Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.     

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of gentically modified crops. 3. Assessing unintended effects

The current procedures to assess the safety of food and feed derived from modern biotechnology include the investigation of possible unintended effects. To improve the probability of detecting unintended effects, profiling techniques such as proteomics are currently tested as complementary analytical tools to the existing safety assessment. An optimized two-dimensional gel electrophoresis (2DE) method was used as a proteomics approach to investigate insertional and pleiotropic effects on the proteome due to genetic engineering. Twelve transgenic Arabidopsis thaliana lines were analyzed by 2DE, and their seed proteomes were compared to that of their parental line as well as to 12 Arabidopsis ecotype lines. The genetic modification of the Arabidopsis lines, using three different genes and three different promoters, did not cause unintended changes to the analyzed seed proteome. Differences in spot quantity between transgenic and nontransgenic lines fell in the range of values found in the 12 Arabidopsis ecotype lines or were related to the introduced gene.     

Exploration of beer proteome using OFFGEL prefractionation in combination with two-dimensional gel electrophoresis with narrow pH range gradients

Two-dimensional gel electrophoresis in combination with mass spectrometry has already been applied successfully to study beer proteome. Due to the abundance of protein Z in beer samples, prefractionation techniques might help to improve beer proteome coverage. Proteins from four lager beers of different origins were separated by two-dimensional electrophoresis (2-DE) followed by tandem mass spectrometric analysis. Initially 52 proteins mostly from Hordeum vulgare (22 proteins) and Saccharomyces species (25 proteins) were identified. Preparative isoelectric focusing by OFFGEL Fractionator was applied prior to 2-DE to improve its resolution power. As a result of this combined approach, a total of 70 beer proteins from Hordeum vulgare (30 proteins), from Saccharomyces species (31 proteins), and from other sources (9 proteins) were identified. Of these, 37 proteins have not been previously reported in beer samples.     

Detection of genetically modified maize by the polymerase chain reaction and capillary gel electrophoresis with UV detection and laser-induced fluorescence

In this paper, the possibilities of capillary gel electrophoresis (CGE) to detect transgenic maize in flours are shown. The method is based on the extraction and amplification by the polymerase chain reaction (PCR) of a specific DNA fragment from transgenic maize and its subsequent analysis by CGE with UV detection or laser-induced fluorescence (LIF). Some useful considerations regarding the optimization of DNA extraction and amplification conditions are given. Also, a comparison is established between the two CGE protocols for DNA detection based on ultraviolet absorption (CGE-UV) and LIF (CGE-LIF). The requirements, advantages, and limitations of both CGE methods are discussed. To our knowledge, this is the first paper on the use of CGE-LIF to detect transgenic food.     

Ultrasensitive detection of genetically modified maize DNA by capillary gel electrophoresis with laser-induced fluorescence using different fluorescent intercalating dyes

In this work, four different fluorescent intercalating dyes are compared for the ultrasensitive CGE-LIF detection of DNA from transgenic maize in flours. The fluorescent intercalating dyes compared are YOPRO-1, SYBR-Green-I, Ethidium bromide (EthBr), and EnhanCE. For all the four dyes optimum concentrations are established, and efficient separations of DNA fragments ranging in size from 80 to 1000 bp are obtained. The comparative study demonstrates that SYBR-Green-I and YOPRO-1 provide better limits of detection (LODs) than EnhanCE or EthBr (i.e., LODs are, respectively, 700, 1000, 11300, and 97400 zmol, calculated for a 200-bp DNA fragment). Separations using YOPRO-1 are faster than those using SYBR-Green-I (30 min vs 47 min for the analysis of the 80-1000 bp DNA fragments). Also, separations using YOPRO-1 are more efficient than those using SYBR-Green-I (e.g., 2.4 x 10(6) plates/m vs 1.6 x 10(6) plates/m, respectively, calculated for the 200-bp fragment). Also, buffer depletion and cost per analysis are worse with SYBR-Green-I than with YOPRO-1. Therefore, YOPRO-1 was selected as the preferred intercalating dye. Using this fluorescent compound, analysis time reproducibility for the CGE-LIF separation of the DNA fragments is determined to be better than 1.7% (% RSD, n = 10) within the same day, and better than 1.9% (% RSD, n = 30) for three different days. Moreover, the fluorescence signal obtained using this dye is shown to vary linearly with the DNA concentration in the range studied, i.e., 1-500 ng/microL. It is demonstrated that by using this method 0.01% of transgenic maize can be detected in flour by direct injection of the PCR-amplified sample.     

