The environmental impacts of genetically modified (GM) plants remain a controversial global issue. To address these issues, comprehensive environmental risk assessments of GM plants is critical for the sustainable development and application of transgenic technology. In this paper, significant differences were not observed between microbial metagenomic and metabolomic profiles in surface waters of the Bt rice (T1C-1, the transgenic line) and non-Bt cultivars (Minghui 63 (the isogenic line) and Zhonghua 11 (the conventional japonica cultivar)). In contrast, differences in these profiles were apparent in the rhizospheres. T1C-1 planting increased soil microbiome diversity and network stability, but did not significantly alter the abundances of potential probiotic or phytopathogenic microorganisms compared with Minghui 63 and Zhonghua 11, which revealed no adverse effects of T1C-1 on soil microbial communities. T1C-1 planting could significantly alter soil C and N, probably via the regulation of the abundances of enzymes related to soil C and N cycling. In addition, integrated multi-omic analysis of root exudate metabolomes and soil microbiomes showed that the abundances of various metabolites released as root exudates were significantly correlated with subsets of microbial populations including the Acidobacteria, Actinobacteria, Chloroflexi, and Gemmatimonadetes that were differentially abundant in T1C-1 and Mnghui 63 soils. Finally, the potential for T1C-1-associated root metabolites to exert growth effects on T1C-1-associated species was experimentally validated by analysis of bacterial cultures, revealing that Bt rice planting could selectively modulate specific root microbiota. Overall, this study indicate that Bt rice can directly modulate rhizosphere microbiome assemblages by altering the metabolic compositions of root exudates that then alters soil metabolite profiles and physiochemical properties. This study unveils the mechanistic associations of Bt plant-microorganism-environment, which provides comprehensive insights into the potential ecological impacts of GM plants. 

/pdf/combined-metagenomic-and-metabolomic-analyses-reveal-that-bt-1ywr5ryt.pdf

Combined metagenomic and metabolomic analyses reveal that Bt rice planting alters soil C-N metabolism

Evaluating response mechanisms of soil microbiomes and metabolomes to Bt toxin additions.

The full-length sequence of integrated Bt gene in genetically modified rice TT51-1,was amplified and cloned into pGEM-T vector. After sequencing was confirmed,the Bt gene was inserted into pET-28a expression vector using HindⅢand BamHⅠ. The recombined pET-28a-cry was transformed into E. coli BL21(DE3)and the recombined target protein(rCRY)was expressed in E. coli BL21(DE3)host cells under 0.1mmol/L IPTG induction. The purified rCRY protein with the relative molecular mass 7.1×104 was recovered from SDS-PAGE gel by denaturation and refolding of inclusive body protein. This work would be helpful for the development of new GMOs detection methods.

Cloning and Prokaryotic Expression of Insect-resistant Gene Cry1Ab/Ac

Soil nutrient cycling and microbiome responses to Bt rice cultivation

25 Years of Pesticidal Cry1Ab/Ac Fusion Proteins in Crop Protection: Advances in Bt Crop Development, Target Pest Management, Safety, Environmental Impact, and Regulatory Frameworks

Synthetic Cry1Ab/Ac proteins expressed by genetically modified (GM) crops have a high potential to control insect pests without utilizing large amounts of chemical insecticides. Before these crops are used in agriculture, the environmental fate and interactions in the soil must be understood. Stable isotope-labeled Cry1Ab/Ac protein is a highly useful tool for collecting such data. We developed a protocol to produce 13 C/15 N single-labeled Cry proteins. The artificially synthesized gene Cry1Ab/Ac of Bt rice Huahui No. 1, which has been certified by the Chinese government to be safe for human consumption, was subcloned into pUC57, and the expression vector pET-28a-CryAb/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM isopropyl thiogalactoside (IPTG) was added to these cells and cultured at 37°C for 4 h to induce the synthesis and formation of inclusion bodies in M9 growth media containing either [U-13 C] glucose (5% 13 C-enriched) or [15 N] ammonium chloride (5% 15 N-enriched). Then, Cry inclusion bodies were dissolved in urea and purified by affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The purities of 13 C/15 N-labeled Cry proteins reached 99% with amounts of 12.6 mg/L and 8.8 mg/L, respectively. The δ 13 C and a 15 N values of 13 C-labeled Cry protein and 15 N-labeled Cry protein were 3,269‰ and 2,854‰, respectively. A bioassay test revealed that the labeled Cry1Ab/Ac proteins had strong insecticidal activity. The stable isotope-labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies on Cry proteins in soil and the characteristics of nitrogen (N) and carbon (C) transformations. Our findings may also be employed as a reference for elucidating the environmental behavior and ecological effects of BT plants and expressed products.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/mbo3.1125

Production and characterization of the 13 C/15 N single-labeled insecticidal protein Cry1Ab/Ac using recombinant Escherichia coli.

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Production and characterization of the 13 C/15 N single-labeled insecticidal protein Cry1Ab/Ac using recombinant Escherichia coli.