转基因玉米目标基因纯合体快速准确的PCR鉴定方法(英文)

An Accurate and Rapid PCR-Based Zygosity Testing Method for Genetically Modified Maize

本文以转基因玉米NK603为例,介绍了一种检测目标基因纯和体的PCR方法。该方法利用4个引物的多重PCR反应,这4个PCR引物分别来自于目标基因的5'端(5'TP)和3'端(3'TP)DNA序列,目标基因5'端一侧的玉米基因组DNA序列(5'GP),以及目标基因3'端一侧的玉米基因组DNA序列(3'GP)。视玉米个体有无目标基因以及目标基因的杂合/纯和状态,PCR扩增可得到三种不同的结果:如果被检测个体无目标基因,5'GP与3'GP间的玉米基因组DNA将被扩增,PCR只产生一条条带;如果被检测个体是目标基因的纯合体,5'GP与5'TP及3'TP与3'GP间的DNA将被扩增,PCR则产生两条条带;如果被检测个体是目标基因的杂合体,5'GP与3'GP、5'GP与5'TP以及3'TP与3'GP间的DNA将都被扩增,PCR则产生三条条带。三条不同长短的PCR产物条带在琼脂糖凝胶上清晰可分,易于辨别。和费时昂贵的定量PCR相比,该方法简单、快速、结果准确,在目标基因位点玉米基因组DNA序列已知的前提下,该方法可扩展到诸如Bt11、Event176、GA21、MON810、MON863 和 TC1507 等任何转基因玉米的回交转育程序中。

Using genetically modified NK603 maize as an example, an accurate and cost-effective PCR-based procedure was described for genetically modified maize zygosity testing. The method involved a simple PCR reaction with four primers. The primers were designed from the inserted transgene at 5＇ and 3＇ ends （5＇TP and 3＇TP）, the 5＇ flanking genomic DNA （5＇GP）, and the 3＇ flanking genomic DNA （3＇GP）, respectively. If the plant was a null （wild type）, genomic DNA between 5＇GP and 3＇GP were amplified and a single PCR product was produced; If the plant was a homozygote, DNA between 5＇GP and 5＇TP, 3＇TP and 3＇GP were amplified and two PCR products were produced; If the plant was a hemizygote, DNA between 5＇GP and 5＇TP, 3＇TP and 3＇GP, 5GP and 3＇GP were all amplified, producing three PCR products. PCR products with different sizes were separated on an agarose gel, and one, two and three bands were easily scored, which represents null, homozygote and hemizygote, respectively. Compared to expensive quantitative PCR and time-consuming bioassay, this method coupled with high throughput genomic DNA extraction is of great assistance to corn breeders for zygosity selection. Hundreds of leaf discs from individual plants can be punched with eppendorf tubes in field and plant zygosity result is readily available. Similar strategy can be applied to develop event-specific zygosity testings for other maize genetically modified events such as Btl 1, Event176, GA21, MON810, MON863 and TC1507 provided that their sequences at both insertion junctions are available.