转基因耐除草剂大豆ZH10-6多重PCR检测方法的建立

doi:10.3969/j.issn.1674-7968.2021.08.021

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摘要转基因耐除草剂大豆(Glycine max) ZH10-6是由中国农业科学院作物科学研究所研究的转草甘膦降解基因(glyphosate acetyltransferase, GAT)和草甘膦抗性基因(pseudomonas fluorescens G2 strain 5-enolpyruvyl shikimate-3-phosphate synthase, G2-EPSPS)的耐除草剂转基因大豆新品系。为建立其分子特征转化体特异性的检测方法,本研究以插入基因GAT、G2-EPSPS和大豆基因组的边界位点为靶标,设计4组插入序列边界位点特异性引物,结合大豆内标准基因Lectin进行多重PCR (multiplex PCR, MPCR)研究。通过引物特异性测试、体系和反应程序优化、灵敏度分析、以及世代遗传稳定性应用,建立了转基因大豆ZH10-6分子特征转化体特异性多重PCR检测方法。结果表明,该方法具有良好的特异性,MPCR检测体系中5种靶标(边界A, 边界B, 边界C, 边界D, 内源基因Lectin)的最适引物终浓度(μmol/L)配比为：0.8∶0.3∶0.1∶0.2∶0.05。每种靶标的检测灵敏度可达到1%,可在3个世代间稳定遗传,适用于ZH10-6插入位点快速高效检测。本研究建立的检测方法可有效提高检测效率,降低检测成本,为转基因耐除草剂大豆ZH10-6新品种的分子特征转化体特异性鉴定提供新的检测技术,为该品种分子特征转化体的生物安全评价及知识产权保护和市场行政监管提供技术支撑。

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关键词 ：转基因耐除草剂大豆ZH10-6, 多重PCR, 分子特征, 转化体特异性, 检测方法

Abstract：Herbicide-tolerant transgenic soybean (Glycine max) ZH10-6 is a new herbicide-tolerant genetically modified soybean line with glyphosate degradation gene (glyphosate acetyltransferase, GAT) and glyphosate resistance gene (pseudomonas fluorescens G2 strain 5-enolpyruvyl shikimate-3-phosphate synthase, G2-EPSPS) studied by the Institute of Crop Science, Chinese Academy of Agricultural Sciences. In order to establish anevent-specific detection method with it, multiplex PCR (MPCR) reaction with 4 sets of PCR primers covering different regions of the insertion and border sequence and 1 primer set for the soybean internal standard gene Lectin were evaluated.. Through primer-specific testing, optimization of PCR reaction system and procedure, assessment of reaction sensitivity and stability for the different generations of the transgenic soybean, a multiplex PCR detection method for the transgenic soybean ZH10-6 was established. The results showed that the method had great specificity. The optimal primer concentration (μmol/L) ratio of the 5 targets in the MPCR detection system (border A, border B, border C, border D, endogenous gene Lectin): 0.8∶0.3∶0.1∶0.2∶0.05. The detection sensitivity of each target could reach 1%, which could be stably inherited among 3 generations, and was suitable for rapid and efficient detection of ZH10-6 insertion sites. The detection method established in this study improved the detection efficiency and reduced the detection cost. It provides a new detection technique for identification of the new variety of transgenic herbicide-tolerant soybean ZH10-6 transformant and its derivatives,which is technical support for its biosafety assessment, administrative supervision and intellectual property protection.

Key words： Genetically modified herbicide tolerant soybean ZH10-6 Multiplex PCR Molecular characteristics Transformant specificity Detection method

收稿日期: 2021-01-10

ZTFLH:

S565.1

基金资助:转基因生物新品种培育专项(2019ZX08013002-004; 2019ZX08004001-005)

通讯作者: * zhaoxin2008999@126.com

引用本文:

李瑞环, 刘双, 兰青阔, 曹际娟, 王永, 赵新. 转基因耐除草剂大豆ZH10-6多重PCR检测方法的建立[J]. 农业生物技术学报, 2021, 29(8): 1640-1648.
LI Rui-Huan, LIU Shuang, LAN Qing-Kuo, CAO Ji-Juan, WANG Yong, ZHAO Xin. Establishment of Multiplex PCR Detection Method for Genetically Modified Herbicide Tolerant Soybean (Glycine max) ZH10-6. 农业生物技术学报, 2021, 29(8): 1640-1648.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2021.08.021 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2021/V29/I8/1640

