Proteomic analysis of the seeds of transgenic rice lines and the corresponding nongenetically modified isogenic variety.

BACKGROUND: An isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis was employed to study the seeds of two genetically modified (GM) rice lines, T2A-1 and T1C-19, and their nontransgenic isogenic variety, MH63, to investigate the unintended effects of genetic modification. RESULTS: A total of 3398 proteins were quantitatively identified. Seventy-seven differentially abundant proteins (DAPs) were identified in the T2A-1/MH63 comparison, and 70 and 7 of these DAPs were upregulated and downregulated, respectively. A pathway enrichment analysis showed that most of these DAPs participated in metabolic pathways and protein processing in endoplasmic reticulum and were ribosome components. Some 181 DAPs were identified from the T1C-19/MH63 comparison, and these included 115 upregulated proteins and 66 downregulated proteins. The subsequent pathway enrichment analysis showed that these DAPs mainly participated in protein processing in endoplasmic reticulum and carbon fixation in photosynthetic organisms and were ribosome components. None of these DAPs were identified as new unintended toxins or allergens, and only changes in abundance were detected. Fifty-four co-DAPs were identified in the seeds of the two GM rice lines, and protein-protein interaction analysis of these co-DAPs demonstrated that some interacting proteins were involved in protein processing in endoplasmic reticulum and metabolic pathways, whereas others were identified as ribosome components. Representative co-DAPs and proteins related to nutrients were analyzed using qRT-PCR to determine their transcriptional levels. CONCLUSIONS: The results suggested that the seed proteomic profiles of the two GM rice lines studied were not substantially altered from those of their natural isogenic control. © 2020 Society of Chemical Industry.

Cry80Aa1, a novel Bacillus thuringiensis toxin with mosquitocidal activity to Culex pipiens pallens.

Bacillus thuringiensis (Bt) can synthesize insecticides to efficiently control insects. In this study, Bt strain S3580-1 with mosquitocidal activity was subjected to whole genome sequencing using an Illumina HiSeq 2000 system. A novel toxin, Cry80Aa1, was identified based on the resulting data. A conserved domain analysis revealed that Cry80Aa1 includes the Ricin_B_lectin domain (located at 10-150) and the Toxin_10 domain (located at 155-353). Phylogenetic tree analysis showed that Cry80Aa1 was in a distinct clade significantly distinguished from the known Cry proteins containing the Toxin_10 domain. Bioassays demonstrated that the Cry80Aa1 protein exhibited toxicity to third instar larvae of Culex pipiens pallens (LC50: 71.9 µg/mL; 95% FL: 59.5-122.7 µg/mL).

Halomonas huangheensis sp. nov., a moderately halophilic bacterium isolated from a saline-alkali soil.

A novel, Gram-stain-negative, aerobic, rod-shaped, non-motile and moderately halophilic bacterium, designated strain BJGMM-B45(T), was isolated from a saline-alkali soil collected from Shandong Province, China. Growth of strain BJGMM-B45(T) occurred at 10-45 °C (optimum, 30 °C) and pH 5.0-12.0 (optimum, pH 7.0) on Luria-Bertani agar medium with 1-20 % (w/v) NaCl (optimum, 7-10 %). The predominant respiratory quinone was Q-9. The major cellular fatty acids (>5 %) were C18 : 1ω7c, C16 : 0, C19 : 0 cyclo ω8c, summed feature 3, C12 : 0 3-OH and C12 : 0. The genomic DNA G+C content was 57.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BJGMM-B45(T) belonged to the genus Halomonas in the class Gammaproteobacteria. The closest relatives were Halomonas cupida DSM 4740(T) (98.2 % 16S rRNA gene sequence similarity) and Halomonas denitrificans M29(T) (97.8 %). Levels of DNA-DNA relatedness between strain BJGMM-B45(T) and Halomonas cupida CGMCC 1.2312(T) and Halomonas denitrificans DSM 18045(T) were 57.0 and 58.9 %, respectively. On the basis of phenotypic, chemotaxonomic and phylogenetic features, strain BJGMM-B45(T) is considered to represent a novel species of the genus Halomonas, for which the name Halomonas huangheensis sp. nov. is proposed. The type strain is BJGMM-B45(T) ( = ACCC 05850(T) = KCTC 32409(T)).

Recombinase polymerase amplification (RPA) of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops.

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37-42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15-25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean). With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops.

Mosquitocidal toxin-like islands in Bacillus thuringiensis S2160-1 revealed by complete-genome sequence and MS proteomic analysis.

