Proteomics analysis reveals that foreign cp4-epsps gene regulates the levels of shikimate and branched pathways in genetically modified soybean line H06-698

Abstract

Although genetically modified (GM) glyphosate-resistant soybeans with cp4-epsps gene have been widely planted all over the world, their proteomic characteristics are not very clear. In this study, the soybean seeds of a GM soybean line H06-698 (H) with cp4-epsps gene and its non-transgenic counterpart Mengdou12 (M), which were collected from two experiment fields in two years and used as 4 sample groups, were analyzed with label-free proteomics technique. A total of 1706 proteins were identified quantitatively by label-free quantification, and a total of 293 proteins were detected as common differential abundance proteins (DAPs, FC is not less than 1.5) both in two groups or more. Functional enrichment analysis of common DAPs identified from four groups, shows that most up-regulated proteins were clustered into stress response, carbon and energy metabolism, and genetic information processing. Further documentary analysis shows that 15 proteins play important roles in shikimate pathways, reactive oxygen species (ROS) and stress response. These results indicated that the change of protein abundance in different samples were affected by various factors, but except shikimate and branched pathways related proteins, only ROS and stress-related proteins were found to be stably regulated by cp4-epsps gene, and no unexpected and safety-related proteins such as antinutritional factors, allergenic proteins, and toxic proteins were found as DAPs. The influence of foreign genes in genetically modified plants is worthy of attention and this work provides new clues for exploring the regulated proteins and pathways in GM plants.

Keywords: Cp4-epsps gene; genetically modified; label-free proteomics; shikimate pathway; soybean.

Figure 1.

Experimental design and workflow for quantitative proteomic analysis of soybean seeds using label-free LC/MS technology.

Figure 2.

Identification of proteins from quantitative proteomics analysis. (a) Principal component analysis of 4 groups of H and M samples. (b) Pearson correlation analysis of 4 groups of samples. (c) The intensity distribution of quantitative identified protein. (d) The sequence coverage distribution of quantitative identified protein. “H1-2” showed the average value of H1 and H2. It was the same as “M1-2,” “H3-4” and “M3-4.” * Indicated p-value < 0.05, ** indicated p-value < 0.01.

Figure 3.

Comparison of proteomics data. (a) Hierarchical cluster analysis of 293 common DAPs in H and M. (b) Venn diagrams represent the overlap of up-regulated DAPs. (c) Venn diagrams represent the overlap of down-regulated DAPs.

Figure 4.

Function classification of differential abundance proteins (DAPs) from H and M. (a) Gene ontology analysis of up-regulated proteins. (b) Gene ontology analysis of down-regulated protein. Y-axis represented each GO term; X-axis represented rich factor, size of bubble represented the number of DAPs in each category, the depth of color represented -log10 (p-value) CC represented cellular component, BP represented biological process, and MF represented molecular function. (c) Pathway analysis of DAPs. TCA, tricarboxylic acid; CHO, carbohydrate.

Figure 5.

The schematic representation general shikimate pathway, down-stream and/or branched metabolic pathway related proteins. Among colored background boxes, green means up-accumulated DAPs, blue means down-accumulated DAPs, yellow means undetected or unchanged proteins, or metabolites. The green dashed arrow indicates shikimate and the downstream pathway, red dashed arrow indicates branched pathway. Abbreviations: ACL, ATP-citrate lyase; CAND1, CULLIN-ASSOCIATED NEDD8-DISSOCIATED 1 (CAND1); DAHPS, 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase; E4P, D-Erythrose 4-phosphate; ENO, Enolase; EPSPS, 5-Enolpyruvylshikimate-3-phosphate synthase; GSTLs, Lambada class glutathione S-transferase; OA, oxaloacetate; PEP, phosphoenolpyruvate; PA, proanthocyanidins.