[1] 白卫滨, 李宵, 朱翠娟, 等. 2016. 通用引物多重PCR新技术对番木瓜转基因成分检测的研究[J]. 现代食品科技, 32(4): 229-234.
(Bai W B, Li X, Zhu C J, et al.2016. Detection of genetically modified ingredients in transgenic papaya using universal primer-multiplex PCR[J]. Modern Food Science and Technology, 32(4): 229-234.)
[2] 董立明, 李葱葱, 邢珍娟, 等. 2016. 利用多重PCR技术快速检测五个转基因大豆品系[J]. 大豆科学, 35(06): 1002-1007.
(Dong L M, Li C C, Xing Z J, et al.2016. Rapid detection of five genetically modified soybean lines by multiplex PCR method[J]. Soybean Science, 35(06): 1002-1007.)
[3] 蔡洪生. 2020. 认识转基因大豆[J]. 大豆科技, 03: 46-47.
(Cai H S.2020. Understanding transgenic soybean[J]. Soybean Since Technology, 03: 46-47.)
[4] 国际农业生物技术应用服务组织. 2019. 2018年全球生物技术/转基因作物商业化发展态势[J]. 中国生物工程杂志, 39(08): 1-6.
(International Service for the Acquisition of Agri-biotech Applications China Biotechnology, 2019. Global commercialization of biotechnology/genetically modified crops in 2018[J]. China Biotechnology, 39(08): 1-6.)
[5] 董悦. 2011. 各国转基因农产品安全管理的国际比较及综合评价[D]. 博士学位论文, 武汉: 华中农业大学, 导师: 柳鹏程. pp. 3-10.
(Dong Y.2011. International comparison and comprehensive evaluation on safety management of genetically modified agricultural products[D]. Thesis for Ph. D., Huazhong Agricultural University, Supervisor: Liu P C. pp. 3-10.)
[6] 郭娜, 黄颜众, 轩慧冬, 等. 2019. 话说大豆转基因[J]. 大豆科技, 05: 56-58.
(Guo N, Huang Y Z, Xuan H D, et al.2019. Talk about transgenic soybean[J]. Soybean Since Technology, 05: 56-58.)
[7] 李飞武, 闫伟, 龙丽坤, 等. 2014. 应用多重PCR技术筛选检测转印基因作物[J]. 现代食品科技, 30(5): 262-266.
(Li F W, Yan W, Long L K, et al.2014. Screening detection of Bt genes in genetically modified crops using a multiplex PCR assay[J]. Modern Food Science and Technology, 30(5): 262-266.)
[8] 李会, 任志莹, 王颖, 等. 2014. 多重PCR法快速检测转基因玉米多种转化体技术优势的比较分析[J]. 江苏农业科学, 42(5): 57-59.
(Li H, Ren Z Y, Wang Y, et al.2014. Comparative analysis of the advantages of multiplex PCR in rapid detection of transgenic maize transformants[J]. Jiangsu Agricultural Sciences, 42(5): 57-59.)
[9] 梁晋刚, 贺晓云, 武玉花, 等. 2020. 中国农业转基因生物安全标准体系现状与展望[J]. 农业生物技术学报, 28(05): 911-917.
(Liang J G, He X Y, Wu Y H, et al.2020. Current status and prospects of safety standard system for agricultural genetically modified organisms in China[J]. Journal of Agricultural Biotechnology, 28(05): 911-917.)
[10] 梁利霞, 宛煜嵩, 金芜军. 2014. 利用复合PCR方法同时检测三种转基因水稻[J]. 基因组学与应用生物学, 33(01): 16-21.
(Liang L X, Wan Y S, Jin W J.2014. The event-specific multiplex pcr detection method for three genetically modified rice[J]. Genomics and Applied Biology, 33(01): 16-21.)
[11] 刘双. 2020.基于PCR技术的转基因耐除草剂大豆ZH10-6检测方法研究[D].硕士学位论文, 河北农业大学,导师: 檀建新. pp.6.
(Liu S.2020.Study on Detection Method of Genetically Modified Herbicide Tolerant Soybean ZH10-6 Based on PCR Technology[D].Thesis for M. S., Hebei Agriculture University,Supervisor: Tan J X. pp.6.)
[12] 刘双, 陈笑芸, 曹际娟, 等. 2020. 转基因耐除草剂大豆ZH10-6实时荧光定量PCR检测方法的建立[J/OL].分子植物育种: 1-13.
(Liu S, Chen X Y, Cao J J, et al.2020. Establishment of real-time quantitative pcr method of transgenetic herbicide-tolerant soybean ZH10-6[J/OL]. Molecular Plant Breeding : 1-13.)
[13] 马硕, 焦悦, 王旭静, 等. 2020. 高通量测序技术在转基因植物分子特征评价中的应用[J]. 中国农业科技导报, 22(05): 6-14.
