茅苍术两个DXR基因(AlDXR)的克隆与分析
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摘要
茅苍术萜类化合物生物合成关键酶1-脱氧-D-木酮糖5-磷酸还原酶异构化酶基因序列(AlDXR)尚未阐明,本研究克隆其基因序列并分析其序列特征,以期为阐明茅苍术萜类化合物生物合成分子机制提供帮助。从已获得的茅苍术转录组序列中筛选出DXR基因,克隆其全长cDNA序列,然后分析基因编码氨基酸序列特征、蛋白亚细胞定位、蛋白结构预测、系统发育等。成功筛选并克隆出两条全长cDNA序列分别为1 431 bp和1 422 bp的DXR基因(GenBank登录号分别为MG859908和MG859909),分别编码476个和473个氨基酸,系统发育分析表明二者分别与橡胶草和野菊花具有较高的同源性。本研究获得两个茅苍术DXR基因的全长cDNA序列,并对其进行初步生物信息学分析,为进一步阐明该基因在茅苍术萜类化合物生物合成中的功能提供指导。
The 1-Deoxy-D-xylulose 5-phosphate Reductase isomerase gene sequence(AlDXR), a key enzyme in terpenoids biosynthesis, has not been elucidated in Atractylodes lancea(Thunb.) DC. In this study, the AlDXR gene sequences were cloned and analyzed to offer help for molecular mechanism of terpenoid biosynthesis in A.lancea. We selected DXR gene from transcriptome sequence in obtained Atractylodes lancea, cloned full length cDNA sequence, and analyzed amino acid sequence characteristics in gene coding, protein subcellular localization,protein structure prediction and phylogeny. Two AlDXR genes were screened and cloned from transcriptome data and their 1 431 bp and 1 422 bp full-length cDNA sequences were obtained, which encode 476 and 473 amino acids, respectively(GenBank accession numbers MG859908 and MG859909). Phylogenetic analysis showed the highest homology with Taraxacum kok-saghyz and Chrysanthemum indicum var. aromaticum respectively. This study obtained two full-length cDNA sequence of DXR gene in Atractylodes lancea, and carried out preliminary bioinformatics analysis for it. These results will provide a guidance for the function of this gene in terpenoid biosynthesis in A. lancea.
The 1-Deoxy-D-xylulose 5-phosphate Reductase isomerase gene sequence(AlDXR), a key enzyme in terpenoids biosynthesis, has not been elucidated in Atractylodes lancea(Thunb.) DC. In this study, the AlDXR gene sequences were cloned and analyzed to offer help for molecular mechanism of terpenoid biosynthesis in A.lancea. We selected DXR gene from transcriptome sequence in obtained Atractylodes lancea, cloned full length cDNA sequence, and analyzed amino acid sequence characteristics in gene coding, protein subcellular localization,protein structure prediction and phylogeny. Two AlDXR genes were screened and cloned from transcriptome data and their 1 431 bp and 1 422 bp full-length cDNA sequences were obtained, which encode 476 and 473 amino acids, respectively(GenBank accession numbers MG859908 and MG859909). Phylogenetic analysis showed the highest homology with Taraxacum kok-saghyz and Chrysanthemum indicum var. aromaticum respectively. This study obtained two full-length cDNA sequence of DXR gene in Atractylodes lancea, and carried out preliminary bioinformatics analysis for it. These results will provide a guidance for the function of this gene in terpenoid biosynthesis in A. lancea.
