烟草5-磷酸脱氧木酮糖还原异构酶（DXR）基因全长cDNA的克隆及序列分析
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摘要
萜类化合物(terpenoids)是自然界存在的一类由异戊二烯为结构单元组成的化合物的统称,也称为类异戊二烯(isoprenoids)。该类化合物广泛分布于古细菌、细菌、真菌、植物等生物体内。烟叶中的致香物质前体主要包括三类,即西柏烷类化合物(Cembranoids)、类胡萝卜素类化合物(Carotenoids)和岩蔷薇类化合物(Labdaniods)。它们都是烟草中与香气相关的萜类化合物,由DOXP/MEP途径合成。5-磷酸脱氧木酮糖还原异构酶(DXR)是萜类化合物合成途径中参与DOXP/MEP途径的第一个关键酶,它催化DOXP生成MEP,是催化萜类合成前体IPP生成的关键步骤。该酶的作用决定了下游萜类化合物的产量。目前,很多细菌、原生动物和高等植物的DXR基因都相继被发现。不同物种中DXR氨基酸水平的同源性很高。
本研究以烟草(Nicotiana tabacum)叶片为材料,应用RT-PCR及RACE的方法首次克隆了烟草DXR基因的全长cDNA序列(DQ839130)。该基因全长为1,804bp,开放阅读框(ORF)由1,422个碱基组成,共编码473个氨基酸,理论分子量约为51.2kDa,等电点为6.04,N-末端有一个富含Pro残基的信号肽;二级结构预测表明,不规则盘绕和α-螺旋是DXR蛋白中主要的结构元件;同源序列比对表明,推断的DXR氨基酸序列与本塞姆氏烟草(Nicotiana benthamiana, AM236596)、番茄(Lycopersicon esculentum, AF331705)、长春花(Catharanthus roseus, AF250235)、金鱼草(Antirrhinum majus, AY770406)、胡椒薄荷(Mentha×piperita, AF116825)DXR的一致性分别达到97%、92%、84%、82%、80%,证实该基因确为DXR基因。进化树分析显示烟草DXR与番茄DXR的亲缘关系最近,且基本反应了10种不同植物DXR之间的进化关系。
烟草DXR基因的克隆与分析为进一步验证DXR在烟草类萜生物合成途径中的分子调控机理,以及构建改良烟叶香气品质的转基因烟草,从而增加烟草中重要香气物质的含量奠定了良好的基础。
Terpenoids, also called isoprenoids, are a group of natural products which all formally built from isoprenic unit. Those kinds of compound are biosynthesized by archaebacterium, bacterium, fungi, and plant. Many of these compounds are of important economic value. There are three key fragrant terpenoids which are synthesized through DOXP/MEP pathway in tobacco, including Cembranoids, Carotenoids, Labdaniods. Their biosynthesis pathway and regulation of concerned emzymes should be elucidated. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is the first key enzyme in the process of the DOXP/MEP pathway for terpenoid biosynthesis. It catalyzes conversion of 1-deoxy-D-xylulose-5-phosphate (DOXP) to 2C-methyl-D-erythritol-4-phosphate (MEP), that represents the committed step in the production of isopentenylallyl diphosphate (IPP). It will determine the outputs of downstream products. DXR gene has been isolated from different species, including bacteria, protozoa and higher plants. The amino acid identity among DXR from different species is high.
In this work, reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques had been applied to clone the DXR gene (GenBank accession No.: DQ839130) using tobacco (Nicotiana tabacum) leaves as the target material for the first time. The full-length cDNA was 1,804bp and the biggest open reading frame (ORF) was 1,422bp and encoded 473 amino acids which had a theoretical molecular weight of 51.2 kDa, an isoelectric point of 6.04 and a plastid transit peptide which was rich in Pro residue at its N-terminal. Secondary structure predicting reveals thatα-helix and random coil were the main structural conformations in DXR protein. Through sequence analysis by Blast P online, the deduced amino acid sequence showed highly homologous to the DXR which was from other plant species. The identification that compared with Nicotiana benthamiana (AM236596), Lycopersicon esculentum(AF331705), Catharanthus roseus(AF250235), Antirrhinum majus(AY770406) and Mentha×piperita(AF116825) were 97%, 92%, 84%, 82% and 80% respectively.The phylogenetic tree showed that tobacco DXR was closely related to tomato DXR, and basically reflect the phylogenetic relationships of these DXR in ten plants.
