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作物学报 ›› 2010, Vol. 36 ›› Issue (3): 365-375.doi: 10.3724/SP.J.1006.2010.00365

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

外源抗草膦EPSPs基因在大豆基因组中的整合与定位

王晓波1,蒋凌雪1,2,魏利1,刘林1,陆伟3,李文欣1,王俊1,陶波2,常汝镇1,邱丽娟1,*   

  1. 1中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程/农业部作物种质资源与生物技术重点开放实验室，北京100081；2东北农业大学，黑龙江哈尔滨，150030；3中国农业科学院生物技术研究所，北京100081
  • 收稿日期:2010-01-25 修回日期:2010-01-31 出版日期:2010-03-12 网络出版日期:2010-02-04
  • 通讯作者: 邱丽娟，E-mail: qiu_lijuan@263.net
  • 基金资助:

    本研究由中国农业科学院作物科学研究所农作物基因资源与基因改良国家重大科学工程开放课题资助。

Integration and Insertion Site of EPSPs Gene on the Soybean Genome in Genetically Modified Glyphosate-Resistant Soybean

WANG Xiao-Bo1,JIANG Ling-Xue1,WEI Li1,LIU Lin,LU Wei2,Li Wen-Xin1,WANG Jun1,CHANG Ru-Zhen1,QIU Li-Juan1*   

  1. 1 The National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Germplasm & Biotechnology / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 Northeast Agricultural University, Haribin 150030, China; 3 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2010-01-25 Revised:2010-01-31 Published:2010-03-12 Published online:2010-02-04
  • Contact: QIU Li-Jiu，E-mail: qiu_lijuan@263.net

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1619

被引次数

10

摘要：

通过基因特异引物扩增和免疫层析试纸法分别对EPSPs基因和其编码的蛋白进行检测。结果表明，EPSPs基因不仅已整合到大豆基因组中，而且EPSPs蛋白可以正常表达。利用染色体步移方法获得了转基因大豆插入位点的侧翼序列，序列比对表明35S上游的大豆DNA序列起始于Gm02:7912740，NOS下游的大豆DNA序列起始于Gm02:7777705。外源基因不是以点插入方式整合，而是导致大豆基因组约135 kb片段的移位和重排。基因组序列重排导致一个编码HEC1和HEAT repeat功能域的基因(Glyma02g09790)结构受到影响，该基因在ABA和PEG处理时下调表达。本研究发现外源基因的插入导致插入位点附近DNA序列发生重排，并鉴定出一个编码HEC1和HEAT repeat功能域的基因可能会在ABA信号通路中参与干旱胁迫应答。本研究通过对抗除草剂EPSPs基因在大豆基因组中的插入位点分析，明确了外源EPSPs基因在大豆基因组中的整合、定位及其侧翼序列，为转基因大豆安全评价提供了依据。

关键词: 转基因大豆, 插入位点, 基因组, EPSPs, 抗草甘膦, 定位, 侧翼序列

Abstract:

The detection of genetically modified crops (GMCs) is becoming both food labels and legal necessity. The aim of this study was to identify integration and insertion locus of foreign EPSPs gene in genetically modified soybean for the safety assessment of genetically modified crop. The EPSPs gene fragment was detected using gene specific primes and immunochromatographic strip was used to detect the EPSPs protein. The result showed that the EPSPs gene was integrated into soybean genome and EPSPs protein could be expressed normally. Genome walking was used to analyze the flanking sequences of both 35S promoter and NOS terminator, and soybean genome database (Phytozome) was used to analyze the insertion site and study the effect of the insertion on soybean genome. The Dra I and EcoR V restriction enzyme were found that they could be used to digest the soybean genome completely, and the library with adaptor was established and nest PCR was used to amplify the flanking sequences of 35S and NOS genes in genetically modified soybean genome. The result showed that the start site of flanking region of either 35S promoter or NOS terminator was Gm02:7912740 or Gm02:7777705, respectively, which means the foreign gene may not insert into a fixed position, and one 135 kb DNA fragment may be translocated. Two unknown sequences and two soybean DNA fragments with physical distance of 24 kb in opposite directions were found at the flanking region of NOS terminator. We also found that there were high AT content (about 70%) and low gene density in 90kb flanking regions of the insertion locus, and one gene coding HEC1 and HEAT repeat domain (Glyma02g09790) was found to be rearranged. RT-PCR and qRT-PCR showed that the gene was down-regulated during PEG and ABA treatment. In this study, we found the genetically engineered soybean genome was rearranged because of the insertion of foreign genes and one gene coding HEC1 and HEAT repeat domain (Glyma02g09790) which may response to drought stress through ABA signal pathway was identified for the first time.

Key words: Genetically modified soybean, Insertion locus, Genome, EPSPs, Glyphosate-Resistance, Location, Flanking sequence

[1] ISAAA .