(Ma S, Jiao Y, Wang X J, et al.2020. Genome sequencing application on molecular characterization assessment of transgenic plant[J]. Journal of Agricultural Science and Technology, 22(05): 6-14.)
[14] 汪智, 卢宝荣. 2018. 生物安全评价系列之三: 各国转基因生物应用的相关法规[J]. 人与生物圈, 6: 65-67.
(Wang Z, Lu B R.2018. Biosafety assessment series 3: Regulations on the application of genetically modified organisms in various countries[J]. Man and The Biosphere, 6: 65-67.)
[15] 向才玉, 凌杏园, 潘广, 等. 2015. 多重PCR-DHPLC法检测转基因棉花品系[J]. 植物检疫, 29(1): 37-41.
(Xiang C Y, Ling X Y, Pan G, et al.2015. Simultaneously detection of three genetically modified cotton events by multiplex PCR coupled with denaturing high performance liquid chromatography (mPCR-DHPLC)[J]. Plant Quarantine, 29(1): 37-41.)
[16] 邢珍娟, 董立明, 刘娜, 等. 2019. 多重PCR检测耐除草剂转基因作物[J]. 核农学报, 33(02): 255-261.
(Xing Z J, Dong L M, Liu N, et al.2019. Multiplex PCR to detect herbicide-tolerant genetically modified crops[J]. Journal of Nuclear Agricultural Sciences, 33(02): 255-261.)
[17] 尹全, 李忆, 宋君, 等. 2016.多重PCR筛查检测进口转基因玉米[J]. 核农学报, 30(6): 1045-1053.
(Yin Q, Li Y, Song J, et al.Multiplex PCR detection of exogenous gene in the imported genetically modified maize[J]. Journal of Nuclear Agricultural Sciences, 30(6): 1045-1053.)
[18] 于滔, 曹士亮, 张建国, 等. 2020. 全球转基因作物商业化种植概况(1996-2018年)[J]. 中国种业, 01: 13-16.
(Yu T, Cao S L, Zhang J G, et al.2020. Global overview of commercial cultivation of genetically modified crops(1996-2018)[J]. China Seed Industry, 01: 13-16.)
[19] 张耀川, 许亮, 张洁, 等. 2012. 利用多重PCR方法检测7种转基因卡诺拉油菜籽品种(系)研究[J]. 粮食与油脂, 9: 30-32.
(Zhang Y C, Xu L, Zhang J, et al.2012. Detection of seven genetically modified canola using multiplex PCR[J]. Cereals and Oils, 9: 30-32.)
[20] 钟泽澄, 王进, 张师音. 2020. 多重PCR技术研究进展[J]. 生物工程学报, 36(2): 171-179.
(Zhong Z C, Wang J, Zhang S Y, 2020. Advances in multiple PCR technology studies[J]. Chinese Journal of Biotechnology, 36(2): 171-179.)
[21] 转基因植物安全评价指南[Z/OL].2017. 北京:农业部办公厅.
(Guidelines for safety assessment of genetically modified plants[Z/OL].2017. Beijing: General Office of the Ministry of Agriculture.)
[22] Datukishvili N, Kutateladze T, Gabriadze I, et al.2015. New multiplex PCR methods for rapid screening of genetically modified organisms in foods[J]. Frontiers in Microbiology, 6: 757.
[23] Li X, Wang X X, Yang J L, et al.2014. A novel quadruplex real-time PCR method for simultaneous detection of Cry2Ae and two genetically modified cotton events (GHB119 and T304-40)[J]. BMC Biotechnology, 14(1): 43.
[24] Liu Y, Yin S Y.Advance of multiplex PCR in rapid detecting transgenic soybean oil[J]. American Journal of Plant Sciences, 2020, 11(03): 382-392.
[25] Luan F X, Tao R, Xu Y G, et al.2012. High-throughput detection of genetically modified rice ingredients in foods using multiplex polymerase chain reaction coupled with high-performance liquid chromatography method[J]. European Food Research & Technology, 234(4): 649-654.
[26] Xu W T, Zhai Z F, Huang K L, et al.2012. A novel universal primer-multiplex-PCR method with sequencing gel electrophoresis analysis[J]. PLOS ONE, 7(1): e22900.