Here, we present the whole genome sequence of Bt S2160-1, a potential alternative to the mosquitocidal model strain, Bti. One chromosome genome and four mega-plasmids were contained in Bt S2160-1, and 13 predicted genes encoding predicted insecticidal crystal proteins were identified clustered on one plasmid pS2160-1p2 containing two pathogenic islands (PAIs) designed as PAI-1 (Cry54Ba, Cry30Ea4, Cry69Aa-like, Cry50Ba2-like, Cry4Ca1-like, Cry30Ga2, Cry71Aa-like, Cry72Aa-like, Cry70Aa-like, Cyt1Da2-like and Vpb4C1-like) and PAI-2 (Cyt1Aa-like, and Tpp80Aa1-like). The clusters appear to represent mosquitocidal toxin islands similar to pathogenicity islands. Transcription/translation of 10 of the 13 predicted genes was confirmed by whole-proteome analysis using LTQ-Orbitrap LC-MS/MS. In summary, the present study identified the existence of a mosquitocidal toxin island in Bacillus thuringiensis, and provides important genomic information for understanding the insecticidal mechanism of B. thuringiensis.

Proteomic and Metabolomic Evaluation of Insect- and Herbicide-Resistant Maize Seeds.

Label-free quantitative proteomic (LFQ) and widely targeted metabolomic analyses were applied in the safety evaluation of three genetically modified (GM) maize varieties, BBL, BFL-1, and BFL-2, in addition to their corresponding non-GM parent maize. A total of 76, 40, and 25 differentially expressed proteins (DEPs) were screened out in BBL, BFL-1, and BFL-2, respectively, and their abundance compared was with that in their non-GM parents. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that most of the DEPs participate in biosynthesis of secondary metabolites, biosynthesis of amino acids, and metabolic pathways. Metabolomic analyses revealed 145, 178, and 88 differentially accumulated metabolites (DAMs) in the BBL/ZH58, BFL-1/ZH58, and BFL-2/ZH58×CH72 comparisons, respectively. KEGG pathway enrichment analysis showed that most of the DAMs are involved in biosynthesis of amino acids, and in arginine and proline metabolism. Three co-DEPs and 11 co-DAMs were identified in the seeds of these GM maize lines. The proteomic profiling of seeds showed that the GM maize varieties were not dramatically different from their non-GM control. Similarly, the metabolomic profiling of seeds showed no dramatic changes in the GM/non-GM maize varieties compared with the GM/GM and non-GM/non-GM maize varieties. The genetic background of the transgenic maize was found to have some influence on its proteomic and metabolomic profiles.

Single universal primer recombinase polymerase amplification-based lateral flow biosensor (SUP-RPA-LFB) for multiplex detection of genetically modified maize.

In this study, an isothermal paper biosensor, combining single universal primer recombinase polymerase amplification (SUP-RPA) and the lateral flow technique was developed for the multiplex detection of genetically modified maize (GMM). In pre-amplification stage, the event-specific primers contain a universal sequence at the 5' end, with a biotin-labeled deoxycytidine triphosphate (dCTP) deoxynucleotide providing additional amplification, which improves their amplification ability and ensures consistent multiplex amplification efficiency. In the signal recognition strategy, the SUP-RPA products are identified visually using the lateral flow biosensor (LFB) through dual hybridization. The accumulation of gold nanoparticles (AuNPs) produces a characteristic red band. Through this biosensor, a limit of detection of at least 50 copies was achieved, which is sensitive enough to detect MON810, MON863 and MON89034 simultaneously. The entire process of analysis was completed within 30 min and without any large-scale instrumentation. This biosensor, therefore, provides a novel rapid and portable multiple detection method for point-of-care applications, especially genetically modified organism (GMO) event-specific detection.

Bacillus thuringiensis novel toxin Epp is toxic to mosquitoes and prodenia litura larvae.

As a pathogenic bacterium, Bacillus thuringiensis (Bt) has become an alternative to chemical insecticides in commercial agricultural to control forestry pests and mosquitoes. To prevent pest resistance, many novel Bt strains have been isolated. Strain S3580-1 (WGS: VHPX0000000) used in this research and originally isolated from Hainan Qixianling National Forest Park (China) showed significant toxicity to Culex pipiens pallens. Here, using whole genome sequencing, assembly, and bioinformatics analysis, the predicted S3580-1CG_5163 (GenBank Accession No. MK124137) gene-encoded protein was found to share low homology with known toxins designated by the Bt toxin nomenclature system. It was considered to be an ETX/MTX2-type toxin and was designated Epp. Bioinformatics analysis showed that the predicted S3580-1CG_5163 gene-encoded protein Epp shared low identity with other known toxic protein sequences containing Cry-ETX/MTX conserved domains at the amino acid level, but significant similarity at the structural level. In addition, bioassays showed that Epp was toxic against Spodoptera litura (LC50 296.133 µg/mL; 95% FL 200.555-471.318 µg/mL) and Cx. pipiens pallens (LC50 322.193 µg/mL; 95% FL 238.217-477.243 µg/mL). On pathological observation, the peritrophic membrane of Cx. pipiens pallens larvae was degraded causing the midgut structure to become incomplete, resulting in larval death. Further bioassays are required to fully elucidate the insecticidal spectrum of the ETX/MTX2-type toxin Epp, and thereby provide future research directions.