Assessment of the nutritional values of genetically modified wheat, corn, and tomato crops

The genetic modification in fruit and vegetables could lead to changes in metabolic pathways and, therefore, to the variation of the molecular pattern, with particular attention to antioxidant compounds not well-described in the literature. The aim of the present study was to compare the quality composition of transgenic wheat ( Triticum durum L.), corn ( Zea mays L.), and tomato ( Lycopersicum esculentum Mill.) to the nontransgenic control with a similar genetic background. In the first experiment, Ofanto wheat cultivar containing the tobacco rab1 gene and nontransgenic Ofanto were used. The second experiment compared two transgenic lines of corn containing Bacillus thuringiensis "Cry toxin" gene (PR33P67 and Pegaso Bt) to their nontransgenic forms. The third experiment was conducted on transgenic tomato ( Lycopersicum esculentum Mill.) containing the Agrobacterium rhizogenes rolD gene and its nontransgenic control (cv. Tondino). Conventional and genetically modified crops were compared in terms of fatty acids content, unsaponifiable fraction of antioxidants, total phenols, polyphenols, carotenoids, vitamin C, total antioxidant activity, and mineral composition. No significant differences were observed for qualitative traits analyzed in wheat and corn samples. In tomato samples, the total antioxidant activity (TAA), measured by FRAP assay, and the naringenin content showed a lower value in genetically modified organism (GMO) samples (0.35 mmol of Fe (2+) 100 g (-1) and 2.82 mg 100 g (-1), respectively), in comparison to its nontransgenic control (0.41 mmol of Fe (2+) 100 g (-1) and 4.17 mg 100 g (-1), respectively). On the basis of the principle of substantial equivalence, as articulated by the World Health Organization, the Organization for Economic Cooperation and Development, and the United Nations Food and Agriculture Organization, these data support the conclusion that GM events are nutritionally similar to conventional varieties of wheat, corn, and tomato on the market today.     

A quantitative immunopolymerase chain reaction method for detection of vegetative insecticidal protein in genetically modified crops

Vegetative insecticidal protein (Vip) is being employed for transgenic expression in selected crops such as cotton, brinjal, and corn. For regulatory compliance, there is a need for a sensitive and reliable detection method, which can distinguish between approved and nonapproved genetically modified (GM) events and quantify GM contents as well. A quantitative immunopolymerase chain reaction (IPCR) method has been developed for the detection and quantification of Vip protein in GM crops. The developed assay displayed a detection limit of 1 ng/mL (1 ppb) and linear quantification range between 10 and 1000 ng/mL of Vip-S protein. The sensitivity of the assay was found to be 10 times higher than an analogous enzyme-linked immunosorbent assay for Vip-S protein. The results suggest that IPCR has the potential to become a standard method to quantify GM proteins.     

Determination of the N-glycosylation patterns of seed proteins: applications to determine the authenticity and substantial equivalence of genetically modified (GM) crops

Methods have been developed to determine the N-glycosylation pattern of proteins at the single-seed level in two different biological systems. These were the well-characterized and widely consumed storage protein phaseolin from several species of Phaseolus (bean) and the α-amylase inhibitor from the same Phaseolus species expressed transgenically in pea. The N-glycosylation pattern of the α-amylase inhibitor expressed transgenically in pea was different from that of the inhibitor present in common bean (P. vulgaris), the species of origin of the gene. However, multivariate analysis showed that the differences in N-glycan patterns between the α-amylase inhibitors from common bean and pea were less than those between the inhibitors from common bean and two related bean species, lima bean (Phaseolus lunatus) and tepary bean (Phaseolus acutifolius).     

