维药新塔花(Ziziphora bungena)zbHDS基因克隆及组织表达分析
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摘要
对维药新塔花(Ziziphora bungena)萜类化合物生物合成途径中,关键酶1-羟基-2甲基-2-(E)-丁烯基-4-二磷酸合成酶HDS基因克隆,进行组织表达特异性分析,为研究新塔花萜类化合物生物合成途径与基因调控提供基础。结合新塔花转录组数据库,根据编码区序列设计引物,通过RT-PCR方法对Z. bungena基因HDS (zbHDS)的编码区cDNA进行克隆,对其进行相关生物信息学分析,通过Real-Time进一步分析其组织表达特异性。RT-PCR扩增了一个长2 465 bp的zbHDS (登录号:MG065856)基因片段,含一个2 229 bp的开放阅读框,编码742个氨基酸。进化树分析结果显示,与丹参(Salvia miltiorrhiza)具有较近的亲缘关系。Real-Time PCR结果显示zbHDS基因在各器官中均有表达,在茎和叶中表达量较高,根和花中表达量相对较低。本研究首次从新塔花中克隆HDS基因并获得其编码区序列,zbHDS基因具有组织表达特异性,为进一步研究新塔花中萜类化合物生物合成途径提供数据支持。
To provide the basis for the further studies on biosynthesis and gene regulation of torpedoed, we tried to clone 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase(HDS) gene that plays an important role in torpedoed biosynthesis pathway in Uighur Medicial plant(Ziziphora bungena). According to the Zb HDS gene sequences of transcriptome of Z. bungena, a pair of primers were designed and the ORF of c DNA of sequences was obtained by RT-PCR, Then TA cloning, sequencing, and sequence analysis were performed in this research. The expression of Zb HDS in different organ of Z. bungena was detected by Real-Time PCR. The Zb HDS gene was obtained in length of2 465 bp gene fragments(GenBank accession number MG065856), with an open reading frame of 2 229 bp in length,encoding 742 amino acids. Phylogenetic analysis on the amino acid sequence of Zb HDS with those of other plants showed that Zb HDS was closely related to Salvia miltiorrhiza. The result of Real-Time PCR showed that Zb HDS gene was expressed in different tissues of Z. bungena. It showed that Zb HDS was high expression in leaves and stems while low expression in root and flower. The Zb HDS gene is cloned from Z. bungena for the first time and the expression of Zb HDS is tissue-specific. This research lays a foundation for studying the gene expression pattern and regulating the functions of Zb HDS in torpedoed biosynthesis.
To provide the basis for the further studies on biosynthesis and gene regulation of torpedoed, we tried to clone 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase(HDS) gene that plays an important role in torpedoed biosynthesis pathway in Uighur Medicial plant(Ziziphora bungena). According to the Zb HDS gene sequences of transcriptome of Z. bungena, a pair of primers were designed and the ORF of c DNA of sequences was obtained by RT-PCR, Then TA cloning, sequencing, and sequence analysis were performed in this research. The expression of Zb HDS in different organ of Z. bungena was detected by Real-Time PCR. The Zb HDS gene was obtained in length of2 465 bp gene fragments(GenBank accession number MG065856), with an open reading frame of 2 229 bp in length,encoding 742 amino acids. Phylogenetic analysis on the amino acid sequence of Zb HDS with those of other plants showed that Zb HDS was closely related to Salvia miltiorrhiza. The result of Real-Time PCR showed that Zb HDS gene was expressed in different tissues of Z. bungena. It showed that Zb HDS was high expression in leaves and stems while low expression in root and flower. The Zb HDS gene is cloned from Z. bungena for the first time and the expression of Zb HDS is tissue-specific. This research lays a foundation for studying the gene expression pattern and regulating the functions of Zb HDS in torpedoed biosynthesis.