iTRAQ-based quantitative tissue proteomic analysis of differentially expressed proteins (DEPs) in non-transgenic and transgenic soybean seeds.

The unintended effects of transgenesis have increased food safety concerns, meriting comprehensive evaluation. Proteomic profiling provides an approach to directly assess the unintended effects. Herein, the isobaric tags for relative and absolute quantitation (iTRAQ) comparative proteomic approach was employed to evaluate proteomic profile differences in seed cotyledons from 4 genetically modified (GM) and 3 natural genotypic soybean lines. Compared with their non-GM parents, there were 67, 61, 13 and 22 differentially expressed proteins (DEPs) in MON87705, MON87701 × MON89788, MON87708, and FG72. Overall, 170 DEPs were identified in the 3 GM soybean lines with the same parents, but 232 DEPs were identified in the 3 natural soybean lines. Thus, the differences in protein expression among the genotypic varieties were greater than those caused by GM. When considering ≥2 replicates, 4 common DEPs (cDEPs) were identified in the 3 different GM soybean lines with the same parents and 6 cDEPs were identified in the 3 natural varieties. However, when considering 3 replicates, no cDEPs were identified. Regardless of whether ≥2 or 3 replicates were considered, no cDEPs were identified among the 4 GM soybean lines. Therefore, no feedback due to GM was observed at the common protein level in this study.

A transcriptomic analysis for identifying the unintended effects of introducing a heterologous glyphosate-tolerant EPSP synthase into Escherichia coli.

Glyphosate is one of the most commonly used broad-spectrum herbicides with little to no hazard to animals, human beings, or the environment. Some microbial 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase variants are not inhibited by glyphosate, and they provide a powerful tool to engineer glyphosate-tolerant plants. However, the unintended effects of EPSP synthase expression patterns on microbes are not yet clear. Here, we use an Affymetrix GeneChip analysis to study how introduction of a heterologous glyphosate-tolerant EPSP synthase into a model microorganism Escherichia coli (E. coli) affects the global gene expression profile. The profile showed that 161 of 4071 genes were differentially expressed after the introduction of the synthase: 19 (0.47%) were up-regulated and 143 (3.49%) were down-regulated. The microarray results, in combination with BiOLOG substrate utilization and amino acid composition assays, suggested that heterologous EPSP synthase expression had very minor effects on E. coli. Although a small number of genes and metabolites were affected by EPSP synthase expression, no functional correlations were identified among the dataset. This study may shed light on the effect of EPSP synthase expression on microbes, which should help in the assessment of environmental safety.

A novel reference plasmid for the qualitative detection of genetically modified rice in food and feed.

Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes (the sucrose phosphate synthase gene, SPS, and the phosphoenolpyruvate carboxylase gene, PEPC). pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice.

Development of a novel reference plasmid for accurate quantification of genetically modified Kefeng6 rice DNA in food and feed samples.

Reference plasmids are an essential tool for the quantification of genetically modified (GM) events. Quantitative real-time PCR (qPCR) is the most commonly used method to characterize and quantify reference plasmids. However, the precision of this method is often limited by calibration curves, and qPCR data can be affected by matrix differences between the standards and samples. Here, we describe a digital PCR (dPCR) approach that can be used to accurately measure the novel reference plasmid pKefeng6 and quantify the unauthorized variety of GM rice Kefeng6, eliminating the issues associated with matrix effects in calibration curves. The pKefeng6 plasmid was used as a calibrant for the quantification of Kefeng6 rice by determining the copy numbers of event- (77 bp) and taxon-specific (68 bp) fragments, their ratios, and their concentrations. The plasmid was diluted to five different concentrations. The third sample (S3) was optimized for the quantification range of dPCR according to previous reports. The ratio between the two fragments was 1.005, which closely approximated the value certified by sequencing, and the concentration was found to be 792 copies/µL. This method was precise, with an RSD of ~3%. These findings demonstrate the advantages of using the dPCR method to characterize reference materials.