植物学报 ›› 2015, Vol. 50 ›› Issue (3): 295-309.doi: 10.3724/SP.J.1259.2015.00295

• 研究报告 • 上一篇    下一篇

植物莽草酸途径EPSPS蛋白的分子进化和基因结构分析

巩元勇1, 郭书巧1, 束红梅1, 倪万潮1*, *, 帕尔哈提·买买提2, 沈新莲1, 徐鹏1, 张香桂1, 郭琪1   

  1. 1江苏省农业科学院经济作物研究所/农业部长江下游棉花和油菜重点实验室, 南京 210014;
    2.国家棉花工程技术研究中心, 乌鲁木齐 830091
  • 收稿日期:2014-05-05 修回日期:2014-08-28 出版日期:2015-05-01 发布日期:2015-04-08
  • 通讯作者: nwchao2002@aliyun.com
  • 基金资助:
    国家自然科学基金(No.31301682)、江苏省农业科技自主创新基金(No; CX(12)3068)、国家转基因重大专项(No.2011- ZX08005-001)和国家科技支撑计划(No.2014BAD11B02)

Analysis of Molecular Evolution and Gene Structure of EPSPS Protein in Plant Shikimate Pathway

Yuanyong Gong1, Shuqiao Guo1, Hongmei Shu1, Wanchao Ni1, *, Paerhati·Maimaiti2, Xinlian Shen1, Peng Xu1, Xianggui Zhang1, Qi Guo1   

  1. 1Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Cotton and Rapeseed in the Lower Reaches of Yangtze River, Ministry of Agriculture, Nanjing 210014, China;
    2.National Engineering Techanical Research Center of Cotton, Urumqi 830091, China
  • Received:2014-05-05 Revised:2014-08-28 Online:2015-05-01 Published:2015-04-08

摘要: EPSPS既是植物、微生物和真菌等生物芳香族氨基酸生物合成途径——莽草酸途径中的关键酶, 也是除草剂草甘膦的靶标酶。EPSPS的克隆能为草甘膦抗性转基因作物的研发提供候选基因。该研究运用比较基因组学方法, 通过对41种不同植物的43条EPSPS蛋白序列进行进化分析, 取得主要结果如下: (1) 不同植物EPSPS蛋白的相似性很高, 且具有相同的结构域、保守基序和保守位点, 但是其叶绿体转运肽序列差异显著; (2) 系统发育分析表明, EPSPS基因按照双子叶植物纲和单子叶植物纲分为2个大的分支, 各个小的分支又按照植物的种属亲缘关系进行分支和聚类; (3) 基因结构分析表明, 植物EPSPS基因基本都含有8个外显子和7个内含子, 且所对应外显子的长度相当, 而内含子的长度差异很大, 说明在植物基因组进化过程中造成EPSPS基因结构差异的主要因素是内含子的改变。研究结果将为揭示植物EPSPS蛋白的结构功能提供参考。

Abstract: 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme with a role in the shikimate pathway of aromatic amino acid biosynthesis in nearly all plants, bacteria, and fungi, is a target enzyme of the herbicide glyphosate [N-(phosphonomethyl) glycine]. The cloning of EPSPS genes can provide candidate gene for the development of glyphosate resistant transgenic crops. Comparative genomic analysis was used to investigate the evolutionary relationship among 43 EPSPS genes in 41 different plant species. The main results are as follows. (1) Various plant EPSPS protein sequences have the same functional domain, conserved motifs and conserved site. However, chloroplast transit peptide sequences significantly differ. (2) Phylogenetic analyses suggested that EPSPS genes correspond to two main branches according to dicotyledoneae and monocotyledoneae, and each small branch was classified based on species kinship of plants. (3) Exon-intron structure analysis revealed that the gene structures of EPSPS basically contained 8 exons and 7 introns, with only a marginal difference in corresponding exons but marked differences in all introns. The change in intron length was the main contributor to the structural diversity of plant EPSPS genes. This study provides valuable information for research into EPSPS proteins in plants.

