Cloning and Sequence Analysis of 1-Hydroxy-2-Methyl-2-E-Butenyl-4-Diphosphate Reductase Gene cDNA from Eucommia ulmoides

LIU Pan-feng; DU Hong-yan; WUYUN Ta-na; DU Lan-ying; SUN Zhi-qiang

Volume 26 Issue 4

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Cloning and Sequence Analysis of 1-Hydroxy-2-Methyl-2-E-Butenyl-4-Diphosphate Reductase Gene cDNA from Eucommia ulmoides

1.
Non-timber Forestry Research and Development Center, Chinese Academy of Forestry, Zhengzhou 450003, He’nan, China
2.
Key Laboratory of Non-wood Forest Products of State Forestry Administration, Changsha 410004, Hu’nan, China

Received Date: 2012-08-28

Abstract

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) synthesizes IPP and DMAPP in the last step of the plant 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Homologous HDR gene cDNA was isolated from the leaves of Eucommia ulmoides by the method of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique, and named as EuHDR. With the highest gene sequence similarity to Camptotheca acuminata (82%), the full-length cDNA of EuHDR was 1 653 bp including 5’non-coding region of 82 bp and 3’non-coding region of 188 bp and encoded 460 amino acids. The transit peptide sequence (A1-A33) and multiple conserved functional sites(A117, A208, A262, A345)of plant HDR protein were found in the deduced coding sequence of EuHDR. The secondary structure of EuHDR protein was predicted with proportion of α-helix to 35.65%,β-sheet to 19.78% and loop/coil to 44.57%. The calculated protein tertiary structure of EuHDR was formed as monomer, which in space displayed asymmetrical shamrock-like shape. Phylogenetic analysis revealed that the evolutionary relationship of EuHDR protein was the closest to Vitis vinifera HDR protein.
- Eucommia ulmoides,
- HDR gene,
- sequence analysis,

References

[1]	Köksal M,Hu R M,Coates R M,et al. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase[J]. Nat Chem Biol, 2011,7: 431-433
[2]	Eric O, Lin F Y. Terpene biosynthesis: modularity rules[J]. Angew Chem,2012,51(5):1124-1137
[3]	Karine B, Yannick E, Alain D,et al. Isopentenyl diphosphate isomerase: a checkpoint to isoprenoid biosynthesis[J]. Biochimi,2012,doi:10.1016/j.biochi.2012.03.021:1-14.
[4]	Daum M, Herrmann S, Wilkinson B,et al.Genes and enzymesinvolved in bacterial isoprenoid biosynthesis[J]. Curr Opin Chem Biol,2009,13:180-188
[5]	Annegret A B, Karnjapan J, Juliusz A W,et al. Biosynthesis of isoprene units:mssobauer spectroscopy of substrate and inhibitor binding to the cluster of the LytB/IspH enzyme[J]. Angew Chem, 2011,50:11976-11979
[6]	Sang-Min K,Tomohisa K,Akio K,et al. 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS)is encoded by multicopy genes in Gymnosperms Ginkgo biloba and Pinus taeda[J]. Planta,2008,227:287-298
[7]	Cunningham Jr F X, Lafond T P, Gantt E. Evidence of a role for LytB in the nonmevalonate pathway of isoprenoid biosynthesis[J]. J Bacteriol,2000,182:5841-5848
[8]	Guevara-Garcia A,San Roman C,Arroyo A,et al. Characterization of the Arabidopsis clb6 mutant illustrates the importance of posttranscriptional regulation of the methyl-D-erythritol 4-phosphate pathway[J]. Plant Cell,2005,17: 628-643
[9]	Hsieh M H, Goodman H M. The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis[J]. Plant Physiol, 2005,138:641-653
[10]	张雯,王玉亮,林娟,等.银杏1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶基因(hdr)转化银杏的研究[J].复旦学报,2008,47(5):598-602
[11]	Botella-Pavía P, óscar B, Phillips M A,et al. Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors[J]. Plant J,2004, 40(2):188-199
[12]	崔克明.杜仲研究的历史、现状和展望[J].西北林学院学报,1994,9(4):51-57
[13]	杜红岩.杜仲优质高产栽培[M]. 北京:中国林业出版社, 1996
[14]	陈建.几种提取杜仲RNA方法的比较[J].林业科技开发,2007,21(5):19-21
[15]	周明兵,王红珍,赵德刚.杜仲叶和树皮总RNA的快速提取法[J].山地农业生物学报,2003,22(5):430-431
[16]	Jones D T. Protein secondary structure prediction based on position-specific scoring matrices [J]. J Mol Biol, 1999, 292(2):195-202
[17]	Emanuelsson O,Nielsen H, von Heijne G. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites[J].Protein Sci,1999,8: 978-984
[18]	Arnold K,Bordoli L, Kopp J,et al. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling[J]. Bioinformatics, 2006,22:195-201
[19]	Tamura K,Peterson D,Peterson N,et al. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Mol Biol Evol,2011,28: 2731-2739
[20]	高文运.同位素标记及未标记的1-脱氧-D-木酮糖-5-磷酸和2-甲基-D-赤藓糖醇-4-磷酸的合成方法[J].有机化学,2010,30 (1):23-37
[21]	金蓉,朱长青,徐昌杰.1-脱氧木酮糖-5-磷酸合成酶(DXS)及其编码基因[J].细胞生物学杂志,2007,29:706-712
[22]	郑洲翔,范燕萍,周纪刚,等. 植物萜类合成酶1-脱氧-D-木酮糖-5-磷酸还原酶研究进展[J]. 安徽农业科学, 2011,39(10):5695-5696
[23]	Tobias G, Ingrid S, Wolfgang E,et al. Probing the reaction mechanism of IspH protein by X-ray structure analysis[J].PNAS,2010,107(3):1077-1081

