Establishment of Multiplex PCR Detection Method for Genetically Modified Herbicide Tolerant Soybean (Glycine max) ZH10-6
LI Rui-Huan1, LIU Shuang1, LAN Qing-Kuo1, CAO Ji-Juan2, WANG Yong1, ZHAO Xin1,*
1. Biotechnology Research Institute, Tianjin Academy of Agricultural Sciences, Tianjin 300381, China; 2. College of Life Sciences, Dalian Minzu University, Dalian 116600, China
Abstract：Herbicide-tolerant transgenic soybean (Glycine max) ZH10-6 is a new herbicide-tolerant genetically modified soybean line with glyphosate degradation gene (glyphosate acetyltransferase, GAT) and glyphosate resistance gene (pseudomonas fluorescens G2 strain 5-enolpyruvyl shikimate-3-phosphate synthase, G2-EPSPS) studied by the Institute of Crop Science, Chinese Academy of Agricultural Sciences. In order to establish anevent-specific detection method with it, multiplex PCR (MPCR) reaction with 4 sets of PCR primers covering different regions of the insertion and border sequence and 1 primer set for the soybean internal standard gene Lectin were evaluated.. Through primer-specific testing, optimization of PCR reaction system and procedure, assessment of reaction sensitivity and stability for the different generations of the transgenic soybean, a multiplex PCR detection method for the transgenic soybean ZH10-6 was established. The results showed that the method had great specificity. The optimal primer concentration (μmol/L) ratio of the 5 targets in the MPCR detection system (border A, border B, border C, border D, endogenous gene Lectin): 0.8∶0.3∶0.1∶0.2∶0.05. The detection sensitivity of each target could reach 1%, which could be stably inherited among 3 generations, and was suitable for rapid and efficient detection of ZH10-6 insertion sites. The detection method established in this study improved the detection efficiency and reduced the detection cost. It provides a new detection technique for identification of the new variety of transgenic herbicide-tolerant soybean ZH10-6 transformant and its derivatives,which is technical support for its biosafety assessment, administrative supervision and intellectual property protection.
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