欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2009, Vol. 35 ›› Issue (8): 1432-1438.doi: 10.3724/SP.J.1006.2009.01432

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转基因玉米转化体特异性寡核苷酸芯片测试方法的研制

路兴波1,武海斌1,2,王敏2,李宝笃2,杨崇良1,孙红炜1,*   

  1. 1山东省农业科学院植物保护研究所，山东济南250100；2青岛农业大学植物保护学院，山东青岛266109
  • 收稿日期:2008-12-08 修回日期:2009-03-20 出版日期:2009-08-12 网络出版日期:2009-06-10
  • 通讯作者: 孙红炜, E-mail: hongweisun@126.com
  • 基金资助:

    本研究由山东省农科院创新基金（200711）和山东省优秀中青年科学家科研奖励基金（2007BS06023）资助。

Developing a Method of Oligonucleotide Microarray for Event Specific Detection of Transgenic Maize（Zea mays）

LU Xing-Bo1,WU Hai-Bin12,WANG Min2,LI Bao-Du2,YANG Chong-Liang1,SUN Hong-Wei1*   

  1. 1Plant Protection Research Institute,Shandong Academy of Agricultural Sciences,Jinan 250100,China;2College of Plant Protection,Qingdao Agricultural University,Qingdao 266109,China
  • Received:2008-12-08 Revised:2009-03-20 Published:2009-08-12 Published online:2009-06-10
  • Contact: SUN Hong-Wei, E-mail: hongweisun@126.com

PDF

1835

被引次数

10

摘要：

根据7种转基因玉米Bt11、Bt176、Mon810、Mon863、TC1507、GA21和NK603的重组DNA结构，利用Primer premier 5.0引物设计软件分别设计转化体特异性引物，优化PCR反应条件，对引物特异性及灵敏度进行验证。结果表明，所设计引物特异性强，灵敏度达0.1%。在植物基因组与外源基因的连接区域，利用Primer premier 5.0和Oligo 6.0软件分别设计和筛选7种转基因玉米40mer oligo转化体特异性探针，制备转基因玉米的寡核苷酸芯片，并对芯片检测的特异性、重复性及检测灵敏度进行检验。证明所设计探针特异性强，灵敏度达0.01%。与PCR检测方法相比，基因芯片检测法灵敏度高、特异性强，可有效检测及鉴定多种转基因玉米，大大提高检测的准确率和效率。

关键词: 转基因玉米, 转化体特异性, 寡核苷酸芯片, Oligo探针, 灵敏度

Abstract:

With the spread of genetically modified organism (GMO), the food and environment securities related to GMO attract great attention to the public. Genetically modified maize (Zea mays L.) is one of the most popular GMO varieties, which accounts for 22.5% of total planting area of transgenic crops. Currently, more than 40 countries and regions govern GMO food products with a compulsory tagging policy. Thus, technique for testing transgenic plants is of great importance, such as screening, gene specific, construct specific, event specific detection method, which have become a mainstay of GMOs detection. Event specific microarray shows a prosperous application due to its advantages of its high specificity, high sensitivity, automation and high efficiency. In this study, on the base of integrated DNA constructs of seven genetically modified maize varieties, Bt11, Bt176, Mon810, Mon863, TC1507, GA21, and NK603, event specific primers were designed for polymerase chain reaction (PCR) using Primer premier 5.0, and the PCR system and program were optimized. The results indicated that the sensitivity of PCR detection was 0.1%. In addition, based on unique and specific integration junction sequences between the host plant genome DNA and the integrated gene, a 40mer event specific oligo probe with high specificity was designed for each variety using Primer premier 5.0 and Oligo 6.0, all the event specific probes were developed in one microarray. To ensure the reliability of microarray detection, we used different kinds of positive and negative controls in oligonucleotide microarray. The results indicate that the sensitivity of microarray detection (0.01%) and the event specific oligonucleotide probes developed in this study meet the requirements of sensitivity and specificity for detecting and identifying genetically modified maize, which enhances the testing accuracy and efficiency.