The cloning and analysis of tobacco DXR gene provided a good base for research on functional expression and potential application in improving the fragrant quality of tobacco leaves, and for construction of high yield key fragrant terpenoids genetic engineering tobacco.
本研究以烟草(Nicotiana tabacum)叶片为材料,应用RT-PCR及RACE的方法首次克隆了烟草DXR基因的全长cDNA序列(DQ839130)。该基因全长为1,804bp,开放阅读框(ORF)由1,422个碱基组成,共编码473个氨基酸,理论分子量约为51.2kDa,等电点为6.04,N-末端有一个富含Pro残基的信号肽;二级结构预测表明,不规则盘绕和α-螺旋是DXR蛋白中主要的结构元件;同源序列比对表明,推断的DXR氨基酸序列与本塞姆氏烟草(Nicotiana benthamiana, AM236596)、番茄(Lycopersicon esculentum, AF331705)、长春花(Catharanthus roseus, AF250235)、金鱼草(Antirrhinum majus, AY770406)、胡椒薄荷(Mentha×piperita, AF116825)DXR的一致性分别达到97%、92%、84%、82%、80%,证实该基因确为DXR基因。进化树分析显示烟草DXR与番茄DXR的亲缘关系最近,且基本反应了10种不同植物DXR之间的进化关系。
烟草DXR基因的克隆与分析为进一步验证DXR在烟草类萜生物合成途径中的分子调控机理,以及构建改良烟叶香气品质的转基因烟草,从而增加烟草中重要香气物质的含量奠定了良好的基础。
Terpenoids, also called isoprenoids, are a group of natural products which all formally built from isoprenic unit. Those kinds of compound are biosynthesized by archaebacterium, bacterium, fungi, and plant. Many of these compounds are of important economic value. There are three key fragrant terpenoids which are synthesized through DOXP/MEP pathway in tobacco, including Cembranoids, Carotenoids, Labdaniods. Their biosynthesis pathway and regulation of concerned emzymes should be elucidated. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is the first key enzyme in the process of the DOXP/MEP pathway for terpenoid biosynthesis. It catalyzes conversion of 1-deoxy-D-xylulose-5-phosphate (DOXP) to 2C-methyl-D-erythritol-4-phosphate (MEP), that represents the committed step in the production of isopentenylallyl diphosphate (IPP). It will determine the outputs of downstream products. DXR gene has been isolated from different species, including bacteria, protozoa and higher plants. The amino acid identity among DXR from different species is high.
In this work, reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques had been applied to clone the DXR gene (GenBank accession No.: DQ839130) using tobacco (Nicotiana tabacum) leaves as the target material for the first time. The full-length cDNA was 1,804bp and the biggest open reading frame (ORF) was 1,422bp and encoded 473 amino acids which had a theoretical molecular weight of 51.2 kDa, an isoelectric point of 6.04 and a plastid transit peptide which was rich in Pro residue at its N-terminal. Secondary structure predicting reveals thatα-helix and random coil were the main structural conformations in DXR protein. Through sequence analysis by Blast P online, the deduced amino acid sequence showed highly homologous to the DXR which was from other plant species. The identification that compared with Nicotiana benthamiana (AM236596), Lycopersicon esculentum(AF331705), Catharanthus roseus(AF250235), Antirrhinum majus(AY770406) and Mentha×piperita(AF116825) were 97%, 92%, 84%, 82% and 80% respectively.The phylogenetic tree showed that tobacco DXR was closely related to tomato DXR, and basically reflect the phylogenetic relationships of these DXR in ten plants.
The cloning and analysis of tobacco DXR gene provided a good base for research on functional expression and potential application in improving the fragrant quality of tobacco leaves, and for construction of high yield key fragrant terpenoids genetic engineering tobacco.