引文
Altincicek B.,Kollas A.K.,Sanderbrand S.,Wiesner J.,Hintz M.,and Beck E.,2001,Gcpe is involved in the 2-c-methyl-derythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli,J.Bacteriol.,183(8):2411-2416
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Chen Q.H.,Li Q.,Yang W.J.,Zhao Y.,Zheng G.L.,and Zhang X.Y.,2015,Effect of ziziphora clinopodioides flavonoids on myocardial ischemia reperfusion injury in isolated rat hearts,Zhongguo Xiandai Yingyong Yaoxue(Chinese Journal of Modern Applied Pharmacy),32(3):264-268(陈秋红,李钦,杨伟俊,赵吟,郑高利,张信岳,2015,芳香新塔花总黄酮对大鼠离体心脏缺血再灌注损伤的保护作用,中国现代应用药学,32(3):264-268)
Dubey V.S.,Bhalla R.,and Luthra R.,2003,An overview of the non-mevalonate pathway for terpenoid biosynthesis in plants,J.Biosci.,28(5):637-646
Ginis O.,Courdavault V.,Melin C.,Lanoue A.,Giglioliguivarc H.N.,and Stpierre B.,2012,Molecular cloning and functional characterization of catharanthus roseus hydroxymethylbutenyl-4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway,Mol.Biol.Rep.,39(5):5433-5447
Jiang D.,Rong Q.X.,Yuan Q.J.,Zhang W.J.,Zhang Y.Q.,and Huang L.Q.,2014,Cloning and prokaryotic expression analysis of HDS from Salvia miltiorrhiza bunge F.alba,Yaoxue Xuebao(Acta Pharmaceutica Sinica),49(11):1614-1620(姜丹,荣齐仙,袁庆军,张文婧,张永清,黄璐琦,2014,白花丹参HDS基因的全长克隆与原核表达分析,药学学报,49(11):1614-1620)
Jiang Z.Z.,2013,Full-length cloning,functional analysis and expression of HDS and HDR gene in the MEP pathway of tea tree,Thesis for M.S.,Anhui Agriculture University,Supervisor:Wei C.L.,pp.21-27(蒋正中,2013,茶树MEP途径中HDS与HDR基因的cDNA全长克隆、功能分析与表达特征研究,硕士学位论文,安徽农业大学,导师:韦朝领,pp.21-27)
Liao J.J.,Xu J.G.,Yang W.J.,Zhao Y.,Dilinuer,and Manerhaba,2011,Antioxidant activity and hypoxiareoxygenation injury protective effects of flavonoids from Ziziphora clinopodioides on neonatal rat cardiomyocytes,Huanqiu Zhongyiyao(Global Traditional Chinese Medicine),4(4):256-259(廖晶晶,徐建国,杨伟俊,刘冲,地力努尔,满尔哈巴,2011,芳香新塔花黄酮抗氧化活性及对大鼠乳鼠心肌细胞缺氧复氧损伤的保护作用,环球中医药,4(4):256-259)
Liao Z.,Chen M.,Gong Y.,Guo L.,Tan Q.,and Feng X.,2004,A new geranylgeranyl diphosphate synthase gene from ginkgo biloba,which intermediates the biosynthesis of the key precursor for ginkgolides,Dna Seq.,15(2):153-158
Liu P.F.,Du H.Y.,Du L.Y.,Wuyuntana.,and Huang H.Y.,2012,Cloning and sequence analysis of 1-hydroxy-2-methyl-2-butenyl-4-diphosphate synthase gene cDNA from Eucommia ulmoides,Zhiwu Yanjiu(Bulletin of Botanical Research),32(4):444-451(刘攀峰,杜红岩,杜兰英,乌云塔娜,黄海燕,2012,杜仲1-羟基-2-甲基-2-(e)-丁烯基-4-二磷酸合酶基因cDNA全长克隆与序列分析,植物研究,32(4):444-451)
