药学学报, 2014, 49(3): 411-418
引用本文:
张琳, 王继涛, 张大为, 张岗, 郭顺星. 珍稀濒危药用铁皮石斛HMGR基因的克隆和特征分析[J]. 药学学报, 2014, 49(3): 411-418.
ZHANG Lin, WANG Ji-tao, ZHANG Da-wei, ZHANG Gang, GUO Shun-xing. Molecular characterization of a HMG-CoA reductase gene from a rare and endangered medicinal plant, Dendrobium officinale[J]. Acta Pharmaceutica Sinica, 2014, 49(3): 411-418.

珍稀濒危药用铁皮石斛HMGR基因的克隆和特征分析
张琳1, 王继涛1, 张大为2, 张岗1,2, 郭顺星2
1. 陕西中医学院药学院, 陕西省中药基础与新药研究重点实验室, 陕西 西安 712046;
2. 中国医学科学院、北京协和医学院药用植物研究所, 北京 100193
摘要:
3-羟基-3-甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme-A reductase,HMGR)是甲羟戊酸途径中的第一个限速酶,在植物细胞萜类物质合成代谢过程中发挥重要作用。但是,HMGR基因在珍稀濒危兰科药用植物铁皮石斛类萜生物合成途径中的作用尚不清楚。本研究利用RT-PCR和RACE技术,首次从铁皮石斛中克隆到一个HMGR基因,命名为DoHMGR1(GenBank注册号JX272632)。DoHMGR1基因cDNA全长2 071 bp,编码一条由562个氨基酸组成的多肽,分子量59.73 kD,等电点6.18。DoHMGR1蛋白包含HMGR蛋白家族的4个保守结构域(63~561、147~551、268~383、124~541)和两个跨膜基序(42~64、85~107)。DoHMGR1基因与多种植物HMGR基因相似性很高(67%~89%),与万代兰、水稻、玉米等单子叶植物亲缘关系较近。DoHMGR1基因具有组织表达特异性,其转录本在石斛根和茎中表达量较高,为叶中的2.13和1.98倍。DoHMGR1基因的分子鉴定为进一步解析该基因在铁皮石斛萜类物质合成代谢途径中的分子调控作用奠定基础。
关键词:    铁皮石斛      3-羟基-3-甲基戊二酰辅酶A还原酶      基因表达      实时定量PCR     
Molecular characterization of a HMG-CoA reductase gene from a rare and endangered medicinal plant, Dendrobium officinale
ZHANG Lin1, WANG Ji-tao1, ZHANG Da-wei2, ZHANG Gang1,2, GUO Shun-xing2
1. Shaanxi Provincial Key Laboratory for Chinese Medicine Basis & New Drugs Research, College of Pharmacy, Shaanxi University of Chinese Medicine, Xi'an 712046, China;
2. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
Abstract:
The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate in mavalonic acid pathway, which is the first committed step for isoprenoid biosynthesis in plants. However, it still remains unclear whether HGMR gene plays a role in the isoprenoid biosynthesis in Dendrobium officinale, an endangered epiphytic orchid species. In the present study, a HMGR encoding gene, designed as DoHMGR1 (GenBank accession JX272632), was identified from D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoHMGR1 was 2 071 bp in length and encoded a 562-aa protein with a molecular weight of 59.73 kD and an isoelectric point (pI) of 6.18. The deduced DoHMGR1 protein, like other HMGR proteins, constituted four conserved domains (63-561, 147-551, 268-383 and 124-541) and two transmembrane motifs (42-64 and 85-107). Multiple sequence alignment and phylogenetic analyses demonstrated that DoHMGR1 had high identity (67%-89%) to a number of HMGR genes from various plants and was closely related to Vanda hybrid cultivar, rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoHMGR1 was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.13 fold over that in the leaves, followed by that in the stems with 1.98 fold. Molecular characterization of DoHMGR1 will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. officinale.
Key words:    Dendrobium officinale    3-hydroxy-3-methylglutaryl-CoA reductase    gene expression    real time quantitative PCR   
收稿日期: 2013-10-28
基金项目: 国家自然科学基金资助项目(31070300,31101608);教育部科学技术研究重点项目(211177);陕西省科技计划青年科技新星项目(2012KJXX-44).
通讯作者: 张岗 Tel/Fax:86-29-38185165,E-mail:zhanggang2006@gmail.com;郭顺星 Tel/Fax:86-10-62829619,E-mail:sxguo1986@163.com
Email: zhanggang2006@gmail.com;sxguo1986@163.com
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参考文献:
[1] Laule O, Fürholz A, Chang HS, et al. Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana [J]. Proc Natl Acad Sci USA, 2003, 100: 6866-6871.
