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作物杂志,2016, 第1期: 69–75 doi: 10.16035/j.issn.1001-7283.2016.01.013

• 遗传育种·种质资源·生物科技 • 上一篇    下一篇

转G2-EPSPS基因玉米D-3侧翼序列分析与转化体特异性检测方法

郭翠1,2,张维2,余桂容3,周正富2,李亮2,冯帅1,2,陈明2,王劲1,2   

  1. 1 西南科技大学生命科学与工程学院,621010,四川绵阳
    2 中国农业科学院生物技术研究所,100081,北京
    3 四川省农业科学院生物技术核技术研究所,610066,四川成都
  • 收稿日期:2015-10-20 修回日期:2015-12-04 出版日期:2016-02-15 发布日期:2018-08-25
  • 通讯作者: 王劲
  • 作者简介:郭翠,硕士研究生,研究方向为特殊环境微生物功能基因资源利用
  • 基金资助:
    转基因重大专项(2014ZX0800301B);国家自然科学基金(31370126);国家“973”计划(2013CB733903);国家“863”计划(2012AA063503)

Analysis of the Flanking Sequence and Event-Specific Detection of Transgenic G2-EPSPS Line of Maize

Guo Cui1,2,Zhang Wei2,Yu Guirong3,Zhou Zhengfu2,Li Liang2,Feng Shuai1,2,Chen Ming2,Wang Jin1,2   

  1. 1 Life Science and Engineering College,Southwest University of Science and Technology,Mianyang 621010,Sichuan,China
    2 Biotechnology Research Institute,Chinese Academy of Agriculture Sciences,Beijing 100081,China
    3 Institute of Biological & Nuclear Technology,Sichuan Academy of Agricultural Sciences,Chengdu 610066,Sichuan,China;
  • Received:2015-10-20 Revised:2015-12-04 Online:2016-02-15 Published:2018-08-25
  • Contact: Jin Wang

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摘要：

通过一次3'端高效热不对称交互PCR（hiTAIL-PCR）和一次长链PCR扩增方法获得了转抗草甘膦基因（G2-EPSPS）玉米品系D-3的外源DNA插入片段的全DNA序列（T-DNA）及两端侧翼序列,并建立了转化体特异性PCR检测方法。结果显示：T-DNA插入片段全长4 318bp,由一个G2-EPSPS基因的表达盒构成。根据T-DNA 5'、3'端侧翼序列设计引物,建立了转化体特异性检测方法,并分析了该方法的灵敏度以及检出限,研究结果表明,3'端定性PCR检测方法特异性强,灵敏度高,检出极限为每100ng模板量的0.05%。本研究结果对转抗草甘膦外源基因检测和生物安全评价及监管具有重要意义。

关键词: 转基因玉米, G2-EPSPS, 侧翼序列, 转化体特异性检测

Abstract:

By hiTAIL-PCR(3'-terminal high-efficiency thermal asymmetric interlaced PCR) and LD-PCR(long distance PCR),the complete sequence and flanking sequence of T-DNA in transgenic maize D-3 were obtained. A specific PCR detection method for transformant was also established. The results revealed that the total length of T-DNA was 4318 bp,which consisted of a G2-EPSPS gene expression cassette. The primers had been designed based on the 3' and 5' terminal flanking sequence,followed by specific PCR to detect the sensitivity as well as the detection limit. The results showed that 3'-qualitative PCR detection method was highly specific and sensitive,with a sensitivity of 0.05% per 100ng DNA template.This research provides the methods for the detection of exogenous gene in transgenic maize and the risk assessment on bio-safety.

Key words: Transgenic maize, G2-EPSPS, Flanking sequence, Event-specific detection

表 1

hiTAIL-PCR中使用的任意简并引物AD1-n与AD-C"

引物Primer 引物序列（5'-3'）Primer sequence（5'-3'）
AD1-1
AD1-2
AD1-3
AD1-4
AD-C		 ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
ACGATGGACTCCAGAGCGGCCGCBDNBNNNAGGT
ACGATGGACTCCAGAG

图 1

侧翼序列获得以及验证 A：转基因玉米D-3 3'端hiTAIL-PCR;M：DNA分子量标记(Trans 2k PlusⅡDNA marker);1-3：hiTAIL-PCR的1、2、3轮扩增结果;B： 3'边界侧翼序列;黑体部分为T-DNA 序列;斜体部分为5'端侧翼序列;C：5'边界侧翼序列;黑体部分为T-DNA 序列,斜体部分为3'端侧翼序列;D：PCR验证侧翼序列;M：DNA分子量标记(Trans 2k PlusⅡDNA marker); 1： LB-F1/LB-R扩增产物;2： LB-F2/LB-R扩增产物;3：LB-F3/LB-R扩增产物; 4： RB-F1/RB-R扩增产物;E：插入片段T-DNA结构示意图以及引物设计示意图"

图 2

不同引物PCR的特异性检测 M：DNA分子量标记Trans 2k plus ⅡDNA Marker;1-5：D-3、14sT133、255、阴性对照、空白对照。"

图 3

转基因玉米D-3特异性PCR灵敏度检测 M: DNA分子量标记Trans 2k plus ⅡDNA Marker;1：Blank;2：negative control;3-10：100.00%、50.00%、10.00%、5.00%、1.00%、0.50%、0.10%、0.05% 的DNA模板"

图 4

转化体特异性检测方法检出极限测试 A、B、C的DNA含量分别为0.10%、0.05%、0.01%;M：DNA分子量标记Trans2k plusⅡDNA Marker;1~10：分别表示10个平行。"

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