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
发布时间：2018-12-28 19:40
【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。
[Abstract]:[objective] cry1Aa gene is an unapproved transgenic component found in cotton commercial seeds in our country in 2011. The purpose of this study was to establish a specific detection method for commercial cotton seeds by integrated structural analysis. [methods] the transgenic cotton transformants with cry1Aa gene (named NN6 transformant, NN6) were isolated by Genome walking, long chain PCR. At the same time, a qualitative polymerase chain reaction (Polymerase chain reaction,PCR) method and a real-time fluorescence quantitative PCR method were established based on the insertion site genome and the exogenous DNA junction region. [results] the integrated structure of NN6 mainly includes cry1Aa,. Aad and Npt II genes were inserted into cotton genome 7 at 37,169,450, resulting in 91 bp genome deletion. The constructed qualitative PCR detection method can identify the pure / heterozygous status of NN6 transformants in cotton plants and seeds quickly. The detection limit of real-time quantitative PCR is 44 copies, which can meet the needs of transgenic identification at home and abroad. The homozygosity of three commercial cotton varieties containing cry1Aa gene was 1.51% and 21.09%, respectively. [conclusion] the integrated structure of NN6 was analyzed and the specific detection method was established. To provide technical means for breeding and safety management of transgenic cotton varieties in China.
【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);
【基金】：国家科技重大专项——转基因生物新品种培育(2016ZX08012-002)
【分类号】：S562
[Abstract]:[objective] cry1Aa gene is an unapproved transgenic component found in cotton commercial seeds in our country in 2011. The purpose of this study was to establish a specific detection method for commercial cotton seeds by integrated structural analysis. [methods] the transgenic cotton transformants with cry1Aa gene (named NN6 transformant, NN6) were isolated by Genome walking, long chain PCR. At the same time, a qualitative polymerase chain reaction (Polymerase chain reaction,PCR) method and a real-time fluorescence quantitative PCR method were established based on the insertion site genome and the exogenous DNA junction region. [results] the integrated structure of NN6 mainly includes cry1Aa,. Aad and Npt II genes were inserted into cotton genome 7 at 37,169,450, resulting in 91 bp genome deletion. The constructed qualitative PCR detection method can identify the pure / heterozygous status of NN6 transformants in cotton plants and seeds quickly. The detection limit of real-time quantitative PCR is 44 copies, which can meet the needs of transgenic identification at home and abroad. The homozygosity of three commercial cotton varieties containing cry1Aa gene was 1.51% and 21.09%, respectively. [conclusion] the integrated structure of NN6 was analyzed and the specific detection method was established. To provide technical means for breeding and safety management of transgenic cotton varieties in China.
【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);
【基金】：国家科技重大专项——转基因生物新品种培育(2016ZX08012-002)
【分类号】：S562
【相似文献】
相关期刊论文 前6条
1 周平兰,梁满中,高健,陈良碧;花粉管通道法转柱头外露棉DNA及转化体RAPD分析[J];湖南师范大学自然科学学报;2004年04期
2 张富丽;宋君;刘勇;尹全;王东;常丽娟;刘文娟;雷绍荣;游米沙;;GTS40-3-2大豆转化体特异性的定性PCR检测[J];西南农业学报;2012年05期
3 刘志生,张坤普,付秀云;芦苇草DNA导入小麦抗盐性转化体的筛选[J];吉林农业科学;1998年04期
4 宋敏;刘娜;任欣欣;卢W，
本文编号：2394347
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2394347.html
最近更新
教材专著
