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作物学报 ›› 2010, Vol. 36 ›› Issue (2): 361-364.doi: 10.3724/SP.J.1006.2010.00361

• 研究简报 • 上一篇    

转基因玉米MON88017旁侧序列分析及定性PCR检测

袁磊1,2，孙红炜1，赵蕾2，杨崇良1，尚佑芬1，路兴波1,*   

  1. 1山东省农业科学院植物保护研究所，山东济南250100；2山东师范大学生命科学学院，山东济南250014
  • 收稿日期:2009-07-06 修回日期:2009-09-06 出版日期:2010-02-10 网络出版日期:2009-11-17
  • 通讯作者: 路兴波, E-mail: luxb99@sina.com, Tel: 0531-83179095
  • 基金资助:

    本研究由国家转基因重大专项(2008ZX08012-001) 和山东省农业科学院博士科研启动基金资助。

Analysis of Junction Sequence in the Transgenic Maize MON8817 and the Methods of Qualitative PCR Detction

YUAN Lei1,2,SUN Hong-Wei1,ZHAO Lei2,YANG Chong-Liang1,SHANG You-Fen1,LU Xing-Bo1*   

  1. 1Institute of Plant Protection，Shandong Academy of Agricultural Sciences，Jinan 250100，China;2College of Life Science,Shandong Normal University,Jinan 250014,China
  • Received:2009-07-06 Revised:2009-09-06 Published:2010-02-10 Published online:2009-11-17
  • Contact: LU Xing-Bao, E-mail: luxb99@sina.com, Tel: 0531-83179095

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2076

被引次数

39

摘要：

采用基因组步移法和巢式PCR方法研究转基因玉米MON 88017外源基因插入位点旁侧序列特征，获得了外源基因插入位点的左边界旁侧序列504 bp，包括336 bp的插入载体序列和168 bp的玉米基因组序列。根据此旁侧序列，设计MON 88017转化事件特异性引物并进行定性PCR扩增，扩增片段为446 bp，建立了转基因玉米MON 88017转化体特异性检测方法。该方法特异性强、灵敏度高(0.1%)，为转基因玉米品种MON 88017进出口检测和标识检测提供了技术基础。

关键词: 转基因生物, 事件特异性检测, 定性PCR, MON88017转化事件, 右边界旁侧序列

Abstract:

The experiment was conducted to investigate the integration site of transgene in maize MON88017 and to establish event specific methods for qualitative detection of MON88017 based on the left border junction fragment, which was isolated with the amended GenomeWalker and Nested-PCR methods. Sequence alignment between the T-DNA sequence and isolated junction fragments showed a 504 bp junction fragment of MON88017 including 336 bp of T-DNA sequence and 168 bp of MON88017 genome DNA. MON88017 event-specific qualitative PCR method was established with the primers (MON88017-1F/R) targeting the junction regions to produce a 446 bp product. The limit of detection for qualitative PCR assay was 0.1%. The method developed in this work is highly specific, sensitive and suitable for MON88017 sample detection.

Key words: GMO, Event-specific detection, Qualitative PCR, MON88017 event, Left border junction fragments

[1] Jin W-J(金芜军), Hao Y(郝旸), Cheng H-M(程红梅), Lu X-B(路兴波), Peng Y-F(彭玉发), Jia S-R(贾士荣). Specific detection of Alien DNA sequences in six transgenic maize by using Multiplex PCR method. J Agric Biotechnol (农业生物技术学报), 2005, 13 (5): 562-567 (in Chinese with English abstract)
[2] Knut J H. Genetically Engineered Food: Methods and Detection. Weinheim: Wiley-VCH, 2003. pp 28-40
[3] Jin W-J(金芜军), Jia S-R(贾士荣), Peng Y-F(彭于发). Comparison of labeling policy of genetically modified products in different countries and territories. J Agric Biotechnol (农业生物技术学报), 2004, 12 (1): 1-7 (in Chinese with English abstract)
[4] Gachet E, Martin G G, Vigneau F, Meyer G. Detection of genetically modified organism (GMOs) by PCR: a brief review of methodologies available. Trends in Food Science and Technology, 1999, 9: 380-388
[5] Forte V T, Di Pinto A, Martino C, Tantillo G M, Grasso G, Schena F P. A general multiplex-PCR assay for the general detection of genetically modified sya and maize. Food Control, 2005, 16: 535-539
[6] Tavemiers I, Windels P, Vaitilingom M, Milcamps A, Van Bockstaele E, Van den Eede G, De Loose M. Event-specific plasmid standards and real-time PCR methods for transgenic Bt11, Bt176, and GA21 maize and transgenic GT73 canola. J Agric Food Chem, 2005, 53: 3041-3052
[7] Xu M-J(徐茂军). Detection of transgenic foods. Food and Fermentation Industries (食品与发酵工业), 2002, 27(12): 69-71 (in Chinese with English abstract)
[8] Zimmermann A, Luthy J, Pauli U. Event specific transgene detection in Bt11 corn by quantitative PCR at the integration site. Lebensm-Wissu-Technol, 2000, 33: 210-216
[9] Lee S H, Min D M, Kim J K. Qualitative and quantitative polymerase chain reaction analysis for genetically modified maize MON863. J Agric Food Chem, 2006, 54: 1124-1129
[10] Yang L, Xu S, Pan A, Yin C, Zhang K, Wang Z, Zhou Z, Zhang D. Event-specific qua1itative and quantitative PCR detection of genetically modified MON863 maize based on the 5'-transgene integration sequence. J Agric Food Chem, 2005, 53: 9312-9318
[11] Nielsen C R, Berdal K G, Holst-Jensen A. Characterisation of the 5'-integration site and development of an event-specific real-time PCR assay for NK603 maize from a low starting copy number. Eur Food Res Technol, 2004, 219: 421-427
[12] Food Safety Commission of Japan. Maize Line MON88017 Tolerant to the Herbicide Glyphosate and Resistant to Coleopteran Pests. Safety Assessment of Genetically Modified Food, 2005. pp 7-10
[13] Jiang G-Z(姜国忠), Xie H(谢华), Guo Y-Z(郭玉忠), Fan T-L(范天黎), Xue L-X(薛乐勋). The comparison between two techniques in isolating 3' flanking region of Dunaliella salina actin gene. J Zhengzhou Univ (Med Sci)(郑州大学学报·医学版), 2004, 39(1): 41-44 (in Chinese with English abstract)
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