The Exogenous Gene Integrated Structure And Event-specific Detection Of Insect Resistant Transgenic Cotton

Cotton is one of the earliest commercially grown genetically-modified crops, and is the firstgenetically-modified plants studied in China. In this paper, a new line of self-cultivated transgeniccotton06N-119was studied. First exogenous cry1A gene copy number in30strains of06N-119sampleswere estimated by real-time quantitative PCR.2cotton samples with about one cry1A gene copynumber were obtained. HiTAIL-PCR and LD-PCR were used to obtain the complete sequence ofexogenous DNA insertion at a new transgenic cotton line06N-119.On this basis, event-specificdetection method and homozygote screening method of line06N-119were established. The maincontents and results were as follows：1. Estimating the exogenous cry1A gene copy number of transgenic insecticidal cotton06N-119.Cotton endogenous reference gene SadⅠand exogenous cry1A gene were cloned into pUC57vector,which was used standard plasmid. By optimization of primers and probes concentration, standard curveof estimating the exogenous cry1A gene copy number was fabricated using TaqMan real-timequantitative PCR.30plants of06N-119were detected by real-time quantitative PCR to determine thecopy number of exogenous gene cry1A. The results showed that two samples with06-1and06-11onecry1A copy number were obtained.2. Determine the flanking sequence of06N-119. Twice of3’-terminus high-efficiency thermalasymmetric interlaced PCR (hiTAIL-PCR), four times of5’-terminus hiTAIL-PCR and one time longdistance PCR (LD-PCR) were used to oBtain the complete sequence of exogenous DNA insertion at anew transgenic cotton line06N-119. The length of the insertion is11578bp and it consists of the NptⅡgene expression cassette and the Bt gene expression cassette in series. A75bp cotton genomic DNAsequence was lost at insertion site and no gene recombination occurred.3. Establishing the event-specific PCR detection of06N-119. Pairs of event-specific detectionprimers were designed between the3’-terminus and cotton genome junction regions. By screening tests,MC1prime was used to detect the06N-119event-specific prime of which the detection limitationreached to0.05%.4. Establishing the homozygote screening method of line06N-119. Two pairs were determined forthe PCR detection. MF1was for cotton genome sequence and MF2was for the determination of3’-terminus specific sequences.30plants of06N-119were detected by this method. The resultsindicated that we got four transgenic homozygote plants were obtained.