Construction Of Transgenic Maize Standard Substance-plasmid Positive Substance And Establishment Of Detection Method For Transgenic Maize

As transgenic crops may have potential safety issues in the medium and long term,GM testing is a very important part of the safety supervision of GM.GM positive substances are essential in the testing process of GM.The research and development of GM positive substances plays a crucial role in the establishment of GM related testing methods and the development of testing equipment.However,there are still problems in the lack of transgenic positive substances,GM testing methods to be optimized,and the vacancy of the purity identification scheme of transgenic corn seeds.This study is for many commercial transgenic maize varieties and transgenic maize varieties independently developed in China,Eight single-target DNA-positive and multitarget DNA-positive substances were constructed,respectively,Enzyme cutting,colony PCR and sequencing of the constructed plasmid DNA molecules were verified;Establish a system of multiple real-time fluorescent PCR screening for maize seeds,Some problems also exist with optimized GM detection methods-real-time fluorescence quantitative PCR,If different genes are amplified without holes,Differences in the amplification results will lead to errors in the GM quantitative process;Establishing a multiple testing method suitable for the purity identification of transgenic maize seeds can provide efficient and accurate purity identification of transgenic seeds at low cost,The proposed method provides a technical reserve for the purity detection of transgenic maize,It also provides a reference for the GM purity identification of other crops.In the first part of this paper,8 new single-target plasmid molecules and the NOS terminator,the ZSSIIB,of the maize endogenous gene were constructed.Through colony PCR、 sequencing and sequencing,nine plasmid positive substances could be normally amplified and in correct sequence and can be used as a positive reference in transgenic testing.The second part of this paper is the establishment of multiple detection methods in GM testing.The multiple real-time fluorescence quantitative PCR system is tested by Ca MV 35 S promoter,NOS termination and maize endogenous gene ZSSIIB in the same reaction system,with good repetition and high sensitivity that can be applied in transgenic quantitative detection;multiple fluorescent capillary system combines7 SSR sites with transgenic element Ca MV 35 S promoter and NOS termination to the purity identification of transgenic maize seeds.In this paper,9 plasmid-positive substances and 2 kinds of transgenic testing systems are constructed.The combination with multiple testing systems can also shorten the error of the process,and provide reference for transgenic testing of other crops.