引文
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[3] Lange BT, Martin W, Croteau R. Isoprenoid biosynthesis; the volution of two ancient and distinct pathways across genomes. Proc Natl Acad Sci USA ,2000,1997(13): 172~175
[4] Davis EM, Croteau R. Cyclization enzymes in the biosynthesis of monoterpenes, sesquiterpenes, and diterpenes. Curr Topics Chem, 2000, 209: 53~95
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[14] Eisenreicha W, Bachera A, Arigonib D, et al. Biosynthesis of isoprenoids via the non-mevalonate pathway. Cell Mol Life Sci, 2004, 61: 1401~1426
[15] Rohmer M. The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Nat Prod Rep, 1999, 16: 565~574
[16] Eisenreicha W, Bachera A, Arigonib D, et al. Biosynthesis of isoprenoids via the non-mevalonate pathway. Cell Mol Life Sci, 2004,61: 1401~1426
[17]兰文智,余龙江,蔡永君等.类异戊二烯非甲羟戊酸代谢途径的分子生物学研究进展.西北植物学报, 2001, 21(5): 1039~1047
[18] Lützow M, Beyer P. The isopentenyl-diphosphate A-isomerase and its relation to the phytoene synthase complex in daffodil chromoplasts. Biochim Biophys Acta, 1988, 959: 118~126
[19] Aubourg S. Genomic analysis of the terpenoid synthase (AtTPS) gene family of Arabidopsis thaliana. Mol Genet Genomics, 2002, 267: 730~745
[20] Kasahara H, Hanada A, Kuzuyama T, et al. Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis. J Biol Chem, 2002, 277: 45188~45194
[21] Arigoni D, Sagner S, Latzel C, et al. Terpenoid biosynthesis from 1-deoxy-Dxylulose in higher plants by intramolecular skeletal rearrangement. Proc Natl Acad Sci USA, 1997, 94: 10600~10605
[22]陈建,赵德刚.植物萜类生物合成相关酶类及其编码基因的研究进展.分子植物育种, 2004, 2(6): 757~764
[23] Proteau PJ. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase: an overview. Bioorg Chem, 2004, 32: 483~493
[24] Carretero-Paulet L, Ahumada I, Cunillera N, et al. 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-erythritol 4-phosphate pathway. Plant Physiol, 2002, 129(4): 1581~1591
[25]郑清平,余龙江,刘智等.红豆杉细胞非甲羟戊酸途径关键酶基因dxr的克隆与分析.生物工程学报, 2004, 20(4): 548~553
[26] Reuter K, Sanderbrand S, Jomaa H, et al. Crystal structure of 1-deoxy--xylulose- -phosphate reductoisomerase, a crucial in the non-mevalonate pathway of isoprenoid biosynthesis. J Biol Chem, 2002, 227: 5378~5384
[27] Yajima S, Nonaka T, Kuzuyama T, et al. 1-deoxy-D-xylulose-5-phosphate Reductoisomerase Complexed with Cofactors: Implications of a Flexible Loop Movement upon Substrate Binding. J. Biochem, 2002, 131: 313~317
[28] Steinbacher S, Kaiser J, Eisenreich W, et al. Structural Basis of Fosmidomycin Action Revealed by the Complex with 2-C-Methyl-d-erythritol 4-phosphate synthase (IspC). J. Biol. Chem, 2003, 278: 18401~18407
[29] Ricagno S, Grolle S, Bringer-Meyer S, et al. Crystal structure of 1-deoxy-d-xylulose-5-phosphate reductoisomerase from Zymomonas mobilis at 1.9A resolution. Biochim. Biophys. Acta, 2004, 1698: 37~44
[30] Takahashi S, Kuzuyama T, Walanabe H, et al. A 1-deoxy-D-xylulose 5-phosphate reductoismerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpeonid biosynthesis. Proc Natl Acad Sci USA, 1998,95: 9879~9 884
[31] Carretero-Paulet L, Ahumada I, Cunillera N, et al. 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-erythritol 4-phosphate pathway. Plant Physiol, 2002, 129(4): 1581~1591
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