Proteomic analysis by two-dimensional gel electrophoresis and starch characterization of Triticum turgidum L. var. durum cultivars for pasta making

Criteria for durum wheat quality are continuously evolving in response to market pressure and consumer's preference. Specific attributes of durum wheat for different end products require more rapid and objective means to grade and classify wheat parcels based on processing potential. A total of 10 durum wheat cultivars were compared for compositional, protein, and starch characteristics. Mean values for the gross composition differed for total protein, gluten, and starch. Two-dimensional electrophoresis (2DE) analysis showed the proteome diversity among the cultivars. As shown by the principal component analysis (PCA) applied to 2DE data of gliadin and glutenin fractions, cultivars differed mainly from the number of proteins and levels of protein expression. As determined by the rapid viscoanalyzer (RVA), swelling power, starch damage, amylose content, and starch pasting properties of 10 cultivars differed significantly. 2DE fingerprinting and amylose content seemed to distinguish specific cultivars being useful tools for selecting suitable durum wheat cultivars for pasta making.     

Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis

In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.     

First application of a microsphere-based immunoassay to the detection of genetically modified organisms (GMOs): quantification of Cry1Ab protein in genetically modified maize

An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis.     

The role of laboratory, glasshouse and field scale experiments in understanding the interactions between genetically modified crops and soil ecosystems: A review of the ECOGEN project

The interactions of genetically modified (GM) crops with soil species and ecosystems is complex, requiring both specific and broad spectrum assessments. In the ECOGEN project we undertook experiments at three scales of increasing complexity, using Bt maize expressing the Cry1Ab protein from Bacillus thuringiensis as an example. Test species were selected for laboratory-scale experiments to represent taxonomic groups that we could also monitor at glasshouse and field scales (e.g., nematodes, protozoa, micro-arthropods, earthworms, and snails). In the laboratory, single species were exposed to purified Cry1Ab protein or to Bt maize leaf powder incorporated into simplified diets under controlled conditions. In the glasshouse, multiple test species and soil microbial communities taken from ECOGEN's field sites were exposed to Bt maize plants growing under glasshouse or mesocosm conditions. In the field, evaluations were conducted on our selected indicator groups over multiple sites and growing seasons. Field evaluation included assessment of effects due to the local environment, crop type, seasonal variation and conventional crop management practice (tillage and pesticide use), which cannot be assessed in the glasshouse. No direct effects of Cry1Ab protein or Bt leaf residues were detected on our laboratory test organisms, but some significant effects were detected in the glasshouse. Total nematode and protozoan numbers increased in field soil under Bt maize relative to conventional maize, whilst microbial community structure and activity were unaffected. Field results for the abundance of nematodes and protozoa showed some negative effects of Bt maize, thus contradicting the glasshouse results. However, these negative results were specific to particular field sites and sampling times and therefore were transient. Taking the overall variation found in maize ecosystems at different sites into account, any negative effects of Bt maize at field scale were judged to be indirect and no greater than the impacts of crop type, tillage and pesticide use. Although the ECOGEN results were not predictive between the three experimental scales, we propose that they have value when used with feedback loops between the scales. This holistic approach can used to address questions raised by results from any level of experimentation and also for putting GM crop risk:benefit into context with current agricultural practices in regionally differing agro-ecosystems.     

