Construction, Quantification And Analysis Of Reference Molecules Of Bt Transgenic Cotton And Rice

Since the success of genetically modified crops, the genetically modified industry had developed rapidly, following the safety suspicion of genetically modified products. However, there were few of standard products which could be used for transgenic detection. In this research, two reference molecules were constructed to alleviate the serious lack of the standard product in detection of the genetically modified product. One reference molecule contained the sequences of cry1Ab gene and the sequences of fsACP gene which was used as an internal standard genetic sequences in cotton. The other contained the sequences of cry1Ab, fsACP,35S, NOS, SPS and NPTII. The correctness of reference molecules were confirmed by analysis of PCR, restriction and sequencing. The reference molecules could be used to detect the genetically modified materials which contained the genes of cry1Ab,35S, NOS and NPTII. Besides, the reference molecules could be used for quantitative analysis of transgenic rice and transgenic cotton. The methods of construction, preparation and correctness verification of reference molecules could be able to provide a reference example for the research of standard plasmid.The method of quantification of reference molecule was not unified. In this research, three quantitative methods of reference molecule were used. The results were different among spectrophotometry, PicoGreen and digital PCR. The quantitative accuracy of reference molecule which quantified by digital PCR was highest, following by the PicoGreen method, and the quantitative accuracy of reference molecule which quantified by spectrophotometry was last.The PCR and real-time PCR system of transgenic detection was optimized and the repeatability, uniformity, homogeneity, stability, specificity and commutability of reference molecules were also analyzed. The results were showed as following:1), The specificity of the sequences of cry1Ab, fsACP,35S, NOS, SPS, NPTII and the primers used in this research were detected very well;2), The repeatability, uniformity and homogeneity of reference molecules were very well, and the reference molecules could be used as the candidate of reference material;3), After three months stored at low temperature (-70℃、-20℃、-4℃), the stability of reference molecules showed very well. But the results of stability at high temperature (20℃、40℃) showed that the reference molecules could not be stored at higher temperature;4), The plasmid with shorter insert segments were more stable than the plasmid with the longer;5), The reference molecules could be used as the substitutes of genomic DNA for the transgenic detection. This study provided a scientific basis and an example of the analysis and storage of reference molecules.The sensitivity of genetically modified detection under the system was analyzed. The results were showed as following:1), The ordinary PCR system could detect the genetically modified materials which contained more than0.5%transgenic component, and the LOD (limit of detection) of reference molecules were50copies/μL.2), The qPCR system could detect the genetically modified materials which contained more than0.5%transgenic component, and the LOD (limit of detection) of reference molecules were10copies/μL. The sensitivity of the reference molecules achieved the requirements of the threshold value of minimum detection (0.9%) in many countries.