The Development Of Molecular Detection Methods For Genetically Modified Crops And Creation Of Salt-tolerant Transgenic Soybean Material With AgGlpF Gene

Aiming at the demand of safety supervision of genetically modified organisms(GMO)in China for fast,accurate,and high throughput detection techniques,this study took several common exogenous insect-resistant genes and new transgenic corn events as the targets.By using conventional polymerase chain reaction(PCR),real-time quantitative PCR,digital PCR,multiplex PCR,and loop-mediated isothermal amplification(LAMP),several molecular detection methods based on exogenous nucleic acid of transgenic crops were developed.Furthermore,to create new salt-tolerant transgenic soybean and provide excellent germplasm resources for the cultivation of salt-tolerant soybean varieties,we transformed AgGlp F gene into cultivated soybean varieties.This gene was cloned early in halophilic Aspergillus and verified with potential salt tolerance by a model organism,Arabidopsis thaliana.The obtained main results are as follows:1.In order to establish gene-specific method for screening transgenic crops,we analyzed the nucleotide sequences of commonly insect resistant genes used in transgenic crops developed by domestic and foreign scientists.According to the consistency of nucleotide sequences,eight common Bt genes,including cry1 Ab,cry1Ac,cry1Ab/Ac,mcry1 Ac,cry1A.105,cry2 Ab,cry3A,and cry3 Bb,were divided into three categories: Cry1 A,Cry2A and Cry3 A.Three simplex-PCR detection method were developed for detecting these Bt gene groups,respectively,and a triplex PCR method also established to screening the three categories of Bt genes simultaneously.Thereinto,the Cry1 A genes method used degenerate PCR strategy.The above methods can accurately detect the target genes from various transgenic samples,and the limit of detection is up to 0.1%.As these methods showed highly specificity and sensitivity,it would be capable to rapidly screening common Bt genes(such as cry1 Ab,cry1Ac,cry1Ab/Ac,etc)in genetically modified crops.2.In addition to conventional PCR method,the rapid LAMP assays for detecting cry2 Ab and cry3 A genes in transgenic crops were also developed in this study.The results shown that using the LAMP method can specifically distinguish transgenic crops containing target gene from other GM and non-GM crop.The absolute detection limit was approximately five copies of haploid genomic DNA,about fourfold greater than that of conventional PCR assays.Moreover,the LAMP assay can be finished within 1 hour,shorten half of the time compared with conventional PCR method.The derived LAMP products can be directly observed by the naked eye through adding SYBR Green I dye,which can provide a quick and reliable approach to detect genetically modified components on-site.3.To strengthen safety assessment,safety supervision and intellectual property protection of transgenic corns IE034 and Bt506 developed by Chinese scientists,we developed qualitative and quantitative PCR assays for detecting these two transgenic corns.Some key parameters of the established methods,such as specificity,sensitivity,limit of detection(LOD),limit of quantitation(LOQ),accuracy and precision,were tested in this study.The results suggested that the qualitative PCR assays of IE034 and Bt506 corns had strict specificity for corresponding event and the detection sensitivity reached 0.05 % which was equivalent to about 20 copies of genomic DNA.The LOD of the IE034 event-specific real-time PCR method was estimated at approximately 8 initial template copies,and the LOQ was estimated at about 40 copies.The accuracy of this method was verified by screening four mixed samples with known levels of the IE034 event(5,1,0.5,and 0.23 %,respectively).The quantified biases were 12.2,3.0,8.0,and 8.7 % respectively.For Bt506 event-specific real-time PCR assay,the LOD was estimated at approximately 4 copies,and the LOQ was estimated at about 35 copies.The accuracy of this method was verified by screening three mixed samples with known levels of the Bt506 event(5,2,and 0.5 %,respectively).The quantified biases were 10.96,18.08,and 12.64 % respectively.These deviations were all in accordance with the criterion issued by ENGL and acknowledged universally in the academic field.Overall,these accurate methods established in this study are applicable to the qualitative identification and quantitative analysis of transgenic maize event IE034 and Bt506.4.The real-time quantitative PCR assay was established for detecting transgenic maize event Bt38 developed by Chinese scientists.The identification results of two practical samples using this method showed that accurate and reliable results were obtained no matter through a simplex or a duplex quantitative PCR system.For simplex q PCR system,the quantified biases were 7.44 % and 14.28 % respectively,and for duplex q PCR system,the biases were 2.50 % and 17.34 % respectively.Furthermore,we transformed the Bt38 eventspecific q PCR method to a droplet digital PCR method(dd PCR).The quantitative results of the two practical samples mentioned above employing the simplex and duplex event-specific dd PCR assays were nearly the same as the results of q PCR.These data indicated that both the new-developed Bt38 event specific q PCR and dd PCR methods are suitable and reliable for the identification and quantification of Bt38 maize and its derivates.5.The AgGlp F gene was transferred to the conventional soybean variety Willams82 using cotyledonary node transformation mediated by Agrobacterium.Being screened by herbicides and identified by PCR,twenty-nine positive soybean seedlings with AgGlp F gene were acquired.Then three transgenic soybean lines with single copy of AgGlp F gene were identified from the T1 generation transgenic plants by Southern blotting.The result of reverse transcription quantitative PCR showed that AgGlp F gene was transcribed to m RNA steadily in different organisms such as leaf,root and stem of transgenic line E8A7027-816 and E8A7027-817.The expression level of the gene in leaves is the highest,followed by that in stems and roots.With salt stress of 200 mmol/L Na Cl,the transgenic soybean line E8A7027-817 grew normally in contrast with the withering and death of non-transgenic soybeans after 3 weeks under the salt stress.The result shown that overexpression of AgGlp F gene can dramatically increase salt tolerance in soybean,which provided an effective way for getting transgenic soybeans varieties with salt tolerance.Furthermore,the fluorescent quantitative PCR method for detecting AgGlp F gene was developed,and provided an efficient approach for targeted gene identification and expression analysis.