RESUMO
Many protein families have numerous members listed in databases as allergens; however, some allergen database entries, herein called "orphan allergens", are members of large families of which all other members are not allergens. These orphan allergens provide an opportunity to assess whether specific structural features render a protein allergenic. Three orphan allergens [Cladosporium herbarum aldehyde dehydrogenase (ChALDH), Alternaria alternata ALDH (AaALDH), and C. herbarum mannitol dehydrogenase (ChMDH)] were recombinantly produced and purified for structure characterization and for clinical skin prick testing (SPT) in mold allergic participants. Examination of the X-ray crystal structures of ChALDH and ChMDH and a homology structure model of AaALDH did not identify any discernable epitopes that distinguish these putative orphan allergens from their non-allergenic protein relatives. SPT results were aligned with ChMDH being an allergen, 53% of the participants were SPT (+). AaALDH did not elicit SPT reactivity above control proteins not in allergen databases (i.e., Psedomonas syringae indole-3-acetaldehyde dehydrogenase and Zea mays ALDH). Although published results showed consequential human IgE reactivity with ChALDH, no SPT reactivity was observed in this study. With only one of these three orphan allergens, ChMDH, eliciting SPT(+) reactions consistent with the protein being included in allergen databases, this underscores the complicated nature of how bioinformatics is used to assess the potential allergenicity of food proteins that could be newly added to human diets and, when needed, the subsequent clinical testing of that bioinformatic assessment.Trial registration number and date of registration AAC-2017-0467, approved as WIRB protocol #20172536 on 07DEC2017 by WIRB-Copernicus (OHRP/FDA Registration #: IRB00000533, organization #: IORG0000432).
Assuntos
Alérgenos , Imunoglobulina E , Aldeído Desidrogenase , Alérgenos/genética , Epitopos , Humanos , Indóis , Manitol DesidrogenasesRESUMO
Site-directed nucleases (SDNs) used for targeted genome editing are powerful new tools to introduce precise genetic changes into plants. Like traditional approaches, such as conventional crossing and induced mutagenesis, genome editing aims to improve crop yield and nutrition. Next-generation sequencing studies demonstrate that across their genomes, populations of crop species typically carry millions of single nucleotide polymorphisms and many copy number and structural variants. Spontaneous mutations occur at rates of â¼10-8 to 10-9 per site per generation, while variation induced by chemical treatment or ionizing radiation results in higher mutation rates. In the context of SDNs, an off-target change or edit is an unintended, nonspecific mutation occurring at a site with sequence similarity to the targeted edit region. SDN-mediated off-target changes can contribute to a small number of additional genetic variants compared to those that occur naturally in breeding populations or are introduced by induced-mutagenesis methods. Recent studies show that using computational algorithms to design genome editing reagents can mitigate off-target edits in plants. Finally, crops are subject to strong selection to eliminate off-type plants through well-established multigenerational breeding, selection, and commercial variety development practices. Within this context, off-target edits in crops present no new safety concerns compared to other breeding practices. The current generation of genome editing technologies is already proving useful to develop new plant varieties with consumer and farmer benefits. Genome editing will likely undergo improved editing specificity along with new developments in SDN delivery and increasing genomic characterization, further improving reagent design and application.
Assuntos
Genoma de Planta/genética , Produtos Agrícolas/genética , Edição de Genes , Taxa de Mutação , Plantas Geneticamente Modificadas/genéticaRESUMO
A challenge to improve an integrative phenotype, like yield, is the interaction between the broad range of possible molecular and physiological traits that contribute to yield and the multitude of potential environmental conditions in which they are expressed. This study collected data on 31 phenotypic traits, 83 annotated metabolites, and nearly 22,000 transcripts from a set of 57 diverse, commercially relevant maize hybrids across three years in central U.S. Corn Belt environments. Although variability in characteristics created a complex picture of how traits interact produce yield, phenotypic traits and gene expression were more consistent across environments, while metabolite levels showed low repeatability. Phenology traits, such as green leaf number and grain moisture and whole plant nitrogen content showed the most consistent correlation with yield. A machine learning predictive analysis of phenotypic traits revealed that ear traits, phenology, and root traits were most important to predicting yield. Analysis suggested little correlation between biomass traits and yield, suggesting there is more of a sink limitation to yield under the conditions studied here. This work suggests that continued improvement of maize yields requires a strong understanding of baseline variation of plant characteristics across commercially-relevant germplasm to drive strategies for consistently improving yield.
Assuntos
Zea mays/genética , Biomassa , Produção Agrícola , Meio Ambiente , Regulação da Expressão Gênica de Plantas/genética , Estudos de Associação Genética , Fenótipo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/crescimento & desenvolvimento , Característica Quantitativa Herdável , Zea mays/anatomia & histologia , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismoRESUMO
BACKGROUND: The safety assessment of foods and feeds from genetically modified (GM) crops includes the comparison of key characteristics, such as crop composition, agronomic phenotype and observations from animal feeding studies compared to conventional counterpart varieties that have a history of safe consumption, often including a near isogenic variety. The comparative compositional analysis of GM crops has been based on targeted, validated, quantitative analytical methods for the key food and feed nutrients and antinutrients for each crop, as identified by Organization of Economic Co-operation and Development (OCED). As technologies for untargeted metabolomic methods have evolved, proposals have emerged for their use to complement or replace targeted compositional analytical methods in regulatory risk assessments of GM crops to increase the number of analyzed metabolites. AIM OF REVIEW: The technical opportunities, challenges and strategies of including untargeted metabolomics analysis in the comparative safety assessment of GM crops are reviewed. The results from metabolomics studies of GM and conventional crops published over the last eight years provide context to enable the discussion of whether metabolomics can materially improve the risk assessment of food and feed from GM crops beyond that possible by the Codex-defined practices used worldwide for more than 25 years. KEY SCIENTIFIC CONCEPTS OF REVIEW: Published studies to date show that environmental and genetic factors affect plant metabolomics profiles. In contrast, the plant biotechnology process used to make GM crops has little, if any consequence, unless the inserted GM trait is intended to alter food or feed composition. The nutritional value and safety of food and feed from GM crops is well informed by the quantitative, validated compositional methods for list of key analytes defined by crop-specific OECD consensus documents. Untargeted metabolic profiling has yet to provide data that better informs the safety assessment of GM crops than the already rigorous Codex-defined quantitative comparative assessment. Furthermore, technical challenges limit the implementation of untargeted metabolomics for regulatory purposes: no single extraction method or analytical technique captures the complete plant metabolome; a large percentage of metabolites features are unknown, requiring additional research to understand if differences for such unknowns affect food/feed safety; and standardized methods are needed to provide reproducible data over time and laboratories.
