Comparative Proteomics Of Phytase-Transgenic Maize And Its Non-transgenic Isogenic Variety

Genetically modified crops(GMCs)have been globally commercialized more than 20 years and have brought great benefits of economic.social and ecological to people.With the increase of the planting area of GMCs.the public violent controversy on the unintended,unexpected,and uncontrolled negative factors of GMCs is still prevailing.It makes the people very anxious that the introduction of exogenous DNA sequences and enzymes into the target plant genome in GMCs might result in some unintended effects,and these negative effects may further affect both human health and the environment safety.Phytase-transgenic(PT)maize is the only GM maize that has been officially issued with a biosafety certificate in China and has great potential in the animal feed industry.Many researches had been done to ensure its safety assessment,there was no research reported yet that was performed to investigate the untargeted effects on omics level.In this paper,we used 2-DE and/or isobaric tags for relative and absolute quantification iTRAQ-based approaches to compare the protein profiles in leaves and seeds between the phytase transgenic maize and its non-transgenic(NT)isogenic variety to investigate the unintended effects in GM maize.We hope to establish a set of scientific proteome technology for the unintended effects’ detection of GM crops and provide theoretical basis and technical support for biosafety assessment of transgenic corn or other GM crops.First,the comparative proteomic analysis of the leaves between the PT maize and the NT plants was performed using two-dimensional gel electrophoresis and mass spectrometry.The 2-DE protein profile of phytase maize leaves were obtained and 57 differentially expressed proteins(DEPS)were successfully identified which accounting for 6.7%of the total isolated protein spots;For these differential protein spots,most were naturally synthesized by the maize kernels,no changes in proteins known to be toxic or allergenic were detected in our study,the PT maize leaves could be judged to be substantially equivalent to the commercial NT maize,and GM didn’t dramatically alter the proteome profiles of the maize leaves.COG functional catalogue showed that about 40%DEPS were related to carbohydrate transport and metabolism,and KEGG analysis revealed that DEPs between WT and PT lines were mainly involved in carbon fixation in photosynthetic organisms;ribulose bisphosphate carboxylase(Rubisco)and sedoheptulose-1,7-two phosphatase(SBPase)showing higher expression in PT maize than in NT line that might be caused by random insertion of the exogenous genes in plant genomes.Then we compared the seed’s proteomics between the PT maize and NT maize which planting in greenhouse using 2-DE and iTRAQ techniques.2-DE protein profiles of phytase maize seeds were obtained and a total of 148 identified DEPS were obtained.Compared with the total identified proteins,the number of DEPS was significantly few(1.83%),a limited number of proteins were affected by gene insertion,and those DEPS were not identified as new proteins,no changes in proteins known to be toxic or allergenic were detected.These results indicate that DEPS were detected but not substantial differences in PT maize seeds and the PT maize seeds were substantially equivalent to the NT maize seeds.COG functional catalogue showed that about 40%DEPS were related to involve in post-transcriptional regulation and post-transcriptional modifications.For the function of post-transcriptional regulation,most of the related DEPS were ribosomal proteins,and the abundance of ribosomal proteins were increased and changes in mRNA expression levels via qRT-PCR analysis were up-regulated in PT maize seeds,these up-regulated effects might be responsed to the insertion of exogenous genes.For the function of post-transcriptional modifications,most of the related DEPS were heat shock proteins,however,some of these proteins were up-regulated and some were down-regulated,this finding suggested that different members of the HSPs had different functions in response to the insertion of exogenous gene.Moreover,we compared the seed proteomics between the PT maize and NT maize which planting in the field using 2D electrophoresis approach.32 DEPS were successfully identified which accounting for 6.7%of the total isolated protein spots.COG functional catalogue showed that about 40%DEPS were related to involve in post-translational modification,protein turnover,and chaperones.These DEPS maybe take part in the regulation of abiotic stresses through processes such as phosphorylation and ubiquitination.Finally,we compared the seed’s proteomics between the PT maize and NT maize which growth under different environmental conditions.2-DE protein profile showed that there were 77 differentially expressed protein spots in NT maize seeds between greenhouse cultivation and field planting and 76 differentially expressed protein spots in PT maize seeds between greenhouse cultivation and field planting.However,at the same cultivating condition,there were 43 different protein points between PT and NT maize seeds which growth under greenhouse or 37 different protein points between PT and NT maize seeds which planting in the field.The data show that the effect of environment on protein expression is greater than the influence of gene insertion.In addition,compared the NT seed’s proteomics between greenhouse cultivation and field planting,it also shows that the occurrence of unintended effects are not specific to transgenic technology,this is a common phenomenon inherent and often occur in traditional breeding.Environmental impact on the traditional crop is much greater than the impact of gene insertion.