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

Integrated Structure of the Modified cry1Aa Gene in Cotton and Its Event-Specific Detection

【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。

[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 cry1Aa 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 transgenic cotton can be bred safely in China.