The Integrated Structure And Event-specific PCR Detection Methods Of The Cry1Ac Transgenic Cotton Line Ezamian 1

The molecular structure of transgenic crop is the basis of the biosafety assessment as well as the basis of the qualitative and quantitative detection methods. Transgenic cry1Ac cotton"Ezamian 1"was analyzed in this study. The insertion structures of foreign DNAs in host genome and the genome flanking sequences were obtained with the molecular biotechnology and methods, and event-specific qualitative and quantitative PCR detection methods were established in accordance with the genome flanking sequences in this cotton line. The relative parameters of the developed qualitative and quantitative PCR system with better stability, higher specificity and sensitivity could fufill the international requirments of transgenic detection. The main results were summarized as follows:1. The insertion structure of the transgenic cry1Ac cotton"Ezamian 1"was analyzed and the genome flanking sequences was obtained by the Genome Walking method. The length of the insertion structure and foreign DNAs in host genome were 13198bp-long, including 1-11901bp were exogenous DNA sequences, and 11902-13198bp were cotton genome sequences; insertion site contained a complete copy of the cry1Ac gene and the nptII marker gene.2. Based on the genome flanking sequence, qualitative and quantitative PCR detection methods were established. The sensitivity of the qualitative PCR detection method was 43 copies, corresponding to 0.1% in 100 ng templates. Obviously, the qualitative PCR detection method designed according to the genome flanking sequences with good stability, high specificity and sensitivity, could be used for the detection of the transgenic cry1Ac cotton"Ezamian 1"event. Quantitative PCR detection limit of quantification was less than 50 copies, the detection limit was 50 copies, this value could meet the current 5% -0.5% international GM labeling system needs, through transgenic and non-transgenic 5% and 3% of the mixed samples testing, found that the accuracy of the stability of quantitative methods fullfiled the international requirements of transgenic detection.In this paper, the analysis of the foreign DNA insertion structure obtained had provided draft information on the molecular characterization of the transgenic cry1Ac cotton"Ezamian 1". The result provided the basis for researching of the foreign DNAs, and the scientific basis and methods for detecting and screening of breeding materials, intellectual property right and regulatory safety evaluation.