Construction And Application Of Positive Plasmid Molecules For Detection Of Genetically Modified Crops

With the constant development of transgenic technique in China, the expansion of the scale of the transgenic crops industry and the increase of the type and quantity of genetically modified crops(GMC), the task of the safety regulation of GMC will become more and more onerous, especially GMC illegally released into the environment occurred at times, so the safety supervision of the trial phase of GMC will be the key and difficult points of the safety management of GMC in China.When the positive sample of genetically modified crops is very limited, how to develop a kind of Reference Material(RM) easily acquired and producted by the genetic information of itself is very critical to meet the safety regulation work of GMC. In order to meet our current detection needs the positive plasmid molecules for GMC detection are the optimal choice.In this paper, we studied on those transgenic events which were in the experimental phage, and urgent need for reference material in safety management. We developed related detection methods and constructed several positive plasmid molecules, in order to solve the difficult problem in GMO detection that a lot of these transgenic positive matrix substances are difficult to obtain. The major research contents and the points of innovation in the study were as follows:1. Developed four detection methodsBased on the sequences of an herbicide resistant gene G2-aroA, a fungal source gene phytase, and the5’-flanking sequence of transgenic event Tlc-19and T2A-1a series of primer combinations were designed separately. The best primer combination was obtained by primer screening, then the PCR reaction system was optimized by improving Mg2+concentration, primer concentration and primer anneal temperature. The results of specificity test showed they had good amplification specificity. And the results of sensitivity test indicated that the target DNA was still observed when the template concentration was down to0.1%, which reaches the national standards for GMO detection.2. Constructed the plasmid pBS Endogenous which is containing the endogenous reference gene of rice, corn, soybean, cotton, wheat and rapeseedSix endogenous reference genes were selected according to China national standards. They were zSSIIb gene for corn, HMG I/Y for rapeseed, waxy-D1for wheat, SPS for rice, Sadl for cotton, and Lectin for soybean. Experiment results indicated that the plasmid pBS Endogenous had good specificity and sensitivity, could be used as positive control for the endogenous reference gene PCR amplification.3. Constructed seven gene-specific screening detection plasmidsSeven expressed genes which were usually used in transgenic events were selected, they were Bar, PAT, CP4-EPSPS(natural), CP4-EPSPS(modified), G2-aroA, Cry1Ab/1Ac and Phytase. These seven genes were cloned into plasmid separately and intended for gene-specific screening detection. Experiment results indicated that the plasmids were specific for identification of expressed gene, and the detection sensitivity reached50copies uL-1, the plasmid pG2-aroA could reach a sensitivity of10copies uL-1.4. Constructed twelve event-specific and two construct-specific(pCV127-G and pBVLA430101-G) positive plasmid molecules for the detection of GM rice, cotton, corn and soybean.These plasmids were constructed with the flanking sequence and specific endogenous reference gene. These plasmids included pKF6, pM12, pTlc-19, pT2A-1, pKMD were used for the detection of GM rice Kefeng6, M12, Tlc-19, T2A-1and Kemingdaol correspondingly; pEZM1, pNN98-7were used for the detection of GM cotton Ezmian1and Nannong98-7correspondingly; pA2704-12, pA5547-127, pCV127-Z were used for the detection of GM soybean A2704-12, A5547-127and CV127correspondingly; pBVLA430101-Z and pMIR162were used for the detection of GM maize BVLA43010and MIR162correspondingly. Experiment results indicated that the positive plasmid molecules were suitable in PCR assays, the detection sensitivity could reach50copies uL-1, the plasmid pM12, pEZMl, pMIR162and pCV127-G could reach a sensitivity of10copies uL-1.5. Organized10domestically qualified testing centers to verify the four detection methods and twenty-two positive plasmid molecules developed in this study. According to the inter-laboratory study, we determined the concentration of plasmids suitable for used in PCR assays. And the validation results showed that all four detection methods had good repeatability and reproducibility, and were specific, sensitive enough for routine work. In addition, the twenty-two plasmids were commutable for matrix reference material in GMO PCR assays.6. Using the results of this study, the plasmids were applied to detect three domestic GM rice Kefeng6, Kemingdaol and TT51-1. The results indicated that the plasmids constructed in this study were suitable for use in GMO detection.