Study On The Detection Technology For Unknown GMOs By Targeted Deep Sequencing

The commercial application of transgenic technology in the field of agriculture has accelerated the global agricultural output and completely subverted the agricultural pattern of the world.In recent years,it has gradually become the focus of agricultural science and technology competition in various countries.With the proliferation of transgenic crop planting area and the increasing investment in technology research and development,the potential harm of genetically modified organisms and their product components to human health and ecological environment has also attracted extensive attention from all walks of life.Therefore,countries all over the world have strengthened the legal supervision of genetically modified products,and put forward more stringent requirements for the detection of genetically modified products.At present,there are great differences in transgenic marking system,threshold range and management mode in different countries and regions,which directly affect the import and export inspection of genetically modified products,international trade mutual recognition and so on.Due to the proliferation of transgenic crop planting area,illegal planting and gene lateral drift,the demand for transgenic detection is becoming more and more diversified and complicated.However,the existing transgenic detection technology based on PCR is inefficient,the strategy is single,and there are many technical limitations,so it is difficult to meet the needs of the rapid development of agricultural transgenic industry.In particular,the lack of transgenic blind inspection technology seriously limits the ability of government supervision,but also restricts the industrialization of transgenic technology.Therefore,the development of accurate,efficient and high-throughput blind detection methods of transgenic genes is of great significance to perfect the existing transgenic detection technology system,promote the healthy development of agricultural transgenic technology,meet the needs of biological supervision and control,and support the management system of transgenic markers.In this study,transgenic soybean was selected as the research object,and the foreign transgenic DNA fragments were enriched by subtractive hybridom.combined with high throughput sequencing and bioinformatics analysis,an accurate and reliable blind detection method for transgenic soybeans was established,and the breakthrough in the field of transgenic blind detection technology was realized.The main research Results of this study are as follows:1.Construction of database of foreign genes and transformation elements.Using literature search,database search and other search methods,integrati ng all available transgenic elements,foreign genes,DNA sequences and detecti on methods of transformation vectors,covering transgenic databases: ISAAA,E U-RLGMFF,CERA,GMDD,CropLife,GMOseek and NCBI.To establish the DNA sequence database of transgenic elements and foreign genes to provide data sup port for the research of transgenic blind detection technology.2.Preliminary establishment of blind detection method for transgenic genes.The suppression subtractive hybrid technique(Suppression Subtractive Hybridization,SSH),was used to enrich the foreign transgenic fragments with the help of 30 times non-transgenic DNA with the help of genomic DNA of 200 ng transgenic soybean(DAS-81419).With the help of 30 times of non-transgenic DNA,the foreign transgenic fragments were enriched by genomic enzyme hydrolysis,two rounds of crossing and moderate nest PCR amplification.3.Blind detection of mixed samples of multiple transgenic soybeans was realized.The mixed samples of eight transgenic soybean lines DAS-81419 ?MON87701,DAS44406,MON87705,MON87769,MON89788,MON87708,GTS40-3-2,with high market share were prepared.The genomic DNA of each soybean 100 ng was used as the starting quantity,combined with PCR-free high throughput sequencing technology and bioinformatics analysis,the detection efficiency was 100% under 10 X high throughput sequencing data,and the blind detection method of transgenic gene was established,compared with the intrinsic gene Lectin,.The enrichment efficiency of foreign genes can reach 40 times,the detection sensitivity is very high,and the detection specificity is good.4.Optimization of technical parameters for blind detection of transgenic genes.Aiming at the preliminary blind detection method of transgenic gene,the parameters were further optimized in the aspects of the ratio of target DNA starting quantity,Driver DNA,the selection of four-base endonuclease,the hydrolysis efficiency of genomic DNA,the enrichment effect,the capture efficiency of streptavidin magnetic beads,and the improvement of bioinformatics data analysis process,so as to reduce the capture of unexpected DNA fragments as much as possible and improve the enrichment efficiency of foreign transgenic fragments.Thus,the cost of high throughput sequencing is reduced,the efficiency of data analysis is improved,and the false positive and false negative Results are minimized.