中图分类号: 

  • 10.3724/SP.J.1259.2015.00295
1 程华, 李琳玲, 王燕, 姜德志, 程水源 (2010). 银杏EPSPS基因克隆及表达分析. 西北植物学报 30, 2365-2372.
2 巩元勇, 郭书巧, 束红梅, 何林池, 倪万潮 (2014). 1株抗草甘膦棉花突变体草甘膦抗性的初步鉴定. 棉花学报 26, 18- 24.
3 郭安源, 朱其慧, 陈新, 罗静初 (2007). GSDS: 基因结构显示系统. 遗传 29, 1023-1026.
4 李雅超, 安泽伟, 谢黎黎, 翟琪麟, 黄华孙 (2013). 橡胶树5-烯醇式丙酮莽草酸-3-磷酸合成酶基因的克隆及其响应非生物胁迫的表达分析. 热带作物学报 34, 807-814.
5 刘东军, 张锐, 郭三堆, 孟志刚, 孙国清, 王成社, 张宏纪 (2008). 棉花品系Y18在草甘膦胁迫下的 EPSPS 基因表达分析研究. 中国生物工程杂志 28, 55-59.
6 路安民, 汤彦承 (2005). 被子植物起源研究中几种观点的思考. 植物分类学报 43, 420-430.
7 童旭宏, 吴玉香, 祝水金 (2009). 陆地棉 EPSPS 基因的克隆及其组织特异性表达分析. 棉花学报 21, 259-264.
8 赵海铭, 宋伟彬, 赖锦盛 (2013). 高粱5-烯醇式丙酮酰莽草酸- 3-磷酸合酶基因( EPSPS )叶绿体转运肽(CTP)的克隆及其在转基因玉米中的功能验证. 农业生物技术学报 21, 1009- 1018.
9 Baerson SR, Rodriguez DJ, Tran M, Feng YM, Best NA, Dill GM (2002). Glyphosate-resistant goosegrass: identification of a mutation in the target enzyme 5-enolpyru- vylshikimate-3-phosphate synthase. Plant Physiol 129, 1265-1275.
10 Bailey TL, Williams N, Misleh C, Li WW (2006). MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res 34, W369-W373.
11 Della-Cioppa G, Bauer SC, Klein BK, Shah DM, Fraley RT, Kishore GM (1986). Translocation of the precursor of 5-enolpyruvylshikimate-3-phosphate synthase into chloro- plasts of higher plants in vitro . Proc Natl Acad Sci USA 83, 6873-6877.
12 Dill GM (2005). Glyphosate-resistant crops: history, status and future. Pest Manag Sci 61, 219-224.
13 Duncan K, Lewendon A, Coggins JR (1984). Mutant EPSP synthase genes from tomato, Arabidopsis thaliana , Brassica napus , Glycine max , E. coli K-12 confer tolerance to glyphosate. FEBS Lett 170, 59-63.
14 Emanuelsson O, Nielsen H, Heijne GV (1999). ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8, 978-984.
15 Fillatti JJ, Kiser J, Rose R, Comai L (1987). Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Biol Technol 5, 726-730.
16 Gong Y, Liao Z, Chen M, Guo B, Jin H, Sun X, Tang K (2006). Characterization of 5-enolpyruvylshikimate 3-pho- sphate synthase gene from Camptotheca acuminata . Biologia Plantarum 50, 542-550.
17 Green JM (2009). Evolution of glyphosate-resistant crop technology. Weed Sci 57, 108-117.
18 Gruys KJ, Walker MC, Sikorski JA (1992). Substrate synergism and the steady-state kinetic reaction mechanism for EPSP synthase from Escherichia coli . Biochemistry 31, 5534-5544.
19 Herrmann KM, Weaver LM (1999). The shikimate pathway. Annu Rev Plant Physiol Plant Mol BioI 50, 473-503.
20 Jones P, Binns D, Chang HY, Fraser M, Li WZ, McAnulla C, McWilliam H, Maslen J, Mitchell A, Nuka G, Pesseat S, Quinn AF, Sangrador-Vegas A, Scheremetjew M, Yong SY, Lopez R, Hunter S (2014). InterProScan 5: genome-scale protein function classification. Bioinformatics 30, 1236-1240.
21 Keeling PJ, Palmer JD, Donal RGK, Roost DS, Waller RF, McFadden GI (1999). Shikimate pathway in Apicomp- lexan parasites . Nature 397, 219-220.