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Cloning and Sequence Analysis of 1-Hydroxy-2-Methyl-2-E-Butenyl-4-Diphosphate Reductase Gene cDNA from Eucommia ulmoides

1. Non-timber Forestry Research and Development Center, Chinese Academy of Forestry, Zhengzhou 450003, He’nan, China

2. Key Laboratory of Non-wood Forest Products of State Forestry Administration, Changsha 410004, Hu’nan, China

Received Date: 2012-08-28

Abstract: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) synthesizes IPP and DMAPP in the last step of the plant 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Homologous HDR gene cDNA was isolated from the leaves of Eucommia ulmoides by the method of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique, and named as EuHDR. With the highest gene sequence similarity to Camptotheca acuminata (82%), the full-length cDNA of EuHDR was 1 653 bp including 5’non-coding region of 82 bp and 3’non-coding region of 188 bp and encoded 460 amino acids. The transit peptide sequence (A1-A33) and multiple conserved functional sites(A117, A208, A262, A345)of plant HDR protein were found in the deduced coding sequence of EuHDR. The secondary structure of EuHDR protein was predicted with proportion of α-helix to 35.65%,β-sheet to 19.78% and loop/coil to 44.57%. The calculated protein tertiary structure of EuHDR was formed as monomer, which in space displayed asymmetrical shamrock-like shape. Phylogenetic analysis revealed that the evolutionary relationship of EuHDR protein was the closest to Vitis vinifera HDR protein.

