Study On Detection Methods Of Transgenic Rice And Reference Material

The technology of transgenic rice is becoming more and more mature,and more and more transformants have obtained safety permits or entered in different countries.Field trials,but as an important staple food,its industrialization and safety issues have caused widespread social concern.In recent years,emergencies related to transgenic rice have occurred from time to time,causing unnecessary trade frictions and public panic,which are highly valued at home and abroad.Safety evaluation and regulatory testing of transgenic rice.Therefore,it is necessary to strengthen the research on transgenic rice detection methods and reference materials and improve the level of regulatory capabilities.This research first optimized the genomic DNA extraction method based on silicon matrix membrane columns,developed a new plant seed genomic DNA extraction kit,and analyzed the relationship between rice seed grinding size and genomic DNA extraction quality to improve genomic DNA quality and The yield provides the basis for the next step of PCR detection.Secondly,the screened genes in rice and the reaction systems and procedures that have been discovered and developed in this research have been optimized and screened.Further research has been conducted to develop the standards for the detection of rice gene standards.The screening elements of transgenic rice were analyzed statistically,a screening and detection method for transgenic rice was established,and a positive standard plasmid for detection was constructed.Finally,a standard material for genomic DNA of transgenic insect-resistant rice and indica rice was successfully developed to provide a material basis for detection.These results provide a data foundation and technical support for the safety evaluation and management of transgenic rice,the implementation of the labeling system,and the supervision and inspection.The main results we achieved in this study are as follows:1.Developed a new plant seed DNA extraction kit.Nucleic acid-based PCR technology is the most stable for transgenic detection and reliable methods,and obtaining high-quality genomic DNA is an important basis for guaranteeing test results.Various biological companies have also developed a variety of DNA extraction kits.However,in practical applications,there are extraction quality and yield.The problem of low cost and high cost.Based on the method of silicon matrix membrane column,this study developed a plant seed DNA extraction kit by optimizing the pH value and salt concentration of the column buffer,which was compared with the evaluation in the commercial industry.Compared with high kits,it has significantly improved the quality and yield of genomic DNA extraction,and carried out research on the genomic DNA extraction efficiency based on the rice seed grinding particle size,providing a basis for the nextPCR detection.2.Developed national standards for standard gene detection in transgenic rice.Various transgenic rice have been established at home and abroad transformant-specific detection methods,but the use of different internal standard genes affects the comparability between the detection results.In this study,domestic and international standards,published rice internal standard genes,and new rice internal standard genes selected through this study were used as targets.Primers and probes were designed to compare the differences between different internal standard genes in different rice varieties.And in-depth analysis and comparison with the sensitivity of detection,screen out the rice standard genes with good interspecific specificity,intraspecific consistency,stable copy number,and high sensitivity,and correlate them with PCR amplification detection Optimize the reaction procedures and reaction system,establish common PCR and real-time fluorescent PCR qualitative detection methods for standardized genes in rice,further provide technical support for the safety management,identification system implementation of genetically modified rice in China,and the standardization of transgene detection.3.A method for screening and detecting transgenic rice was established,and a positive standard plasmid was constructed.In the daily testing of genetically modified rice,each testing agency has not determined uniform testing parameters,and sometimes the same sample will have different testing results between different testing agencies,bringing uncertainty to customers and government law enforcement,and unnecessary disputes to the testing agency.This study collected and counted the foreign components of transgenic rice reported at home and abroad,and analyzed the frequency and coverage of these components,and further established a screening test method for transgenic rice.At the same time,a positive for screening and specific transformant detection Standard plasmid,which provides a unified standard for the regulatory testing of transgenic rice;4.A genomic DNA reference material for transgenic rice and indica rice was developed.Genetically modified insect-resistant rice indica rice has been introduced in China at the stage of productive test research,both at home and abroad are very concerned.The detection methods and standards have been released one after another,but there is no standard substance or standard for detection.In this study,the indica rice was used as the basic material,and the indica rice digital PCR detection method was established.The genomic DNA was extracted,and the homogeneity and stability tests were performed.The digital PCR method was used to jointly determine the values in eight laboratories.The indica rice genomic DNA standard material has filled the domestic gap and also provided an important material basis for the regulatory inspection of rice.