Study On Molecular Characterization And Detection Method Of Insect-resistant And Herbicide-tolerant Maize With Cry1Ab And Epsps Genes

With the commercial production of transgenic crops,the planting area has been continuously expanded.Taking turns cry1 Ab and epsps gene insect-resistant herbicideresistant corn as material,through the determination of the genetically modified maize exogenous insert fragment sequence,insertion site,flanking sequences and copy number and other information,for safety assessment of genetically modified corn to provide the basis of molecular characteristics.The results showed that:1.The molecular characteristic information of the gene resistant to insect-resistant maize of the transgenic cry1 Ab and epsps gene was clarified.(1)The insertion element was detected by conventional PCR technology.Based on the principle of long chain PCR,high fidelity enzyme was used to determine the sequence of insertion segment.After PCR amplification and sequencing result analysis,the total length of insertion sequence was 12499 bp,and the transfer sequence was inserted into the maize Chr01 chromosome,at 271,871,046.(2)Using the Southern bloting and micro flow drops digital PCR two methods of transgenic insect-resistant herbicide-resistant corn exogenous insertion sequence of copy number.The target gene was identified as a single copy.2.The transformation body specific qualitative PCR method was established for the conversion of cry1 Ab and epsps gene resistance to insect resistance.The specific primers were designed,and the specificity,reaction system and reaction procedure were optimized and determined.Mainly for:(1)Using ordinary PCR method,to determine the specific primers,upstream primer for5'-CAGTACTAAAATCCAGATCCCCCGA-3,downstreamprimers for 5'-CAGACGACGGTCCGCTAA-3,expected amplified fragment size of 259 bp.(2)Using ordinary PCR method,to determine the most suitable primer concentration of0.4 mu mol/L,58 ? annealing temperature.(3)Using ordinary PCR method,determines the specificity of the good detection method,detection limit of 0.1%,meet the needs of genetically modified organisms safety inspection regulation.