[2] Learned RM, Fink GR. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis thaliana is structurally distinct from the yeast and animal enzymes [J]. Proc Natl Acad Sci USA, 1989, 86: 2779-2783.
[3] Kato-Emori S, Higashi K, Hosoya K, et al. Cloning and characterization of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in melon (Cucumis melo L. reticulatus) [J]. Mol Genet Genomics, 2001, 265: 135-142.
[4] Ha SH, Kim JB, Hwang YS, et al. Molecular characterization of three 3-hydroxy-3-methylglutaryl-CoA reductase genes including pathogen-induced Hmg2 from pepper (Capsicum annuum) [J]. Biochim Biophys Acta, 2003, 1625: 253-260.
[5] Wang YC, Guo BH, Zhang F, et al. Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3- methylglutaryl coenzyme A reductase from hazel (Corylus avellana L. Gasaway) [J]. Biochem Mol Biol, 2007, 40: 861-869.
[6] Sando T, Takaoka C, Mukai Y, et al. Cloning and characterization of mevalonate pathway genes in a natural rubber producing plant, Hevea brasiliensis [J]. Biosci Biotechnol Biochem, 2008, 72: 2049-2060.
[7] Zheng ZJ, Cao XY, Liu Q, et al. Molecular cloning and function analysis of HMGR gene from Tilia miqueliana Maxim [J]. Bull Bot Res (植物研究), 2012, 32: 198-203.
[8] Maldenado-Mendoza IE, Burnett RJ, Nessler CL. Nucleotide sequence of a cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Catharanthus roseus [J]. Plant Physiol, 1992, 100: 1613-1614.
[9] Liao ZH, Tan QM, Chai YR, et al. Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast [J]. Funct Plant Biol, 2004, 31: 73-81.
[10] Jiang JH, Kai GY, Cao XY, et al. Molecular cloning of a HMG-CoA reductase gene from Eucommia ulmoides oliver [J]. Biosci Rep, 2006, 26: 171-181.
[11] Shen GA, Pant YZ, Wu WS, et al. Cloning and characterization of a root-specific expressing gene encoding 3-hydroxy-3- methylglutaryl coenzyme A reductase from Ginkgo biloba [J]. Mol Biol Rep, 2006, 33: 117-127.
[12] Cao XY, Zong ZM, Ju XY, et al. Molecular cloning, characterization and function analysis of the gene encoding HMG-CoA reductase from Euphorbia pekinensis Rupr [J]. Mol Biol Rep, 2010, 37: 1559-1567.
[13] Liao P, Zhou W, Zhang L, et al. Molecular cloning, characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from Salvia miltiorrhiza [J]. Acta Physiol Plant, 2009, 31: 565-572.
[14] Dai ZB, Cui GH, Zhou SF, et al. Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation [J]. J Plant Physiol, 2011, 168: 148-157.
[15] Liu Y, Xu QX, Xi PY, et al. Cloning and characterization of a cDNA coding 3-hydroxy-3-methylglutary CoA reductase involved in glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis [J]. Acta Pharm Sin (药学学报), 2013, 48: 773- 779.
[16] Luo HM, Song JY, Li XY, et al. Cloning and expression analysis of a key device of HMGR gene involved in ginsenoside biosynthesis of Panax ginseng via synthetic biology approach [J]. Acta Pharm Sin (药学学报), 2013, 48: 219-227.
[17] Zhang G, Zhang DW, Zhao MM, et al. Progress in molecular biology research of Dendrobium officinale [J]. Chin Pharm J (中国药学杂志), 2012, 48: 1614-1619.
[18] Li L, Deng XL, Zhao XB, et al. Advances in studies on chemical constituents in Dendrobium candidum and their pharmacological effects [J]. Anti-Tumor Pharm (肿瘤药学), 2011, 1: 90-94.
[19] Onaka T, Kameta S, Maeda T, et al. The structure of dendrobine [J]. Chem Pharm Bull, 1964, 12: 506-512.
[20] Zhang G, Song C, Zhao MM, et al. Characterization of an A-type cyclin-dependent kinase gene from Dendrobium candidum [J]. Biologia, 2012, 67: 360-368.
[21] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR [J]. Nucleic Acids Res, 2001, 29: e45.
[22] Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs [J]. Nucleic Acids Res, 1987, 15: 8125-8148.
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