Liu Y.M.,ed.,1999,Weiwuer Yaozhi(Uighur medicine),Xinjiang Science and Technology Press,Urmuqi,China,pp.446-451(刘勇民,主编,1999,维吾尔药志(上册),新疆科技卫生出版社,中国,乌鲁木齐,pp.446-451)
Querol J.,Campos N.,Imperial S.,Boronat A.,and Rodríguezconcepción M.,2002,Functional analysis of the arabidopsis thaliana gcpe protein involved in plastid isoprenoid biosynthesis,FEBS Letters,514(2-3):343-346
Rodríguez-Concepción M.,Querol J.,Lois L.M.,Imperial S.,and Boronat A.,2003,Bioinformatic and molecular analysis of hydroxymethylbutenyl diphosphate synthase(GCPE)gene expression during carotenoid accumulation in ripening tomato fruit,Planta,217(3):476-482
Shi F.,Yang W.J.,Gu Z.Y.,He C.H.,Liu J.L.,and Zhang J.,2009,Analysis of chemical composition of volatile oil from Z.clinopodioides,Xinjiang Zhongyiyao(Xinjiang Journal of Traditional Chinese medicine),27(1):23-24(石峰,杨伟俊,顾政一,何承辉,刘君琳,张洁,2009,芳香新塔花挥发油的化学成份分析,新疆中医药,27(1):23-24)
Unal E.L.,Mavi A.,Kara A.A.,Cakir A.,Sengül M.,and Yildirim A.,2008,Antimicrobial and antioxidant activities of some plants used as remedies in turkish traditional medicine,Pharmaceutical Biology,46(3):207-224
Wang X.,Wu L.S.,Wu Q.J.,Lin Y.,Cai Y.P.,and Fang H.H.,2016,Cloning and preliminary expression analysis on HDSgene in Dendrobium officinale,Zhongcaoyao(Chinese Traditional and Herbal Drugs),47(5):803-809(王翔,吴林松,吴秋菊,林毅,蔡永萍,樊洪泓,2016,铁皮石斛HDS基因的克隆与初步表达分析,中草药,47(5):803-809)
Yang W.J.,Chong L.,Gu Z.Y.,Zhang X.Y.,Bo C.,and Yan M.,2014,Protective effects of acacetin isolated from Ziziphora clinopodioides,lam.(xintahua)on neonatal rat cardiomyocytes,Chinese Medicine,9(1):1-6
Yang W.J.,Hamulati.,Sun Y.H.,He J.,Manerhaba.,and Abudushalamu.,2010,Protective effects of Ziziphora clinopodiodes on rat acute myocardial ischemia and neonatal rat cardiomyocytes,Xiandai Yaowu Yu Linchuang(Drugs&Clinic),25(3):194-198(杨伟俊,哈木拉提,孙玉华,何江,满尔哈巴,阿不都沙拉木,2010,芳香新塔花对大鼠急性心肌缺血和乳鼠心肌细胞的保护作用,现代药物与临床,25(3):194-198)
Yue Z.,Min C.,Yang C.X.,Ming X.J.,Liu X.Q.,and Lan X.Z.,2011,Cloning and characterization of MECS and HDS genes from rauvolfia verticillata,Plant Omics Journal of Plant Molecular Biology&Omics,4(2):82-90
Zhang Z.R.,Liao Z.H.,and Peng M.F.,2012,cloning and function analyses of HDS gene from artemisia annua,Zhongcaoyao(Chinese Traditional and Herbal Drugs),43(1):148-154(张祖荣,廖志华,彭梅芳,2012,黄花蒿HDS基因的克隆与功能分析,中草药,43(1):148-154)
Zheng Y.,2011,Cloning of MCT,HDS,and SGD gene from Rauvolfia verticillata and establishment of genetic transformation system,Thesis for M.S.,Southwestern University,Supervisor:Liao Z.H.,pp.26-45(郑月,2011,萝芙木MCT、HDS、SGD基因的克隆与遗传转化体系的建立,硕士学位论文,西南大学,导师:廖志华,pp.26-45)