Applicability of the quantification of genetically modified organisms to foods processed from maize and soy

The applicability of quantifying genetically modified (GM) maize and soy to processed foods was investigated using heat treatment processing models. The detection methods were based on real-time quantitative polymerase chain reaction (PCR) analysis. Ground seeds of insect resistant GM maize (MON810) and glyphosate tolerant Roundup Ready (RR) soy were dissolved in water and were heat treated by autoclaving for various time intervals. The calculated copy numbers of the recombinant and taxon specific deoxyribonucleic acid (DNA) sequences in the extracted DNA solution were found to decrease with time. This decrease was influenced by the PCR-amplified size. The conversion factor (Cf), which is the ratio of the recombinant DNA sequence to the taxon specific DNA sequence and is used as a constant number for calculating GM% at each event, tended to be stable when the sizes of PCR products of two DNA sequences were nearly equal. The results suggested that the size of the PCR product plays a key role in the quantification of GM organisms in processed foods. It is believed that the Cf of the endosperm (3n) is influenced by whether the GM originated from a paternal or maternal source. The embryos and endosperms were separated from the F1 generation seeds of five GM maize events, and their Cf values were measured. Both paternal and maternal GM events were identified. In these, the endosperm Cf was lower than that of the embryo, and the embryo Cf was lower than that of the endosperm. These results demonstrate the difficulties encountered in the determination of GM% in maize grains (F2 generation) and in processed foods from maize and soy.     

Sampling and modeling for the quantification of adventitious genetically modified presence in maize

The coexistence of genetically modified (GM) and non-GM crops is an important economic and political issue in the European Union. We examined the GM content in non-GM maize crops in Spain in 2005. Both the standing crop and the harvest were tested, and the %GM DNA was quantified by real-time polymerase chain reaction. We compared the level of GM as a function of distance from known GM source fields in a 1.2 km2 landscape. The distribution of GM was compared to predictions from previous studies, and good agreement was found. Control and monitoring of adventitious GM presence in non-GM crops can only be achieved by fit-for-purpose sampling and testing schemes. We used a GM dispersal function to simulate non-GM crops in the studied zone and tested the accuracy of five different sampling schemes. Random sampling was found to be the most accurate and least susceptible to bias by GM spatial structure or gradients. Simulations showed that to achieve greater than 95% confidence in a GM labeling decision of a harvest (when treated as a single marketed lot), 34 samples would be needed when the harvest was outside 50% of the GM threshold value. The number of samples required increased rapidly as the harvest approached the GM threshold, implying that accurate labeling when the harvest is within +/-17% of the threshold may not be possible with high confidence.     

Use of murine models to detect the allergenicity of genetically modified Lactococcus lactis NZ9000/pNZPNK

By introducing aprN into Lactococcus lactis NZ9000, the genetically modified L. lactis NZ9000/pNZPNK successfully expressed the nattokinase. The safety assessment of this novel strain was based on allergenicity of pepsin digestion stability and murine model serologic identity. Subjecting to the GM strain and host to pepsin digestion, the soluble fractions and cell debris were fast degraded completely. Feeding with ovalbumin resulted in significantly higher production of IgG1 and IgE as compared to that of L. lactis NZ9000/pNZPNK or L. lactis NZ9000. Further, the serum IgG2a level increased dose-dependently at week 2 and induced immune reaction toward Th1 pathway. Secretion of cytokines IL-4 and IL-10 fed with lactococci was significantly lower than that of the OVA group. L. lactis NZ9000/pNZPNK did not increase the proliferation of type 2 helper T cells in spleen or induce allergenicity in BALB/c mice. On the basis of the results, the new GM lactic acid bacterium is regarded as safe to use.     

Plantations of Convallaria majalis L. as a threat to the natural stands of the species: Genetic variability of the cultivated plants and natural populations

The level of polymorphism, genetic variability and relatedness of Convallaria majalis-populations (species native in Poland, under partial legal protection) obtained from three Polish regions and from commercial producers (Polish and Dutch) were studied. In addition the differences between the cultivated plants and those occurring in natural stands were analyzed. Each region was represented by at least 20 populations among which half were collected in natural stands and half from cultivation sites (botanical gardens, private gardens and cemeteries), and compared with samples obtained directly from commercial producers. Seven primer pairs used for AFLP profiling amplified 466 scoreable DNA fragments that were used for multidimensional scaling and clustering. The above analyses make it possible to clearly distinguish among individuals and revealed groups of populations according to their geographic origin. Samples from populations collected in natural stands and cultivated in the same region did not differ from each other significantly.These results suggested that cultivated plants were probably obtained directly from the natural stand and the influence of plant cultures on natural populations was rather small.     

