Novel Approaches To Molecular Characterization Identification And Multiplex Detection Of Transgenic Crops

The detailed molecular characterization and specific detection technology are the key factors for commercialization and market supervision of genetically modified(GM)crops.Meanwhile,the detection of the GM crops is based on the accurate molecular characterization.In order to develop better and more comprehensive molecular characterization identification technology,in this study,randomly broken fragment PCR with 5' end-directed adaptor was established for obtaining the flanking sequence of single or low copy insertion;next generation sequencing with bioinformatics analysis method was established for indentifying the molecular characterization of a transgene crop generated by microparticle bombardment.Simultaneously,multiplex PCR technology was conbimed with the newly obtained molecular characterization information of GM crops,two high-throughput multiplex detection methods with high efficiency,specificity and sensitivity were established.The main contents and conclusions of this study were as follows:1.Randomly broken fragment PCR(RBF-PCR)with 5' end-directed adaptor for genome walking was established.The genome was fragmented randomly without restriction enzyme,and the developed 5' end-directed adaptor effectively enhanced the specificity of PCR and prevented the self-ligation between the adaptors.All of the steps in this new method were the basic techniques of molecular biology with straightforward operation.Experimental results in GM maize LY038 showed that the left and right flanking sequences could be obtained by employing RBF-PCR.Through the study,RBF-PCR was proved to be a gemone walking method with high specificity and reliability,an unknown sequence could be precisely and confidently obtained by applying the method just once,and it could be applied to obtaining the flanking sequence of single or low copy insertion in GM crop and other aspects related to genome walking.2.A strategy of identifying the molecular characterization of GM crops generated via microparticle bombardment was established based on next generation sequencing and bioinformatics analysis.Two sequence alignment and analysis patterns according to the different reference sequences were put forward to reveal the molecular characterization information,and the information in different sequencing depthes was also discussed.By using this method,13 insertion sites and flanking sequences of GM maize BT176 were obtained,which broke the consensus that only one definite insertion site exsited in BT176.Furthermore,in order to analyze and indentify the molecular characterization of a GM crop,the sequencing depth should be as high as possible,at least 30×;if the sequencing data was used to detect the GM crop compounds,the 15× sequencing depth was enough to provide the event-specific information.These results proved that this novel method was a highly efficient and accurate strategy of identifying the molecular characterization,and it offered a support for the safety assessment and commercialization of GM crop generated via microparticle bombardment.3.A novel single universal primer multiplex ligation-dependent probe amplification(SUP-MLPA)technique was established.The complexity of the reaction system was reduced through transforming the structure of ligation probes,two reversely complementary common sequences were designed on the 5'or 3' end of the ligation probes,which also preserved the high specificity of the original method.SUP-MLPA products were analyzed on sequencing gel electrophoresis with the extraordinary resolution.As much as seven target genes,including event-specific regions,screen genes,endogenous reference genes of GM crops MON810,MON863 and GTS 40-3-2,were successfully detected by applying this novel method,and the low detection limit was 0.1 ng.Moreover,through comparing the results of quantitative PCR,the ligation probes with different stuffer sequences did not affect the ligation efficiency,which provided the possibility of increasing the multiplicity of this assay in future study.4.The qualitative and quantitative universal primer-multiplex-PCR with pyrosequencing using only one sequencing primer detection technology was established.Software of base sequence acquisition and sequencing result calculation was self-compiled based on the principle of pyrosequencing for the first time,which realized the quantitative detection.In this novel method,the detection did not depend on any electrophoresis ways or probes labeled with fluorescence,which saved the cost and time,and offered a new technical support for the GM crop detection.The molecular characterization indentification and multiple qualitative and quantitative detection technologies establided in this study will not only offer a powerful technical support for the safety assessment,qualitative and quantitative screening of GM crop,but also provide a new idea for other related fields.