardiac glycosides[J].Nature Reviews Drug Discovery,2008,7(11):926-935.
[20]Tripathi P K,Awasthi S,Kanojiya S,et al.Callus culture and in vitro biosynthesis of cardiac glycosides from Calotropis gigantea(L.)Ait[J].In Vitro Cellular and Developmental Biology - Plant,2013,49(4):455-460.
[21]Pérez-Bermúdez P,Moya García A A,Tunón I,et al.Digitalis purpurea P5 beta R2,encoding steroid 5 beta-reductase,is a novel defense-related gene involved in cardenolide biosynthesis [J].New Phytologist,2010,185(3):687-700.
[22]Thitima L,Somyote S.19-Nor- and 18,20-epoxy-cardenolides from the leaves of Calotropis gigantea[J].Journal of Natural Products,2006,69(8):1249-1251.
[23] Hagimori M,Matsumoto T,Obi Y.effects of mineral salts,initial pH and precursors on digitoxin formation by shoot-forming cultures of Digitalis purpurea L.grown in liquid media[J].Journal of the Agricultural Chemical Society of Japan,1983,47(3):565-571.
[24]Gartner D E,Ulrich Seitz H.Enzyme activities in Cardenolide-accumulating,mixotrophic shoot cultures of Digitalis purpurea L[J].Journal of Plant Physiology,1993,141(3):269-275.
[25]Grtner D E,Keilholz W,Seitz H U.Purification,characterization and partial peptide microsequencing of progesterone 5 beta-reductase from shoot cultures of Digitalis purpurea[J].Febs Journal,2010,225(3):1125-1132.
[26]Herl V,Fischer G,Mulleruri F,et al.Molecular cloning and heterologous expression of progesterone 5beta-reductase from Digitalis lanata Ehrh[J].Phytochemistry,2006,67(3):225-231.
[27]Herl V,Albach D C,MüllerUriF,et al.Using progesterone 5β-reductase,a gene encoding a key enzyme in the cardenolide biosynthesis,to infer the phylogeny of the genus Digitalis[J].Plant Systematics and Evolution,2008,271(1/2):65-78.
[28]Bauer P,Munkert J,Brydziun M,et al.Highly conserved progesterone 5 beta-reductase genes(P5 beta R)from 5 beta-cardenolide-free and 5 beta-cardenolide-producing angiosperms [J].Phytochemistry,2010,71(13):1495-1505.
[29]Newman R A,Yang P,Pawlus A D,et al.Cardiac glycosides as novel cancer therapeutic agents[J].Molecular Interventions,2008,8(1):36-49.
[30]Sun Y,Huang Y H,Huang F Y,et al.3’-epi-12 beta-hydroxyfroside,a new cardenolide,induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells [J].Theranostics,2018,8(7):2044-2060.
[31]Gavidia I,Tarr O R,Rodr G F,et al.Plant progesterone 5β-reductase is not homologous to the animal enzyme.Molecular evolutionary characterization of P5βR from Digitalis purpurea [J].Phytochemistry,2007,68(6):853-864.
[32]Muriira N G,Xu W,Muchugi A,et al.De novo sequencing and assembly analysis of transcriptome in the Sodom apple(Calotropis gigantea)[J].Bmc Genomics,2015,16(1):723.
[33]Munkert J,Pollier J,Miettinen K,et al.Iridoid synthase activity is common among the plant progesterone 5 beta-reductase family[J].Molecular Plant,2015,8(1):136-152.