应用微核试验和单细胞凝胶电泳技术检测农药对蜘蛛的遗传毒性

应用蜘蛛血细胞微核试验和单细胞凝胶电泳试验研究了两种杀虫剂——吡虫啉和阿维菌素对蜘蛛头胸部和腹部的遗传毒性。结果表明:各供试浓度吡虫啉和阿维菌素对蜘蛛血细胞微核率均有明显影响,与对照组相比有显著性差异(P<0.05,P<0.01);且随着两种农药浓度升高,血细胞微核率显著增加,存在明显的剂量效应关系(吡虫啉浓度与星豹蛛头胸部血细胞微核率相关系数r=0.672 8,腹部为r=0.800 6;阿维菌素与星豹蛛头胸部血细胞微核率相关系数r=0.989 9,腹部为r=0.985 8)。吡虫啉和阿维菌素对星豹蛛血细胞DNA损伤有明显的剂量效应关系(吡虫啉浓度与星豹蛛头胸部血细胞DNA损伤相关系数r=0.948 2,腹部为r=0.970 4;阿维菌素与星豹蛛头胸部血细胞DNA损伤相关系数r=0.978 1,腹部为r=0.975 6)。两种农药在同一浓度下,对星豹蛛腹部血细胞微核率和DNA损伤程度明显大于头胸部。     

Degradation of Cry1Ab protein from genetically modified maize in the bovine gastrointestinal tract

Immunoblotting assays using commercial antibodies were established to investigate the unexpected persistence of the immunoactive Cry1Ab protein in the bovine gastrointestinal tract (GIT) previously suggested by enzyme-linked immunosorbent assay (ELISA). Samples of two different feeding experiments in cattle were analyzed with both ELISA and immunoblotting methods. Whereas results obtained by ELISA suggested that the concentration of the Cry1Ab protein increased during the GIT passage, the immunoblotting assays revealed a significant degradation of the protein in the bovine GIT. Samples showing a positive signal in the ELISA consisted of fragmented Cry1Ab protein of approximately 17 and 34 kDa size. Two independent sets of gastrointestinal samples revealed the apparent discrepancy between the results obtained by ELISA and immunoblotting, suggesting that the antibody used in the ELISA reacts with fragmented yet immunoactive epitopes of the Cry1Ab protein. It was concluded that Cry1Ab protein is degraded during digestion in cattle. To avoid misinterpretation, samples tested positive for Cry1Ab protein by ELISA should be reassessed by another technique.     

转基因抗除草剂水稻对稻田叶冠层节肢动物群落多样性的影响

转基因抗除草剂水稻可能对稻田节肢动物产生影响。对转Bar基因抗除草剂籼稻"Bar68-1"和非转基因对照"D68"稻田叶冠层节肢动物群落的结构和特征进行调查、比较。结果显示,转基因水稻和非转基因对照水稻间的冠层节肢动物各亚群落中相对丰盛度高的物种(科)具有较大相似性,但在相对丰盛度低的科上有所差别;除2007年不喷药处理区天敌亚群落的Simpson指数(D)有差异外,转基因与非转基因对照稻田之间叶冠层节肢动物群落、亚群落的多样性指数(物种丰富度、Shannon-Wiener多样性指数、Pielou均匀度指数、Simpson优势集中性指数)差异不显著;在水稻分蘖中期、分蘖末期、齐穗期和乳熟期等4个生育期,95.3%的稻田节肢动物群落主要参数时间动态的品种间差异不显著。研究结果表明转Bar基因抗除草剂水稻"Bar68-1"对稻田叶冠层节肢动物的群落组成和多样性无显著影响。