引文
Carretero-Paulet L.,Ahumada I.,Cunillera N.,Concepcion M.R.,Ferrer A.,Boronat A.,and Campos N.,2002,Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-Deoxy-D-Xylulose 5-Phosphate reductoisomerase,the first committed enzyme of the 2-C-Methyl-D-Erythritol4-Phosphate pathway,Plant Physiol.,129(4):1581-1591
Deng J.,Wan Q.Y.,Gong M.,Liu H.G.,and Yu K.,2017,Cloning and analysis of DXS gene in Atractylodes lancea operation,Zhongguo Shiyan Fangjixue Zazhi(Chinese Journal of Experimental Formulae),23(16):39-44(邓娟,万倩芸,龚玲,刘合刚,余坤,2017,茅苍术DXS基因的克隆与分析,中国实验方剂学杂志,23(16):39-44)
Lange B.M.,and Croteau R.,1999,Isoprenoid biosynthesis via a mevalonate-independent pathway in plants:cloning and heterologous expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from pepper mint,Arch.Biochem.Biophys.,365(1):170-174
Li X.J.,Liu H.B.,Lin X.Q.,Wu Z.D.,Xu C.H.,and Liu X.L.,2015,In Silico cloning and bioinformatics analysis of KNOXgene in sugarcane(Sckn1),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):136-142(李旭娟,刘洪博,林秀琴,吴转娣,徐超华,刘新龙,2015,甘蔗KNOX基因(Sckn1)的电子克隆及生物信息学分析,基因组学与应用生物学,34(1):136-142)
Ma Z.Z.,Pang X.Q.,Chen R.,Pei X.L.,Wang Q.Y.,Xie T.,and Yin X.P.,2015,Research progress of key enzymes in biosynthesis pathway of terpene compounds,Hangzhou Shifan Daxue Xuebao(Journal of Hangzhou Normal University(Natural Science Edition)),14(6):608-615(马转转,庞潇卿,谌容,裴晓林,王秋岩,谢恬,殷晓浦,2015,萜类化合物生物合成途径中关键酶研究进展,杭州师范大学学报(自然科学版),14(6):608-615)
Mahmoud S.S.,and Croteau R.B.,2001,Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase,Proc.Natl.Acad.Sci.USA,98(15):8915-8920
Schwender J.,Muller C.,Zeidler J.,and Lichtenthaler H.K.,1999,Cloning and heterologous expression of a cDNA encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase of Arabidopsis thaliana,FEBS Lett.,455(1-2):140-144
Singh N.,ChevaG.,Avery M.A.,and Mccurdy C.R.,2007,Targeting the methyl erythritol phosphate(MEP)pathway for novel antimalarial,antibacterial and herbicidal drug discovery:inhibition of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase(DXR)enzymej,Cnrr.Pharm.Design,13(11):1161-1177
Tong Y.R.,Li G.G.,Cheng Q.Q.,and Gao W.,2013,Advances in the study of the gene of 5-phosphate reductase for the reduction of heterogeneous enzymes in medicinal plants,Zhongcaoyao(Chinese Herbal Medicine),44(4):488-496(童宇茹,李桂桂,程琪庆,高伟,2013,药用植物5-磷酸脱氧木酮糖还原异构酶基因研究进展,中草药,44(4):488-496)
Veau B.,Courtois M.,Oudin A.,Chenieux J.C.,and Rideau M.,2000,Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus,Biochemistry Biophysical Acta,1517(1):159-163
VranováE.,Coman D.,and Gruissem W.,2013,Network analysis of the MVA and MEP pathways for isoprenoid synthesis,Annu.Rev.Plant Biol.,64(1):665-700
Wang C.H.,Lei X.Y.,Xia J.,and Wang J.W.,2018,Effect of down-regulating 1-deoxy-d-xylulose 5-phosphate reductoisomerase by RNAi on growth and artemisinin biosynthesis in Artemisia annua L.,Plant Growth Regul.,84(3):549-559
