Molecule Characterization And Event-specific Detection Of Glyphosate-resistant Transgenic Maize Harboring 2mG2-epsps

The genetically modified(GM) corn was one of the initial launched and marketed crops in the history of agriculture, is also the most widely planted GM grain crops in the world. However, till the date of this study there was no transgenic maize approved to be produced in China on the industrial large scale, one of the reasons is that the lack of feasible protocol for molecule characterization,to distinguish the wild type from the GMO plant, which blocked the progress of safety evaluation. G2-epsps, a glyphosate resistant gene(isolated by our group) has been codon optimized to 2m G2-epsps, consequently, The transgenic 2m G2-epsps maize showed high glyphosate resistance in the field. Toward molecular characterize the transgenic maize we studied the strain to find out the copy-number and the location of the exogenous T-DNA along the whole genome of GM strain. The RT-PCR, Hi-TAIL-PCR and molecular hybridization techniques were used, besides establishing the trans-formant specific PCR detection method based on the flanking sequences. Not only did this study provide molecule characterization information and detection method for safety evaluation of the GM Maize, but also provide strong guarantee to apply for safety certificates as well as inspection and supervision after the commercial cultivating. The main contents and results were as follows:(1) Q-PCR and Western bolt were used to analysis the expression of 2m G2-epsps gene in the strains D?T255?T254?T257 respectively, the results indicated that all the strains can express the 2m G2-epsps normaly, which show the same result as Rapid diagnosis strip.(2) Southern bolt and was used to analysis the copies of T-DNA integrated into transgenic maize, the results indicated that strain D has 2 copies of 2m G2-epsps while T254 and T257 have 3 copies and strain T255 has 4 copies. So we regard strain D as the sample for the following study.(3) 3'-terminal high-efficiency thermal asymmetric interlaced PCR(hi TAIL-PCR) and long distance PCR(LD-PCR) methods were used to obtain the complete s equence and flanking sequence of T-DNA in transgenic maize D. The length of the T-DNA was 3827 bp, which contained a 2m G2-epsps gene derived by the ubiquitin promoter and a Nos-Ter and a CTP. The two exogenous T-DNA loc ated in the chromosome?(227622408..227656679) and chromosome?(113077961-113077617) respectively.(4) The event-specific PCR detection method has been established by designing primers based on the flanking sequences and sequence of T-DNA. And using th e wild type and transgenic maize, rape, rice soybean as control to analysis the sensitivity and specificity. The results indicated that the detection method has se nsitivity of which was 0.05% in per 100 ng template with high specificity.