Study Of New GMO Reference Material And Qualitative Detection Method Targeting Marker Genes

Screening detection system includes GMO (genetically modified organism) reference material and screening detection methods. Reference material is the material basis of the screening detection system, while screening detection methods are the technology base of the screening system.The matrix reference material and the plasmid reference material are two major forms of reference materials applied in GMO detection. However, several problems have restricted their application, such as difficulty in access to the source materials and high cost in development of matrix reference materials, and contamination problem found in application of plasmid reference materials. In order to overcome the above problems, this study attempts to combine the advantages of the matrix reference material and the plasmid standard molecule and overcome their weaknesses to develop an alternative reference material for GMO detection. This alternative reference material is developed by transforming the target sequences into tobacco with the plasmid pBI121-RM containing 11 universal elements from transgenic crops and the endogenous reference genes (ERGs) of rape, maize, rice, sugar beets, soybean, and potato. The positive tobacco plants were obtained under the selection pressure of Kanamycin, and the reference materials were multiplied by tobacco seed. PCR analysis confirmed that the transgenic tobacco contained all the target sequences of universal elements and one endogenous gene transformed. The transgenic tobacco was used as positive control in GMO screening detection and showed good specificity and accuracy.The study also analyzed the molecular characteristics and components distribution of the commercial transgenic varieties, finding that seven marker genes, including NPTII, PAT, CP4-EPSPS, Bar, HPTII, GUS and PMI, are used in high frequency currently or have potential applications in future. Then the conventional PCR and real-time PCR detection methods were developed with these seven genes as detecting targets.This study provided a new model for the preparation of reference materials used for GMO detection. The first transgenic tobacco-based reference material was successfully developed for screening detection, providing the possibility to overcome the shortcomings of the existing standard material. This study also established a standardized system for qualitative PCR detection targeting the marker genes, which has become a national standard.This research has made the following major findings: 1) Cloned 10 endogenous reference genes from 6 different crop species;2) Successfully constructed a binary vector (positive plasmid) pBI-121RM, containing 11 gene elements and 10 reference genes from 6 crops;3) Obtained transgenic tobacco with exogenous target sequences;4) Completed the commutablity test of the transgenic tobacco, confirming that the transgenic tobacco behaved well as positive control in GMO screening detection;5)Established the effective screening method targeting marker genes, which is specific, sensitive, reproducible, and suitable for large-scale screening detection of genetically modified agricultural products.