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[1]	Köksal M,Hu R M,Coates R M,et al. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase[J]. Nat Chem Biol, 2011,7: 431-433
[2]	Eric O, Lin F Y. Terpene biosynthesis: modularity rules[J]. Angew Chem,2012,51(5):1124-1137
[3]	Karine B, Yannick E, Alain D,et al. Isopentenyl diphosphate isomerase: a checkpoint to isoprenoid biosynthesis[J]. Biochimi,2012,doi:10.1016/j.biochi.2012.03.021:1-14.
[4]	Daum M, Herrmann S, Wilkinson B,et al.Genes and enzymesinvolved in bacterial isoprenoid biosynthesis[J]. Curr Opin Chem Biol,2009,13:180-188
[5]	Annegret A B, Karnjapan J, Juliusz A W,et al. Biosynthesis of isoprene units:mssobauer spectroscopy of substrate and inhibitor binding to the cluster of the LytB/IspH enzyme[J]. Angew Chem, 2011,50:11976-11979
[6]	Sang-Min K,Tomohisa K,Akio K,et al. 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS)is encoded by multicopy genes in Gymnosperms Ginkgo biloba and Pinus taeda[J]. Planta,2008,227:287-298
[7]	Cunningham Jr F X, Lafond T P, Gantt E. Evidence of a role for LytB in the nonmevalonate pathway of isoprenoid biosynthesis[J]. J Bacteriol,2000,182:5841-5848
[8]	Guevara-Garcia A,San Roman C,Arroyo A,et al. Characterization of the Arabidopsis clb6 mutant illustrates the importance of posttranscriptional regulation of the methyl-D-erythritol 4-phosphate pathway[J]. Plant Cell,2005,17: 628-643
[9]	Hsieh M H, Goodman H M. The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis[J]. Plant Physiol, 2005,138:641-653
[10]	张雯,王玉亮,林娟,等.银杏1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶基因(hdr)转化银杏的研究[J].复旦学报,2008,47(5):598-602
[11]	Botella-Pavía P, óscar B, Phillips M A,et al. Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors[J]. Plant J,2004, 40(2):188-199
[12]	崔克明.杜仲研究的历史、现状和展望[J].西北林学院学报,1994,9(4):51-57
[13]	杜红岩.杜仲优质高产栽培[M]. 北京:中国林业出版社, 1996
[14]	陈建.几种提取杜仲RNA方法的比较[J].林业科技开发,2007,21(5):19-21
[15]	周明兵,王红珍,赵德刚.杜仲叶和树皮总RNA的快速提取法[J].山地农业生物学报,2003,22(5):430-431
[16]	Jones D T. Protein secondary structure prediction based on position-specific scoring matrices [J]. J Mol Biol, 1999, 292(2):195-202
[17]	Emanuelsson O,Nielsen H, von Heijne G. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites[J].Protein Sci,1999,8: 978-984
[18]	Arnold K,Bordoli L, Kopp J,et al. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling[J]. Bioinformatics, 2006,22:195-201
[19]	Tamura K,Peterson D,Peterson N,et al. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Mol Biol Evol,2011,28: 2731-2739
[20]	高文运.同位素标记及未标记的1-脱氧-D-木酮糖-5-磷酸和2-甲基-D-赤藓糖醇-4-磷酸的合成方法[J].有机化学,2010,30 (1):23-37
[21]	金蓉,朱长青,徐昌杰.1-脱氧木酮糖-5-磷酸合成酶(DXS)及其编码基因[J].细胞生物学杂志,2007,29:706-712
[22]	郑洲翔,范燕萍,周纪刚,等. 植物萜类合成酶1-脱氧-D-木酮糖-5-磷酸还原酶研究进展[J]. 安徽农业科学, 2011,39(10):5695-5696
[23]	Tobias G, Ingrid S, Wolfgang E,et al. Probing the reaction mechanism of IspH protein by X-ray structure analysis[J].PNAS,2010,107(3):1077-1081

Cloning and Sequence Analysis of 1-Hydroxy-2-Methyl-2-E-Butenyl-4-Diphosphate Reductase Gene cDNA from Eucommia ulmoides

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通讯作者: 陈斌, bchen63@163.com

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Cloning and Sequence Analysis of 1-Hydroxy-2-Methyl-2-E-Butenyl-4-Diphosphate Reductase Gene cDNA from Eucommia ulmoides

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