Detection Of Transgenic Bt Rice And The Degradation Of Bt DNA And Proteins In The Thermal Processing

Transgenic technology has been widely employed in rice breeding along with the rapid development of transgenic technology.China led the world in transgenic rice research,especially in the field of Bt gene application in the rice.In order to meet the needs of the genetically modified(GM)labeling and safety assessment of GM crops,in this paper,two aspects about the related research were conducted.Firstly,multiplex event-specific qualitative polymerase chain reaction to detect the main GM rice lines in China and application of a standard plasmid as a quantitative reference molecule.Omthe other side,degradation and detection of transgenic Bt DNA and proteins in genetically modified rice submitted to various thermal processes was studied.1.Multiplex event-specific qualitative polymerase chain reaction to detect the the transgenic rice lines of TT51-1,KMD1 and KF6The three most well-known genetically modified(GM)rice lines in China are TT51-1,KMD1,and KF6.Due to different degree of inadvertently released,and the elements of the three GM rice were detected in the Chinese food market.Meanwhile,the European Union committee of food and feed reported many times for the check out rice insect-resistant Bt genes of the three kinds of GM rice.In the three GM rice only TT51-1 has obtained the safety certificates from the Chinese government,but not be allowed the commercial cultivation.The purposes of this study were to establish a multiplex event-specific qualitative polymerase chain reaction(meqPCR)system for simultaneous detection of the three transgenic rice events.Event-specific primers for each event were selected or designed by focusing on the transgene borders between the inserted DNA and the flanking rice DNA.The developed meqPCR was anticipated to detect distinct amplicons as 454,398,301,and 250 bp from KF6,KMD1,TT51-1,and the rice endogenous reference gene,respectively.The robustness of the meqPCR was tested with different levels of the three transgenic rice genomic DNAs and the sensitivity threshold of the meqPCR was at least 50 ng 0.1%rice DNA for each event when the three transgenic rice events present and with other GM materials together.2.Construction and application of plasmids as the reference molecule for detection of Genetically modified(gm)ricePositive referenc materials are need for detection of GM ingredients in the routiing work,however,there is a relative lack of positive matrix reference materials.In this study,firstly,we contrsucted a plasmid for quantitative analysis of TT51-1,KMD1 and KF6,respectively.The plasmid was contained part sequcence of rice endogenous gene PLD and the events spesifical sequence of TT51-1,KMD1 and KF6.The resulsts of the analysis of the quantitative repeatability,limit of the detection(LOD)and the limit of quantification(LOQ)demonstrated that the constructed plasmid was suit for quantitative analysis.Then the constructed plasmid was evaluated using mixed samples with known GM contents in real-time quantitative PCR.The results indicated that the constructed plasmid was acceptable and suitable for TT51-1,KMD1 and KF6 quantitative analysis,and meet the need of reference materials for quantitative analysis of the three kinds of GM rice.Further,in order to solve the problem of lack of reference materials in the screening tests of transgenic rice in routing detection.In this work,we integrated the rice endogenous gene of sucrose phosphate synthase(SPS),CaMV35S promoters,NOS terminator,Bt gene,Bar,Hpt and NPT ? genes into a plasmid,the experimental results show that build plasmid molecules can be a very good application in screening tests of genetically modified(gm)rice.3.Degradation and detection of transgenic Bt DNA and proteins in genetically modified rice submitted to various thermal processes.Considering the Bt genes in the widespread use of GM crops,and especially for the degradation of exogenous Bt gene and protein of the GM rice in food processing is not in-depth study.In the present study,three lines of insect-resistant transgenic rice,KMD1,KF6,and TT51-1,harboring CrylAb,CrylAc,or the fused Cty1Ac/Cry1Ab genes,respectively,were selected for studies on the effect of thermal processing on the degradation and detection of these Bt genes and proteins in rice.At present,CrylAb,CrylAc,and Cry1Ac/Cry1Ab are the most commonly used Bt genes in transgenic crops,especially in Chin.Based on previous studies,temperature plays an essential role in DNA and protein degradation in the processing of transgenic crops.To mimic the temperature effects on the degradation of Bt genes and proteins,different thermal treatments,including autoclaving,cooking,baking,and microwaving,were used.To eliminate matrix effects,KMD1,KF6,and TT51-1 rice seed powders were directly subjected to these thermal treatments without other components.Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase(SPS)genes,and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes.Autoclaving,cooking(boiling in water,30 min),and baking(200?,30 min)induced the most severe Bt protein degradation effects,and Cryl Ab protein was more stable than Cry1Ac and Cryl Ab/Ac protein,which was further confirmed by baking samples at 180? for different periods of time.Microwaving induced mild degradation of the Bt and SPS genes,and Bt proteins,whereas cooking and baking led to further degradation,and autoclaving induced the most severe degradation.The findings of the present study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensityFinally,conventional PCR methods,combined with methods based on detection of proteins by immunoassays such as enzyme-linked immunosorbent assay(ELISA)and lateral flow tests(LFT),were used to determine the degradation and detection of Bt genes and proteins.