22 Klee HJ, Muskopf YM, Gasser CS (1987). Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshi- kimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants. Mol Gen Genet 210, 437-442.
23 Macheroux P, Schmid J, Amrhein N, Schaller A (1999). A unique reaction in a common pathway: mechanism and function of chorismate synthase in the shikimate pathway. Planta 207, 325-334.
24 McDowell LM, Poliks B, Studelska DR, O’Connor RD, Beusen DD, Schaefer J (2004). Rotational-echo double-resonance NMR-restrained model of the ternary com- plex of 5-enolpyruvylshikimate-3-phosphate synthase. J Biomol NMR 28, 11-29.
25 Nicholas KB, Nicholas HB Jr, Deerfield II (1997). GeneDoc: a tool for editing and annotating multiple sequence alignments. Embnew News 4, 1-4.
26 Rogers SG, Brand LA, Holder SB, Sharps ES, Brackin MJ (1983). Amplification of the aroA gene from Escherichia coli results in tolerance to the herbicide glyphosate. Appl Environ Microbiol 46, 37-43.
27 Saitou N, Nei M (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.
28 Schönbrunn E, Eschenburg S, Shuttleworth WA, Schloss JV, Amrhein N, Evans JNS, Kabsch W (2001). Interaction of the herbicide glyphosate with its target enzyme 5-enol- pyruvylshikimate-3-phosphate synthase in atomic detail. Proc Natl Acad Sci USA 98, 1376-1380.
29 Smart CC, Amrhein N (1987). Ultrastructural localisation by protein A-gold immunocytochemistry of 5-enolpyruvylshi- kimic acid-3-phosphate synthase in a plant cell culture which overproduces the enzyme. Planta 170, 1-6.
30 Stalker DM, Hiatt WR, Comai L (1985). A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3- phosphate synthase confers resistance to the herbicide glyphosate. J Biol Chem 260, 4724-4728.
31 Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596-1599.
32 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876- 4882.
33 Wang YX, Jones JD, Weller SC, Goldsbrough PB (1991). Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glypho- satetolerant tobacco cells. Plant Mol Biol 17, 1127-1138.
34 Xu JW, Wei XL, Li XG, Chen L, Feng DJ, Zhu Z (2002). Isolation of rice EPSP synthase cDNA and its sequence analysis and copy number determination. Acta Bot Sin 44, 188-192.
No related articles found!
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
[1] 高翠;陈玉霞;包颖;冯旻;路安民. 白穗花有性器官与胚胎发育形态的研究 [J]. 植物学报, 2010, 45(06): 705 -712 .
[2] 蒋高明. 全球大气二氧化碳浓度升高对植物的影响[J]. 植物学报, 1995, 12(04): 1 -7 .
[3] 张军 韩碧文. 植物激素细胞和组织化学定位的研究进展[J]. 植物学报, 1995, 12(专辑3): 131 -142 .
[4] 汤彦承. 国际植物命名法规简介(V)[J]. 植物学报, 1984, 2(04): 51 -57 .
[5] 徐继. 保护固氮酶免受氧破坏的蛋白Fe—S蛋白Ⅱ[J]. 植物学报, 1986, 4(12): 1 -4 .
[6] . 国外大刊重要论文简介[J]. 植物学报, 2001, 18(05): 633 .
[7] 黄兆祥 郑珍贵 朱笃. 池杉—水稻系统的生态效应(1)系统中池杉的生长特性[J]. 植物学报, 1996, 13(02): 48 -51 .
[8] 谷瑞升 刘群录 陈雪梅 蒋湘宁. 木本植物蛋白提取和SDS-PACE分析方法的比较和优化[J]. 植物学报, 1999, 16(02): 171 -177 .
[9] 蒋高明. LI-6400光合作用测定系统:原理、性能、基本操作与常见故障的排除[J]. 植物学报, 1996, 13(增刊): 72 -76 .
[10] 李玲 罗蕴秀 何建辉 潘瑞炽. GL生根剂促进木本植物插枝生根[J]. 植物学报, 1996, 13(增刊): 63 -65 .