转基因棉花MON757转化体特异性PCR检测方法及应用

发布时间：2018-06-02 09:13

本文选题：转基因棉花 + MON　；参考：《农业生物技术学报》2016年06期

【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 real-time PCR,q RT-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q RT-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q RT-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q RT-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。
[Abstract]:Transgenic cotton Gossypium hirsutum)MON757 transformant was developed by Monsanto Company and developed by Monsanto. It has been approved to grow or be used as feedstuff in USA, Canada, Australia and other countries, but it has not been approved and applied in our country. At present, there are no pure heterozygosity detection and quantitative detection methods for MON757 specificity of transgenic cotton at home and abroad. In order to detect the existence of MON757 transformants in Chinese cotton, the genomic sequence of insertion site and exogenous insert fragment / genomic linkage region of transgenic cotton MON757 transformants were used as targets in this study. A specific PCR detection method for pure heterozygosity of MON757 transformants and a real-time fluorescent quantitative PCR(quantitative real-time PCRQ RT-PCR method were established. The detection primers for pure heterozygous PCR detection of MON757 transformants were composed of three primers located on the genomes of the two sides of the insertion site of the exogenous gene of MON757 and on the inserted fragments of the exogenous gene, respectively. The homozygosity and heterozygosity of MON757 transformants in cotton plants and seeds can be identified accurately. The established Q RT-PCR assay has good reproducibility and sensitivity. The detection limit of detection limit is 11 copies, and the limit of quantitative limit is 44 copies. This method was used to detect 49 commercial cotton seeds in Hubei province in 2014. The results showed that MON757 transformants were detected in 5 seed samples. The pure heterozygosity qualitative PCR assay of MON757 transformants was used to detect 60 single seeds of the 5 samples, and the Q RT-PCR method was used to detect the 5 samples quantitatively. The results showed that the content of 1 phr was 1.5% or so and the content of 4 phr exceeded 20 phr. The results of the two methods were basically consistent. The qualitative PCR and Q RT-PCR detection methods for pure heterozygosity of MON757 transformants in cotton plants and seeds were developed in this study. They were used to determine the homozygous status of MON757 transformants in cotton plants and seeds and the content of MON757 transformants in mixed samples. It has important application value.
【作者单位】：中国农业科学院植物保护研究所/农业部转基因环境安全及植物抗性监督检验测试中心(北京)/植物病虫害生物学国家重点实验室;
【基金】：转基因新品种培育重大科技项目(No.2014ZX08012-01B)
【分类号】：S562

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