转基因棉花MON757转化体特异性PCR检测方法及应用

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摘要转基因棉花(Gossypium hirsutum)MON757转化体是孟山都公司研发的转cry1Ac基因的抗虫棉，在美国、加拿大、澳大利亚等国被批准种植或允许用作食品饲料原料，但在我国尚未获得批准种植和应用。目前国内外尚没有转基因棉花MON757特异性的纯杂合检测和定量检测方法报道。为了检测我国棉花中MON757转化体的存在情况，本研究以转基因棉花MON757转化体的插入位点基因组序列和外源插入片段/基因组连接区序列为靶标，建立了特异性的MON757转化体纯杂合定性PCR检测方法和实时荧光定量PCR(quantitative realtime PCR, qRT-PCR)检测方法。所建立的MON757转化体纯杂合定性PCR检测方法的检测引物由分别位于MON757外源基因插入位点两侧基因组和外源插入片段上的3条引物组成，能准确地鉴别棉花植株和种子中MON757转化体的纯合、杂合状态。建立的MON757转化体特异性的qRT-PCR检测方法具有良好的可重复性和灵敏度，其检测下限(Limit of Detection, LOD)为11个拷贝，定量下限(Limit of Quantitative, LOQ)为44个拷贝。用此方法对2014年湖北省的49份商品棉花种子进行了检测，结果有5份种子样品检测到MON757转化体。采用MON757转化体纯杂合定性PCR检测方法对这5份种子样品各60个单粒分别进行了检测，同时采用qRT-PCR检测方法对这5份种子样品进行定量测定。结果表明，有1份含量在1.5%左右，4份含量超过了20%，两种方法的测定结果基本一致。本研究建立的MON757转化体纯杂合定性PCR检测方法和qRT-PCR检测方法在田间棉花植株和种子中MON757转化体纯杂合状态的测定以及混合样品中MON757转化体含量的测定方面都具有重要的应用价值。

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	杨阳
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关键词 ：转基因棉花, MON757, 定性PCR, 定量PCR, 纯杂合

Abstract：Genetically modified cotton (Gossypium hirsutum) event MON757 expressing Cry1Ac protein was developed by U.S. Monsanto Company targeting Lepidoptera pests. It has already been approved for planting or using for food/feed in the United States, Canada, Australia and other countries, but has not yet been permitted for planting or application in China. At present, there are not specific methods for Genetically Modified (GM) cotton MON757 reported to identify the homozygous/heterozygous states or to quantify its content in mixed samples. In order to detect GM cotton MON757 in commercial cotton seeds of China, we established the specific homozygous/heterozygous qualitative PCR method and the event-specific quantitative Real-time PCR (qRT-PCR) method for MON757 based on the DNA sequences of the junctions between exogenous DNA fragments and cotton genomic DNA. The homozygous/heterozygous qualitative PCR method using 3 primers respectively located in the exogenous DNA segments and the cotton genomic DNA sequence on both sides of the insertion fragments could accurately identify homozygous and heterozygous states of MON757. The qRT-PCR method established in this study could specifically detect the samples of MON757 with the limit of detection (LOD) about 11 copies and the limit of quantitative (LOQ) about 44 copies. We tested 49 samples of cotton seeds from the market in Hubei province of China, and 5 samples were identified having MON757. In order to quantify the MON757 content of those 5 samples, 600 seeds of each sample were mixed for quantitative test using event-specific qRT-PCR method. At the same time, we tested 60 seeds from each samples using the homozygous/heterozygous qualitative PCR method to calculate the MON757 content. The results of both methods showed that the content of MON757 of one sample was about 1.5% and other 4 samples are all over 20%. The homozygous/heterozygous PCR method and the qRT-PCR method for MON757 that we developed are suitable and useful for homozygous and heterozygous states identification of cotton plants and seeds in the field and for quantifying the content MON757 from mixed samples.

Key words： Transgenic cotton MON757 Qualitative PCR Quantitative PCR Homozygous/heterozygous

收稿日期: 2015-12-16 出版日期: 2016-05-06

基金资助:转基因新品种培育重大科技项目

通讯作者: 谢家建 E-mail: jjxie@ippcaas.cn

引用本文:

杨阳王叶范金杰谢家建彭于发. 转基因棉花MON757转化体特异性PCR检测方法及应用[J]. , 2016, 24(6): 908-918.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I6/908

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