Study On Detection Method Of Genetically Modified Herbicide Tolerant Soybean ZH10-6 Based On PCR Technology

In recent years,with the increase of the types and planting areas of genetically modified(GM)crops,the kinds of foods containing GM ingredients are constantly enriched.GM soybean is the most important GM crop with huge economic benefits.It is of great significance to establish detection methods for the effective management of GM food and food safety.GM soybean ZH10-6 is a new herbicide-tolerant GM soybean line with G2-EPSPS and GAT gene,which is resistance to broad-spectrum herbicides.Glyphosate has an important industrial application prospect in China.In this study,we used this new herbicide-tolerant GM soybean line as the test material,determined molecular characteristics and designed primers to establish a qualitative and quantitative detection protocol for herbicide-tolerant GM soybean ZH10-6.The main results are as follows:(1)The molecular characteristics of GM soybean ZH10-6 were determined.Exogenous genes include G2-EPSPS gene and GAT gene,as well as regulatory elements include four copies of P-35S,two copies of T-35S and T-NOS.GAT gene,3’flanking sequence and 5’flanking sequence are all single copy.The exogenous gene insertion site is between 7980527-7980541(Chromosome 17)of soybean genome.(2)A PCR qualitative detection method of ZH10-6 was Established.The amplification sensitivity to the GM herbicide-tolerant soybean ZH10-6 can reach to 0.025 ng/μL when the primer temperature is 0.4 pmol/L and the annealing temperature is 54℃.The detection limit is 0.1%.After testing on processed products,this protocol with a high specificity meets requirements for the establishment of qualitative methods in Announcement No.2259-4-2015.It can be used for the qualitative detection of herbicide-tolerant soybean ZH10-6 transformants.(3)A multiple PCR qualitative detection method for transgenic soybean ZH10-6 was established.The concentrations for boundary A,boundary B,boundary C,boundary D,and endogenous Lectin primers were 0.6 μmol/L,0.3 μmol/L,0.1μmol/L,0.2μmol/L,and 0.05μmol/L separately.The optimum annealing temperature is 56℃.This protocol can be used since the concentration of ZH10-6 is higher than 1%.Specificity test has proved that this protocol has a good specificity and can be used for qualitative detection of transgenic herbicide-tolerant soybean ZH10-6 transformants.(4)A real-time fluorescent PCR(qPCR)detection method for genetically modified herbicide-tolerant soybean ZH10-6 was established.The primer concentration of the system is 0.4μmol/L and the probe is 0.2 μmol/L.The method has a high specificity and sensitivity with LOD=0.032%and LOQ=0.16%.The standard curve is:Y=-3.416X+30.257.The linearity is greater than 0.995 within the range of 0.0064%to 100%template content,and the amplification efficiency is 95.4%to 96.2%.It meets the requirements of Announcement No.2259-5-2015 about the establishment of real-time fluorescent PCR detection methods It is suitable for further quantitative analysis of genetically modified herbicide-tolerant soybean ZH10-6.(5)A droplet digital PCR accurate quantitative detection method for genetically modified herbicide-tolerant soybean ZH10-6 was established.The concentration of primer probe is 0.5μmol/L and the annealing temperature is 60℃.The detection limit of this method is 0.016%when the RSD is less than 25%,and the quantification limit is 0.08%.The linear relationship between DNA content and copy number is:Y=23.593X+40.205,with linearity greater than 0.998.The test of processed products proved that this method can be applied to the precise quantitative study of the specific sequence of the transgenic herbicide-tolerant soybean ZH10-6 transformant.In summary,this study determined the accurate sequence information of the genetically modified herbicide-tolerant soybean ZH10-6 through molecular characterization.Primers were designed and precise quantitative detection method such as single PCR,multiplex PCR,qPCR and droplet PCR were established.It provides new technical methods for qualitative and quantitative detection of genetically modified herbicide-tolerant soybean ZH10-6,Furthermore,this project provides technical support for the detection of GM products after the commercialization of the GM soybean lines.