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| Establishment of an Event-specific Method to Detect Transgenic Rice(Oryza sativa) EB7001S |
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Abstract As more and more genetically modified organisms (GMOs) and their products coming into all areas of human life, to detect GMOs and their products accurately, quickly and efficiently becomes basic requirements for the research and safety management of GMOs. The transgenic rice(Oryza sativa) EB7001S resistant to two kinds of non-selective herbicide was developed recently by our research team using Agrobacterium-mediated transformation method. In order to establish the event-specific detection method of transgenic rice EB7001S, the high efficient thermal asymmetric interlaced PCR (hiTAIL-PCR) and long distance PCR (LD-PCR) were adopted to reveal the flanking sequences of foreign genes in this research. Firstly, the right flanking sequence in length of 1 515 bp was revealed by hiTAIL-PCR, in which the upstream sequence from the 1st to 169th bp belonged to the vector sequence and the remaining was the rice genomic sequence of Chromosome 7. Secondly, the left flanking sequences in length of 1 460 bp was amplified by LD-PCR, which included a 777-bp (the 684th to 1460th bp) vector sequence and a 683-bp (the 1st to 683th bp) rice genomic sequence of chromosome 7. The BLAST analysis in NCBI database showed that the foreign genes were inserted into the position 1 470 725 of short arm of chromosome 7. The insertion of foreign genes resulted in 3 bases deletion (1 470 726~1 470 728). The event-specific qualitative PCR method were successfully developed by designing specific primer pairs based on the right and left flanking sequence, respectively. In the specific primer pair, one primer was designed based on the rice genomic sequence, and the other primer based on the foreign gene sequence. The event-specific detection methods could amplify a 458 bp and a 629 bp fragments from right border and left border respectively in transgenic rice EB7001S, but could not amplify in any other transgenic or non-transgenic rice, which showed strong specificity and high stability. When the DNA templates to be amplified by event-specific detection methods were the mixture of different percentage of genomic DNA of EB7001S, the sample only contained 0.1% EB7001S genomic DNA could be detected, of which the sensitivity could satisfy the detection demands of the Customs, Administration for Industry and Commerce, Ministry of Agriculture and so on. Then, the tri-primer PCR method based on the flanking sequence was also established to select homozygous genotype from transgenic offspring quickly and accurately. One primer GEB-F was designed according to the upstream of the insertion site on Chromosome 7, the 2nd primer and the 3rd primer were the event-specific primers (RB-F, RB-R) of right flanking sequence. When the tri-primer PCR run, the homozygous plants of foreign genes amplified a 458 bp fragment, the plants free of foreign genes showed a 808 bp fragment, and the heterozygous plants displayed both 458 bp and 808 bp fragments simultaneously, which made the homozygous plants identified from one segregative generation. The detection method established in this research will provide key technical supports for the utilization and detection of transgenic rice EB7001S.
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Received: 24 December 2013
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SHEN Xue-Jing, ZHANG Er-Qin, XU Qian-Ru, LIU Lin-Ke, WAN Bo, LIU Yun-Chao, SUN Ya-Ning, ZHAO Dong, NIU Xiang-Xiang, DENG Rui-Guang, ZHANG Gai-Ping. Expression of Rabies virus G Protein in Rice (Oryza sativa) and Identification of Its Genetic Stability[J]. 农业生物技术学报, 2019, 27(2): 204-211. |
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