Key words: Transgenic maize, Event specific, Oligonucletide microarray, Oligo probe, Sensitivity

[1]James C. Global status of commercialized biotech: GM crop. ISAAA Brief, 2006, 35:192-193

[2] Vaïtilingom M, Pijnenburg H, Gendre F, Brignon P. Real-time quantitative PCR detection of genetically modified maize and roundup ready soybean in some representative foods. J Agric Food Chem, 1999, 5: 55-64

[3]Matsuoka T, Kuribara H, Akiyama H, Godu Y, Kusakabe Y, Isshiki K, Toyoda M, Hino A. A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize. J Food Hygiene Soc Jpn, 2001, 42: 24-32

[4] Zimmermann A, Lüthy J, Pauli U. Event specific transgene detection in BT11 corn by quantitative PCR at the integration site. 2000, 33: 210-216

[5]Xu X-D(许小丹), Wen S-Y(文思远), Wang S-Q(王升启), Huang K-L(黄昆仑), Luo Y-B(罗云波). Preparation of oligonucleotide microarray for detecting and identifying roundup ready soybean. J Agric Biotechnol (农业生物技术学报), 2005, 13(4): 429-434 (in Chinese with English abstract)

[6]Zhang H-Y(张海燕), Ma W-L(马文丽), Li L(李凌), Wu Q-H(吴清华), Xu Q-L(徐秋林), Wen Y(温颖), Zheng W-L(郑文岭). Study on oligonucleotide microarray for detecting encephalitis B virus. J Practical Med (实用医学志), 2005, 21(12): 1244-1247 (in Chinese with English abstract)

[7]Wang Y(王艳), Ma W-L(马文丽), Mao X-M(毛向明), Wu Q-H(吴清华), Li L(李凌), Wang H-M(王洪敏), Xiao W-W(肖维威), Zheng W-L(郑文岭). Design and preparation of oligonucleotide microarray for vaccinia virus detection. J First Military Med Univ (第一军医大学学报), 2004, 24(2): 180-183 (in Chinese with English abstract)

[8] Hernández M, Pla M, Esteve T, Prat S, Puigdomènech P, Ferrando A. A specific real-time quantitative PCR detection system for event MON810 in maize YieldGard based on the 3’-transgene integration sequence. Transgenic Res, 2003, 12: 179-189

[9] Yang L T, Xu S C, Pan A H, Yin C S, Zhang K W, Wang Z Y, Zhou Z G, Zhang D B. Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence. J Agric Food Chem, 2005, 53: 9312-9318

[10] Yang L T, Guo J C, Pan A H, Zhang H B, Zhang K W, Wang Z M, Zhang D B. Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule. J Agric Food Chem, 2007, 55: 15-24

[11] Kok E J, Aarts H J, Van Hoef A M, Kuiper H A. DNA methods: Critical review of innovative approaches. J AOAC Intl,2002, 85: 797-800

[12] Huang Y-C(黄迎春), Sun C-J(孙春昀), Feng H(冯红), Hu X-D(胡晓东), Yin H-B(尹海滨). Detection of transgenic crop with gene chip. Hereditas (遗传), 2003, 25(3): 307-310 (in Chinese with English abstract)

[13] Liu X(刘烜), Zheng W-J(郑文杰), Zhao W-D(赵卫东), He Y(贺艳), Tang D-Z(唐丹舟), Liu H(刘辉). Research of gene chip for detection of genetically modified soybean. Chin J Food Hygiene (中国食品卫生杂志), 2005, 17(2): 85-89 (in Chinese with English abstract)

[14] Call D R, Brockman F J, Chandler D P. Detecting and genotyping Escherichia coli 0157: H7 using multiplexed PCR and nucleic acid microarrays. Intl J Food Microbiol, 2001, 67: 71-80
[1] 单露英, 李俊, 李亮, 张丽, 王颢潜, 高佳琪, 吴刚, 武玉花, 张秀杰. 转基因玉米NK603基体标准物质研制[J]. 作物学报, 2022, 48(5): 1059-1070.
[2] 李俊,李亮,李夏莹,宋贵文,沈平,张丽,翟杉杉,柳方方,吴刚,张秀杰,武玉花. 转基因玉米MIR604基体标准物质研制[J]. 作物学报, 2020, 46(4): 473-483.
[3] 张广远,孙红炜,李凡,杨淑珂,路兴波,赵蕾. 转基因玉米MIR162转化事件特异性检测方法及其标准化[J]. 作物学报, 2013, 39(07): 1141-1147.
[4] 陈颖;徐宝梁;苏宁;葛毅强;王曙光. 实时荧光定量PCR技术在转基因玉米检测中的应用研究[J]. 作物学报, 2004, 30(06): 602-607.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2