Chappell J.,1995,Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants,Annual Review of Plant Physiology&Plant Molecular Biology,46(1):521-547
Chen Q.H.,Li Q.,Yang W.J.,Zhao Y.,Zheng G.L.,and Zhang X.Y.,2015,Effect of ziziphora clinopodioides flavonoids on myocardial ischemia reperfusion injury in isolated rat hearts,Zhongguo Xiandai Yingyong Yaoxue(Chinese Journal of Modern Applied Pharmacy),32(3):264-268(陈秋红,李钦,杨伟俊,赵吟,郑高利,张信岳,2015,芳香新塔花总黄酮对大鼠离体心脏缺血再灌注损伤的保护作用,中国现代应用药学,32(3):264-268)
Dubey V.S.,Bhalla R.,and Luthra R.,2003,An overview of the non-mevalonate pathway for terpenoid biosynthesis in plants,J.Biosci.,28(5):637-646
Ginis O.,Courdavault V.,Melin C.,Lanoue A.,Giglioliguivarc H.N.,and Stpierre B.,2012,Molecular cloning and functional characterization of catharanthus roseus hydroxymethylbutenyl-4-diphosphate synthase gene promoter from the methyl erythritol phosphate pathway,Mol.Biol.Rep.,39(5):5433-5447
Jiang D.,Rong Q.X.,Yuan Q.J.,Zhang W.J.,Zhang Y.Q.,and Huang L.Q.,2014,Cloning and prokaryotic expression analysis of HDS from Salvia miltiorrhiza bunge F.alba,Yaoxue Xuebao(Acta Pharmaceutica Sinica),49(11):1614-1620(姜丹,荣齐仙,袁庆军,张文婧,张永清,黄璐琦,2014,白花丹参HDS基因的全长克隆与原核表达分析,药学学报,49(11):1614-1620)
Jiang Z.Z.,2013,Full-length cloning,functional analysis and expression of HDS and HDR gene in the MEP pathway of tea tree,Thesis for M.S.,Anhui Agriculture University,Supervisor:Wei C.L.,pp.21-27(蒋正中,2013,茶树MEP途径中HDS与HDR基因的cDNA全长克隆、功能分析与表达特征研究,硕士学位论文,安徽农业大学,导师:韦朝领,pp.21-27)
Liao J.J.,Xu J.G.,Yang W.J.,Zhao Y.,Dilinuer,and Manerhaba,2011,Antioxidant activity and hypoxiareoxygenation injury protective effects of flavonoids from Ziziphora clinopodioides on neonatal rat cardiomyocytes,Huanqiu Zhongyiyao(Global Traditional Chinese Medicine),4(4):256-259(廖晶晶,徐建国,杨伟俊,刘冲,地力努尔,满尔哈巴,2011,芳香新塔花黄酮抗氧化活性及对大鼠乳鼠心肌细胞缺氧复氧损伤的保护作用,环球中医药,4(4):256-259)
Liao Z.,Chen M.,Gong Y.,Guo L.,Tan Q.,and Feng X.,2004,A new geranylgeranyl diphosphate synthase gene from ginkgo biloba,which intermediates the biosynthesis of the key precursor for ginkgolides,Dna Seq.,15(2):153-158
Liu P.F.,Du H.Y.,Du L.Y.,Wuyuntana.,and Huang H.Y.,2012,Cloning and sequence analysis of 1-hydroxy-2-methyl-2-butenyl-4-diphosphate synthase gene cDNA from Eucommia ulmoides,Zhiwu Yanjiu(Bulletin of Botanical Research),32(4):444-451(刘攀峰,杜红岩,杜兰英,乌云塔娜,黄海燕,2012,杜仲1-羟基-2-甲基-2-(e)-丁烯基-4-二磷酸合酶基因cDNA全长克隆与序列分析,植物研究,32(4):444-451)
