Study On Genetic Stability Of Transgenic Crops With Stacked Traits

The transgenic crops with stacked traits,which were obtained by combining two or more transgenic events together by hybridization,extending the function of transgenic crops and improving resource utilization,have become the main trend of transgenic crops development.Safety evaluation system for transgenic crops with stacked traits needed to be formulated as soon as possible to regulate transgenic crops in China.In order to study the genetic stability of transgenic maize with complex traits of insect-resistance and herbicide-resistance,SW-1,which harboring both of the traits was obtained through hybridization of event IE034 containing cry1 Ie and 12-5 containing cry1Ab/cry2Aj/ G10evo-EPSPs.The integration and expression of foreign genes of SW-1 were analyzed,and the insect and herbicide resistance were tested.Transcriptome and metabolism were used to analyze the unintended effects resulting from combining transgenic crops.The main results were as bellow:1.The multiplex PCR detection system was established by screening of primer specificity and the optimization of reaction conditions.In the established PCR system,three target genes and endogenous reference gene zSSIIb,could be detected simultaneously with sensitivity of 0.5%.2.Detection of the insertion site of target genes in the SW-1 with event-specific primers: the integration sites of three target genes cry1 Ie,cry1Ab / cry2 Aj and G10evo-EPSPs in the SW genome were same as those in the parents 12-5 and IE034.Event-specific PCR showed that the three target genes could be stably inherited in SW1 and its offspring SW-2 and SW-3.3.The exogenous genes of the transgenic maize were quantified through droplet digital PCR(ddPCR)to determine the copy number of the exogenous genes.The results showed that the copy number of cry1 Ie,cry1Ab / cry2 Aj and G10evo-EPSPs in the transgenic maize SW-1 did not change compared with the parents.4.The transcriptional level of three exogenous genes of SW-1 was studied by RT-PCR and qPCR.The results showed that three exogenous genes in SW-1 were stable transcript.But the expression level of mRNA was different between SW-1 and parents depending on copy number.5.The three target genes in SW-1,SW-2 and SW-3 could be stably expressed at the protein level.The expression of Cry1 Ab was analyzed by ELISA,and the expression levels of 12-5 and SW-1 were 32.22 μg/g and 31.88μg/g respectively.Hybrids SW-1,SW-2,SW-3 protein expression level were 31.88 μg/g,30.30 μg/g and 31.22 μg/g,respectively,and did not reach significant difference.6.Bioassay in lab showed that there was no difference in the insect resistance between SW-1 and its parents 12-5 and IE034.In the herbicide resistance test,12-5 and SW-1 spray the same concentration of herbicide 4000 ppm(about the recommended concentration of 2 times),12-5 and SW-1 presented a good herbicide resistance.7.A total of nine maize samples including SW-1,12-5,IE034,transgenic receptor and conventional cultivars were transcriptome analysis.The differences in gene expression between different maize cultivars were 3263-12595,and the differences in gene expression between transgenic maize and their receptor were less than that between different cultivars,differences between SW-1 and its parents were less than that between different cultivars.These results suggest that stacked transgenic crop through breeding does not cause greater variation in the maize transcriptome and does not have an unintended effect on the stability of the genome.8.A total of 10 maize samples including SW-1,12-5,IE034,transgenic receptors and conventional cultivars were sequenced.A total of 1185 metabolites were obtained.PCA and correlation analysis showed that the difference between hybrids SW-1 and the parents was less than that between conventional cultivars,and the similarity between the parents was above 99%;Cluster analysis showed that although there were some differences in the metabolites(P <0.05),there were some differences between 10 samples,but difference between samples did not bring change above the lines.The difference between the SW-1 and parents was less than that between the cultivars.The above results indicate that the breeding compound will not have unintended effects on the maize metabolic group.