Studies On Detection Of Transgenic Soybean MON87705and Its Products

In1983，the world’s ifrst genetically modiifed crop-transgenic tobacco was bornin American. Because of the fast development of transgenic technology, manycountries promulgated laws and regulation on controlling the safety of geneticallymodiifed crops and their products. The prerequisite to implement it is to establishaccurate detection methods of genetically modiifed organisms. Monsanto usedagrobacterium-mediated transformation. The genetically modiifed soybeanMON87705is discovered by the Monsanto Company through agrobacteriummediated transformation method of soybean meristem tissue to introduce DNA intosoybean A3525. It had transferred the plasmid vector PV-GMPQ/HT4404containingtwo genes cassettes. FATB1-A and FAD2-1A gene segments were assembled in onesuppression cassette under the control of a seed promoter. And the other is cp4-epsps(Herbicide resistance gene) expression cassette.According to the revealed lfanking sequences of transgenic soybean MON87705，four speciifc primers were designed. Select the most appropriate a pair of primersthrough PCR method. And obtain the optimum reaction system by optimizing thereaction conditions. As a result, this study established a speciifc test method ofMON87705. With the development of the research on transgenic safety, qualitativeresearch could not meet the requirement of testing. Thus, it is a demand to studyfurther design appropriate probe and primer of Real-time quantitative PCR. Astandard curve was established for the alignment of GMO detection. Meanwhile, it isimportant to construct a calibrator plasmid to solve the problem which the CertiifedReference Material is diiffcult to obtain. The main results were as follows:1.Comparing the primers that reported in related literatures and designed bysoftware, the result indicated that the eiffciency of MON877Q5GF/GR (318bp) is higher than other primers, and its optimal annealin°g temperature was58C.Qualitative PCR detection method of MON87705could speciifcally detect thesamples with the detection sensitivity about0.1%.7different GMO safety testingorganizations also got this result. It means that the detection qualitative PCR methodwas suitable to be the national standard of GM soybean MON87705and its products.2.Theprimers and probes were designed based upon the right border sequencesof MON87705. The primer MON87705qF/qR and probe MON87705qP had the bestsensitivity and speciifcity. Real-time lfuorescence quantitative PCR detection showedthe limit of detection was15copies.3.Thelectin gene and MON877053’end of insertion sequence part wereconnected by overlapping PCR method. The new fragment was inserted into thevector pMD18-T by designing speciifc primers. The detection positive standardplasmid molecule was named pMD-87705. The results indicated that the pMD-87705plasmid can be used as standards for MON87705soybean detection.