Liu Y.M.,ed.,1999,Weiwuer Yaozhi(Uighur medicine),Xinjiang Science and Technology Press,Urmuqi,China,pp.446-451(刘勇民,主编,1999,维吾尔药志(上册),新疆科技卫生出版社,中国,乌鲁木齐,pp.446-451)
Querol J.,Campos N.,Imperial S.,Boronat A.,and Rodríguezconcepción M.,2002,Functional analysis of the arabidopsis thaliana gcpe protein involved in plastid isoprenoid biosynthesis,FEBS Letters,514(2-3):343-346
Rodríguez-Concepción M.,Querol J.,Lois L.M.,Imperial S.,and Boronat A.,2003,Bioinformatic and molecular analysis of hydroxymethylbutenyl diphosphate synthase(GCPE)gene expression during carotenoid accumulation in ripening tomato fruit,Planta,217(3):476-482
Shi F.,Yang W.J.,Gu Z.Y.,He C.H.,Liu J.L.,and Zhang J.,2009,Analysis of chemical composition of volatile oil from Z.clinopodioides,Xinjiang Zhongyiyao(Xinjiang Journal of Traditional Chinese medicine),27(1):23-24(石峰,杨伟俊,顾政一,何承辉,刘君琳,张洁,2009,芳香新塔花挥发油的化学成份分析,新疆中医药,27(1):23-24)
Unal E.L.,Mavi A.,Kara A.A.,Cakir A.,Sengül M.,and Yildirim A.,2008,Antimicrobial and antioxidant activities of some plants used as remedies in turkish traditional medicine,Pharmaceutical Biology,46(3):207-224
Wang X.,Wu L.S.,Wu Q.J.,Lin Y.,Cai Y.P.,and Fang H.H.,2016,Cloning and preliminary expression analysis on HDSgene in Dendrobium officinale,Zhongcaoyao(Chinese Traditional and Herbal Drugs),47(5):803-809(王翔,吴林松,吴秋菊,林毅,蔡永萍,樊洪泓,2016,铁皮石斛HDS基因的克隆与初步表达分析,中草药,47(5):803-809)
Yang W.J.,Chong L.,Gu Z.Y.,Zhang X.Y.,Bo C.,and Yan M.,2014,Protective effects of acacetin isolated from Ziziphora clinopodioides,lam.(xintahua)on neonatal rat cardiomyocytes,Chinese Medicine,9(1):1-6
Yang W.J.,Hamulati.,Sun Y.H.,He J.,Manerhaba.,and Abudushalamu.,2010,Protective effects of Ziziphora clinopodiodes on rat acute myocardial ischemia and neonatal rat cardiomyocytes,Xiandai Yaowu Yu Linchuang(Drugs&Clinic),25(3):194-198(杨伟俊,哈木拉提,孙玉华,何江,满尔哈巴,阿不都沙拉木,2010,芳香新塔花对大鼠急性心肌缺血和乳鼠心肌细胞的保护作用,现代药物与临床,25(3):194-198)
Yue Z.,Min C.,Yang C.X.,Ming X.J.,Liu X.Q.,and Lan X.Z.,2011,Cloning and characterization of MECS and HDS genes from rauvolfia verticillata,Plant Omics Journal of Plant Molecular Biology&Omics,4(2):82-90
Zhang Z.R.,Liao Z.H.,and Peng M.F.,2012,cloning and function analyses of HDS gene from artemisia annua,Zhongcaoyao(Chinese Traditional and Herbal Drugs),43(1):148-154(张祖荣,廖志华,彭梅芳,2012,黄花蒿HDS基因的克隆与功能分析,中草药,43(1):148-154)
Zheng Y.,2011,Cloning of MCT,HDS,and SGD gene from Rauvolfia verticillata and establishment of genetic transformation system,Thesis for M.S.,Southwestern University,Supervisor:Liao Z.H.,pp.26-45(郑月,2011,萝芙木MCT、HDS、SGD基因的克隆与遗传转化体系的建立,硕士学位论文,西南大学,导师:廖志华,pp.26-45)