Xing C.B.,Long Y.H.,He S.,Liang N.S.,and Li B.C.,2012,Cloning,bioinformatics and expression analysis of Acanthopanax senticosus farnesyl diphosphate synthase gene,Zhongguo Zhongyao Zazhi(Chinese Journal of Chinese Medicine),37(12):1725-1729(邢朝斌,龙月红,何闪,梁能松,李宝财,2012,刺五加法尼基焦磷酸合酶基因的克隆、生物信息学及表达分析,中国中药杂志,37(12):1725-1729)
Xing S.F.,Miao J.,Li S.,Qin G.J.,Tang S.,Li H.N.,Gu H.Y.,and Qu L.J.,2010,Disruption of the 1-deoxy-d-xylulose5-phosphate reductoisomerase(DXR)gene results in albino,dwarf and defects in trichome initiation and stomata closure in Arabidopsis,Cell Res.,20:688-700
Yu Z.F.,Li R.F.,and Huang Q.,2014,Effects of partial codon of bacillus subtilis on protein folding rate,Shengwu Xinxixue(Bioinformatics),4(12):292-299(于志芬,李瑞芳,黄俏,2014,枯草芽孢杆菌同义密码子使用偏性对蛋白质折叠速率的影响,生物信息学,4(12):292-299)
Zhang L.,Ouyang Z.,Zhao M.,Wang P.X.,and Fang J.,2010,Simultaneous determination and cluster analysis of atractylodes ketone,atractylodes alcohol,β-eucalyptus and atractylodes atractylodes lancea in the process of operation,Zhong guo Zhiwu Fangjixue(Chinese Journal of Traditional Chinese Medicine),35(6):725-728(张磊,欧阳臻,赵明,王佩香,方静,2010,茅苍术中苍术酮、苍术醇、β-桉叶醇和苍术素的同时含量测定及其聚类分析,中国中药杂志,35(6):725-728)
Zhu X.Y.,Wang J.,Zhao E.W.,and Cui H.,2011,Cloning and expression analysis of a tobacco 5-phosphate reductase gene(DXR),Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),19(2):263-269(朱晓宇,王景,赵二卫,姚姗姗,崔红,2011,烟草5-磷酸脱氧木酮糖还原异构酶基因(DXR)的克隆和表达分析,农业生物技术学报,19(2):263-269)
Deng J.,Wan Q.Y.,Gong M.,Liu H.G.,and Yu K.,2017,Cloning and analysis of DXS gene in Atractylodes lancea operation,Zhongguo Shiyan Fangjixue Zazhi(Chinese Journal of Experimental Formulae),23(16):39-44(邓娟,万倩芸,龚玲,刘合刚,余坤,2017,茅苍术DXS基因的克隆与分析,中国实验方剂学杂志,23(16):39-44)
Lange B.M.,and Croteau R.,1999,Isoprenoid biosynthesis via a mevalonate-independent pathway in plants:cloning and heterologous expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from pepper mint,Arch.Biochem.Biophys.,365(1):170-174
Li X.J.,Liu H.B.,Lin X.Q.,Wu Z.D.,Xu C.H.,and Liu X.L.,2015,In Silico cloning and bioinformatics analysis of KNOXgene in sugarcane(Sckn1),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):136-142(李旭娟,刘洪博,林秀琴,吴转娣,徐超华,刘新龙,2015,甘蔗KNOX基因(Sckn1)的电子克隆及生物信息学分析,基因组学与应用生物学,34(1):136-142)
Ma Z.Z.,Pang X.Q.,Chen R.,Pei X.L.,Wang Q.Y.,Xie T.,and Yin X.P.,2015,Research progress of key enzymes in biosynthesis pathway of terpene compounds,Hangzhou Shifan Daxue Xuebao(Journal of Hangzhou Normal University(Natural Science Edition)),14(6):608-615(马转转,庞潇卿,谌容,裴晓林,王秋岩,谢恬,殷晓浦,2015,萜类化合物生物合成途径中关键酶研究进展,杭州师范大学学报(自然科学版),14(6):608-615)
Mahmoud S.S.,and Croteau R.B.,2001,Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase,Proc.Natl.Acad.Sci.USA,98(15):8915-8920
