| 转cry1Aa基因抗虫棉整合结构解析及转化体特异性检测方法的建立 |
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| 引用本文： | 王叶,谢家建,黄春蒙,彭于发.转cry1Aa基因抗虫棉整合结构解析及转化体特异性检测方法的建立[J].棉花学报,2017,29(4):307-315. |
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| 作者姓名： | 王叶 谢家建 黄春蒙 彭于发 |
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| 作者单位： | 中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室/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),北京,100193 |
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| 基金项目： | 国家科技重大专项——转基因生物新品种培育(2016ZX08012-002) |
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| 摘 要： | 【目的】cry1Aa基因是2011年本实验室在我国棉花商品种子中发现的1种未经批准的转基因成分,本研究旨在通过整合结构解析,建立特异性检测方法,对市售棉种进行初测,明确该外源基因在我国棉种中的存在情况。【方法】通过Genome walking、长链PCR分离获得了转cry1Aa基因棉花转化体(定名为NN6转化体,简称NN6)的外源基因整合结构,同时基于插入位点基因组及外源DNA连接区,建立了NN6转化体特异性的定性聚合酶链式反应(Polymerase chain reaction,PCR)方法和实时荧光定量PCR方法。【结果】NN6整合结构主要包括cry1Aa、aad和Npt II基因,插入棉花基因组7号染色体37 169 450位,产生91 bp基因组缺失;所构建的定性PCR检测方法能特异、快速地对棉花单株和种子单粒中NN6转化体的纯/杂合状态进行鉴定,实时荧光定量PCR检测限为44拷贝,能满足国内外对转基因标识的需要;对含有cry1Aa基因的3个市售棉种进行检测,其纯合度分别为1.51%,5.21%和21.09%。【结论】本研究解析了NN6的整合结构,并建立特异性检测方法,为我国转基因棉花新品种选育及转基因安全管理提供技术手段。
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| 关 键 词： | 转基因棉花 NN6转化体 cry1Aa基因 定性PCR 定量PCR |
Integrated Structure of the Modified cry1Aa Gene in Cotton and Its Event-Specific Detection |
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| Wang Ye,Xie Jiajian,Huang Chunmeng,Peng Yufa.Integrated Structure of the Modified cry1Aa Gene in Cotton and Its Event-Specific Detection[J].Cotton Science,2017,29(4):307-315. |
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| Authors: | Wang Ye Xie Jiajian Huang Chunmeng Peng Yufa |
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| Institution: | Institute of Plant Protection, Chinese Academy of Agricultural Sciences/State Key Laboratory for Biology of Plant Diseases and Insect Pests/Inspection Test Center for Environmental Safety of Transgenic Crops and Plant Resistance (Beijing), Ministry of Agriculture, Beijing 100193, China |
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| Abstract: | Objective] We detected a modified cry1Aa gene in a Chinese cotton line in 2011.The objectives of this study were to establish a specific detection method applicable for commercial cotton varieties by integrating the results of structural analyses and to confirm the existence of the exogenous gene in cotton.Method] The integrated structure of the modified cry1Aa gene (NN6) was determined by genome walking and a long-chain polymerase chain reaction (PCR).An event-specific qualitative PCR and a quantitative real-time PCR were established based on the linkage region between genomic DNA and the exogenous DNA of NN6.Result] The NN6 structure mainly comprised the cry1Aa,aad,and nptⅡ genes,which had been inserted into chromosome 7 at position 37 169 450,with a 91 bp deletion in the genome.The highly sensitive qualitative PCR was able to quickly identify the heterozygous and homozygous NN6.The limit of detection for the NN6-specific quantitative real-time PCR was 44 copies,which satisfied the requirements for analyzing Chinese and international cotton varieties.The seeds of three commercial cotton varieties carrying the cry1 Aa gene were tested,with a resulting purity of 1.51％,5.21％,and 21.09％.Conclusion]We analyzed the integrated structure of NN6 and established a specific detection method that may be useful for ensuring transgenie cotton can be bred safely in China. |
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| Keywords: | transgenic cotton NN6 event cry1Aa gene event-specific qualitative PCR quantitative real-time PCR |
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