Study On The Establishment Of DNA Extraction Method And Study On Its Distribution In The Processing Of Transgenic Soybean Oil

In recent years,as one of the most important genetically modified crops in the world,the application rate of genetically modified soybeans is high.Genetically modified soybeans are mainly used for oil production,and the foods made from them have become increasingly widely involved in people’s lives.As the most widely used genetically modified soybean products,whether or not foreign genes exist in soybean oil and how they exist have become issues of common concern to consumers and scholars.The existence status of DNA in oil is closely related to DNA extraction methods and oil processing technology.Currently,DNA extraction methods in soybean oil still need to be improved,and the distribution pattern and degradation mechanism of DNA in raw soybean during oil processing are still unclear.Magnetic nanoparticles are a new type of polymer material,and detection techniques based on magnetic nanoparticles have been successfully applied to various fields of scientific research.Therefore,this paper first introduces magnetic nanoparticles into the process of extracting DNA from oil,establishes a magnetic bead method for extracting DNA from oil,evaluates its extraction efficiency,and applies it to the soybean oil processing process to explore the distribution and degradation of DNA during the soybean oil processing process.Firstly,this article establishes a magnetic bead method for extracting DNA from genetically modified soybean oil,which can successfully extract DNA for PCR positive amplification from genetically modified refined soybean oil.In this paper,the extraction magnetic beads with surface modification groups,the composition of the adsorption system,and the key extraction steps were discussed and optimized.The results showed that compared to hydroxyl groups,magnetic nanoparticles with carboxyl groups as surface modification groups had stronger adsorption capacity for soybean oil DNA;Silicon dioxide magnetic beads modified with carboxyl groups have the strongest adsorption capacity when the particle size is 300 nm;In the adsorption system that promotes the binding of magnetic beads to DNA,the salt type is preferably guanidine isothiocyanate,and its concentration is preferably 1 mol/L,and the p H of the adsorption system is preferably 6.0.After determining the above conditions,the polarity of the adsorption system is optimized.It is found that the magnetic beads have the highest adsorption efficiency for DNA after adding anhydrous ethanol of the same volume as the adsorption system;The presence of oil in the adsorption system before adding magnetic beads significantly affects the stability of the magnetic bead DNA complex.The use of chloroform isoamyl alcohol extraction can greatly improve the efficiency of magnetic bead DNA adsorption;In the DNA washing step,magnetic separation is superior to centrifugal separation.Secondly,this article compares the magnetic bead method established in the previous article with Announcement No.1485 of the Ministry of Agriculture of the People’s Republic of China-4-2010 A.5.3 method,the national standard GB/T 19495.3-2004 C.6.3 CTAB-3method,the commercially available resin based oil DNA extraction kit method,and the CTAB–Nucleo Spin Food Kit method reported in current representative literature to investigate and verify the efficiency of the magnetic bead method in extracting DNA.The results showed that for DNA extracted from soybean oil,the agarose gel electrophoresis results of its genomic DNA may be false negative and misjudged;Among the five methods,the magnetic bead method has the highest efficiency in extracting DNA from soybean oil,followed by the CTAB–Nucleo Spin Food Kit method.This is mainly manifested in 1)only the double nested PCR test using the magnetic bead method and CTAB–Nucleo Spin Food Kit method can amplify positive electrophoresis bands,and 2)real-time fluorescence quantitative PCR results show that 0.093 ng and 0.088 ng DNA can be extracted from 1 m L of soybean oil using the magnetic bead method and CTAB–Nucleo Spin Food Kit method,respectively,However,DNA cannot be extracted using the other three methods.3)A DNA labeling recovery test was conducted on refined soybean oil,and it was found that the extraction recovery rate of the magnetic bead method was 76.38%,the extraction recovery rate of the CTAB–Nucleo Spin Food Kit method was 62.15%,and the recovery rate of the GB/T 19495.3-2004 C.6.3 method was only 6.69%.The other two methods did not measure recovery.Thirdly,this article explores the distribution of DNA during soybean oil processing.At present,the distribution pattern of DNA during soybean oil processing is not clear,and related research lacks systematicity.The magnetic bead method established in the previous article was applied to the processing of soybean oil to extract DNA from various main intermediate products in the process,and the distribution of DNA in the process was discussed and studied.The results showed that DNA in the raw soybean（3.56×10⁶ng/g and 3.27×10⁶ng/g）during the processes of crushing,tempering,embryo rolling,and extrusion,there was no significant change in the total amount of DNA at 10⁶ ng/g.After being extracted by organic solvents,a small portion was separated into soybean crude oil（2×10²ng/g）;After degumming treatment,a small amount of DNA in soybean crude oil is transferred into the degumming oil(7.43×10^-1ng/g and 6.93×10^-1 ng/g);There was no significant change in the mass concentration of DNA in degummed oil,deacidified oil,and decolored oil;Therefore,DNA in deodorized oil(3.6×10^-2 ng/g and 2.6×10^-2 ng/g)mass concentration is relatively low;Organic solvent extraction and degumming are key steps for DNA removal from refined oil;Obtaining key intermediate products from different soybean oil processing factories,research has found that although there are differences in the mass concentration of DNA from the same intermediate product,the differences are not significant.The distribution pattern of DNA during soybean oil processing in different factories is consistent.Finally,this article explored the mechanism of DNA degradation during soybean oil processing.There are few reports on the mechanism of DNA degradation in food substrates during processing.In this paper,the chain lengths of lectin and cp4epsps genes,which are key intermediate products in the process of soybean oil production and refining,were studied,and the morphological changes of DNA during soybean oil processing were analyzed.The results showed that during the process of soybean oil production,n-hexane extraction could degrade1067 bp and 1589 bp fragments of lectin gene and 1099 bp and 2070 bp fragments of cp4epsps gene;During the refining process of soybean oil,deodorization treatment can degrade 438 bp fragments of lectin gene and 370 bp fragments of cp4epsps gene;Fragments of internal and external genes below 200 bp are present in degummed oil,deacidified oil,decolorized oil,and deodorized oil;As the processing of soybean oil deepens,DNA strands in raw soybean break,resulting in shorter DNA strands and weaker entanglement;During the processing of soybean oil,organic solvent extraction and deodorization are the key steps to initiate DNA degradation;Obtaining key intermediate products from different soybean oil processing factories,although there are differences in the detection rate of degraded fragments for the same key intermediate product DNA,the differences are not significant.The degradation mechanism of DNA during soybean oil processing in different factories is consistent.