Schwender J.,Muller C.,Zeidler J.,and Lichtenthaler H.K.,1999,Cloning and heterologous expression of a cDNA encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase of Arabidopsis thaliana,FEBS Lett.,455(1-2):140-144
Singh N.,ChevaG.,Avery M.A.,and Mccurdy C.R.,2007,Targeting the methyl erythritol phosphate(MEP)pathway for novel antimalarial,antibacterial and herbicidal drug discovery:inhibition of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase(DXR)enzymej,Cnrr.Pharm.Design,13(11):1161-1177
Tong Y.R.,Li G.G.,Cheng Q.Q.,and Gao W.,2013,Advances in the study of the gene of 5-phosphate reductase for the reduction of heterogeneous enzymes in medicinal plants,Zhongcaoyao(Chinese Herbal Medicine),44(4):488-496(童宇茹,李桂桂,程琪庆,高伟,2013,药用植物5-磷酸脱氧木酮糖还原异构酶基因研究进展,中草药,44(4):488-496)
Veau B.,Courtois M.,Oudin A.,Chenieux J.C.,and Rideau M.,2000,Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus,Biochemistry Biophysical Acta,1517(1):159-163
VranováE.,Coman D.,and Gruissem W.,2013,Network analysis of the MVA and MEP pathways for isoprenoid synthesis,Annu.Rev.Plant Biol.,64(1):665-700
Wang C.H.,Lei X.Y.,Xia J.,and Wang J.W.,2018,Effect of down-regulating 1-deoxy-d-xylulose 5-phosphate reductoisomerase by RNAi on growth and artemisinin biosynthesis in Artemisia annua L.,Plant Growth Regul.,84(3):549-559
Xing C.B.,Long Y.H.,He S.,Liang N.S.,and Li B.C.,2012,Cloning,bioinformatics and expression analysis of Acanthopanax senticosus farnesyl diphosphate synthase gene,Zhongguo Zhongyao Zazhi(Chinese Journal of Chinese Medicine),37(12):1725-1729(邢朝斌,龙月红,何闪,梁能松,李宝财,2012,刺五加法尼基焦磷酸合酶基因的克隆、生物信息学及表达分析,中国中药杂志,37(12):1725-1729)
Xing S.F.,Miao J.,Li S.,Qin G.J.,Tang S.,Li H.N.,Gu H.Y.,and Qu L.J.,2010,Disruption of the 1-deoxy-d-xylulose5-phosphate reductoisomerase(DXR)gene results in albino,dwarf and defects in trichome initiation and stomata closure in Arabidopsis,Cell Res.,20:688-700
Yu Z.F.,Li R.F.,and Huang Q.,2014,Effects of partial codon of bacillus subtilis on protein folding rate,Shengwu Xinxixue(Bioinformatics),4(12):292-299(于志芬,李瑞芳,黄俏,2014,枯草芽孢杆菌同义密码子使用偏性对蛋白质折叠速率的影响,生物信息学,4(12):292-299)
Zhang L.,Ouyang Z.,Zhao M.,Wang P.X.,and Fang J.,2010,Simultaneous determination and cluster analysis of atractylodes ketone,atractylodes alcohol,β-eucalyptus and atractylodes atractylodes lancea in the process of operation,Zhong guo Zhiwu Fangjixue(Chinese Journal of Traditional Chinese Medicine),35(6):725-728(张磊,欧阳臻,赵明,王佩香,方静,2010,茅苍术中苍术酮、苍术醇、β-桉叶醇和苍术素的同时含量测定及其聚类分析,中国中药杂志,35(6):725-728)
Zhu X.Y.,Wang J.,Zhao E.W.,and Cui H.,2011,Cloning and expression analysis of a tobacco 5-phosphate reductase gene(DXR),Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),19(2):263-269(朱晓宇,王景,赵二卫,姚姗姗,崔红,2011,烟草5-磷酸脱氧木酮糖还原异构酶基因(DXR)的克隆和表达分析,农业生物技术学报,19(2):263-269)