[2009-07-15] http://www.isaaa.org/resources/publications/briefs/37/pressrelease/default.html. 2008

[2] Hardegger M, Brodmann P, Herrmann A. Quantitative detection of the 35S promoter and the NOS terminator using quantitative competitive PCR. Eur Food Res Technol, 1999, 209: 83−87

[3] Hübner P, Studer E, Lüthy J. Quantitative competitive PCR for the detection of genetically modified organisms in food. Food Control, 1999, 10: 353−358

[4] Studer E, Rhyner C, Lüthy J, Hübner P. Quantitative competitive PCR for the detection of genetically modified soybean and maize. Z Lebensm Unters Forsch, 1998, 207: 207−213

[5] Zheng W-J(郑文杰), Liu X(刘烜), Liu W(刘伟), Tang D-Z(唐丹舟), He L(何霖), Zhang H-W(张宏伟). Qualitative analysis of the processed genetically modified soybean products by PCR-based methods. J Agric Biotech (农业生物技术学报), 2003, 11 (5): 467−471 (in Chinese with English abstract)

[6] Hernández M, Esteve T, Prat S, Pla M. Development of Real-Time PCR Systems based on SYBR Green I, Amplifluor and TaqMan technologies for specific quantitative detection of the transgenic maize event GA21. J Cereal Sci, 2004, 39: 99−107

[7] Chaouachi M, El Malki R, Berard A, Romaniuk M, Laval V, Brunel D, Bertheau Y. Development of a real-time PCR method for the differential detection and quantification of four solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum). J Agric Food Chem, 2008, 56: 1818−1828

[9] Brunnert H-J, Spener F, Börchers T. PCR-ELISA for the CaMV-35S promoter as a screening method for genetically modified roundup ready soybeans. Eur Food Res Technol, 2001, 213: 366−371

[10] Wang Y(王永), Lan Q-K(兰青阔), Zhao X(赵新), Zhu Z(朱珠), Cheng Y(程奕). Development and application of loop-mediated isothermal amplification for detection of genetically modified crops. Sci Agric Sin (中国农业科学), 2009, 42(4): 1473−1477 (in Chinese with English abstract)

[11] Service of Biosafety and Biotechnology, Scientific Institute of Public Health Belgium. Biosafety in Europe, 2000. SBB.

[2009-07-15] http://biosafety.ihe.be/Menu/BiosEur.html

[12] APHIS. Agricultural biotechnology: Permitting, notification, and deregulation. Animal and Plant Health Inspection Service (APHIS). 2000,

[2009-07-15] http://www.aphis.usda.gov/biotechnology/permits.html

[13] Haslberger A G. Monitoring and labeling for genetically modified. Science, 2000, 287:431−432

[14] Liu B(刘博), Su Q(苏乔), Tang M-Q(汤敏谦), Yuan X-D(袁晓东), An L-J(安利佳). Progress of the PCR amplification techniques for chromosome walking. Hereditas, 2006, 28(5): 587−595 (in Chinese with English abstract)

[15] Wang C-L(王春连), Chen L-T(陈乐天), Zeng C-Z(曾超珍), Zhang Q-Y(张群宇), Liu P-Q(刘丕庆), Liu Y-G(刘耀光), Fan Y-L(樊颖伦), Zhang Q(章琦), Zhao K-J(赵开军). Chromosome walking for fine mapping of Xa23 gene locus by using genomic libraries. Chin J Rice Sci (中国水稻科学), 2006, 20(4): 355−360 (in Chinese with English abstract)

[16] Yang C-Y(杨存义), Chen Z-Z(陈忠正), Zhuang C-X(庄楚雄), Mei M-T(梅曼彤), Liu Y-G(刘耀光). Genetic and physical fine-mapping of the Sc locus conferring indica-japonica hybrid sterility in rice (Oryza sativa L.). Chin Sci Bull, 2004, 49(16): 1718−1721 (in Chinese with English abstract)

[17] Lü S-H(吕山花), Chang R-Z(常汝镇), Tao B(陶波), Li X-H(李向华), Luan F-X(栾凤侠), Guo S-H(郭珊花), Qiu L-J(邱丽娟). Methodological research on PCR based detection of genetically modified soybean resistant to glyphosate. Sci Agric Sin (中国农业科学), 2003, 36(8): 883−887 (in Chinese with English abstract)

[18] Wu A-M(吴蔼民), Liu J-Y(刘进元). An improved method of chromosome walking for promoter sequences cloning. Chin J Biochem Mol Biol (中国生物化学与分子生物学报), 2006, 22(3): 243−246 (in Chinese with English abstract)

[19] Forsbach A, Schubert D, Lechtenberg B, Gils M, Schmidt R. A comprehensive characterization of single-copy TDNA insertions in the Arabidopsis thaliana genome. Plant Mol Biol, 2003, 52: 161−176

[20] Fladung M. Gene stability in transgenic aspen (Populus): Flanking DNA sequences and T-DNA structure. Mol Gen Genet, 1999, 260: 574−581

[21] Kumar S, Fladung M., Transgene repeats in aspen: molecular characterization suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome. Mol Gen Genet, 2000, 264: 20−28

[22] Zuo K-J(左开井), Zhang X-L(张献龙), Nie Y-C(聂以春), Liu J-L(刘金兰), Sun J-Z(孙济中). Sequence analysis of Bt insertion flanking fragments in transgenic Bt cotton. Acta Genet Sin (遗传学报), 2002, 29(8): 735−740 (in Chinese with English abstract)

[23] Muller A E, Kamisugi Y, Gruneberg R, Niedenhof I, Horold R J, Meyer P. Palindromic sequences and A+T-rich DNA elements promote illegitimate recombination in Nicotiana tabaeum. J Mol Biol, 1999, 291: 29−46

[24] Schmidt M, Haas W, Crosas B, Santamaria P G, Gygi S P, Walz T, Finley D. The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle. Nat Struct Mol Biol, 2005, 12: 294−303
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