Assuntos
Inocuidade dos Alimentos/métodos , Metabolômica/métodos , Plantas Geneticamente Modificadas/metabolismo , Ração Animal/análise , Animais , Biotecnologia , Produtos Agrícolas/genética , Alimentos Geneticamente Modificados , Humanos , Metaboloma , Plantas Geneticamente Modificadas/genética , Medição de Risco/métodosRESUMO
Assessing the safety of genetically engineered crops includes evaluating the risk (hazard and exposure) of consuming their newly expressed proteins. The dicamba monooxygenase (DMO) protein, introduced into soybeans to confer tolerance (DT) to dicamba herbicide, was previously characterized and identified to pose no food or feed safety hazards. Most agricultural commodities (e.g., soybeans, maize) enter the food supply after processing methods that can include exposure to high temperatures, harsh solvents or pH extremes that can adversely impact the structure and function of proteins. To understand the likelihood of exposure to DMO in foods from DT soy, enzymatically active and/or immunodetectable forms of DMO were measured in pilot-scale productions of two soy foods (soymilk and tofu), and eight processed fractions (full fat flour, inactivated full fat flour, defatted flour, toasted meal, protein isolate, protein concentrate, crude lecithin, and refined, bleached and deodorized oil). Western blot analysis detected DMO in tofu and in five of the eight processed fractions. DMO activity was not detected in either soymilk or tofu, nor in six of the eight processed fractions. Therefore, many commercial soy processing methods can denature and/or degrade introduced proteins, like DMO. Although the DMO protein has shown no evidence of hazard, this study demonstrates that processing further reduces any food or feed risk by limiting dietary exposure to intact DMO protein.
Assuntos
Dicamba , Manipulação de Alimentos , Glycine max , Herbicidas , Oxigenases de Função Mista , Plantas Geneticamente Modificadas/enzimologia , Alimentos de Soja/análise , Exposição Dietética/prevenção & controle , Resistência a Medicamentos , Oxigenases de Função Mista/análise , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Glycine max/enzimologia , Glycine max/genéticaRESUMO
The expression of the CP4 EPSPS protein in genetically engineered (GE) soybean confers tolerance to the Roundup® family of agricultural herbicides. This study evaluated the variability of CP4 EPSPS expression using an enzyme-linked immunosorbent assay in soybean tissues collected across diverse germplasm and 74 different environments in Argentina, Brazil and the USA. Evaluated material included single and combined (stacked) trait products with other GE traits in entries with cp4 epsps gene at one or two loci. The highest level of CP4 EPSPS was observed in leaf tissues, intermediate in forage and seed, and lowest in root tissues. Varieties with two loci had approximately twice the level of CP4 EPSPS expression compared to one locus entries. Variable and non-directional level of CP4 EPSPS was observed with other factors like genetic background, trait stacking, growing region or season. The maximum and average CP4 EPSPS expression levels in seed provided large margins of exposure (MOE of approximately 4000 and 11,000, respectively), mitigating concerns over exposure to this protein in food and feed from soybean varieties tolerant to Roundup® herbicides.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/metabolismo , Agrobacterium/enzimologia , Tolerância a Medicamentos , Glycine max/enzimologia , Plantas Geneticamente Modificadas/enzimologia , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Glicina/análogos & derivados , Glicina/farmacologia , Herbicidas/farmacologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Glycine max/classificação , Glycine max/efeitos dos fármacos , Glycine max/crescimento & desenvolvimento , GlifosatoRESUMO
The lepidopteran-active Cry1A.105 protein is a chimeric three-domain insecticidal toxin with distinct structural domains derived from the naturally occurring Cry1Ab, Cry1Ac and Cry1F proteins from the soil bacterium Bacillus thuringiensis (Bt). The X-ray crystal structure of the Cry1A.105 tryptic core at 3.0â¯Å resolution demonstrates its high structural similarity to the tryptic core of Cry1Ac. Bioinformatics analyses demonstrate that Cry1A.105 has no significant amino acid sequence similarity to known allergens or mammalian toxins, which is the same conclusion reached for its component domains. Like its intact donor proteins, Cry1A.105 was heat labile at temperatures ≥75⯰C and degraded upon exposure to gastrointestinal proteases. No adverse effects were observed in mice when Cry1A.105 was dosed orally at 2451â¯mg/kg body weight. Therefore, the weight of evidence supports that Cry1A.105 is safe for human and animal consumption. These results support the conclusion that the safety of a chimeric protein for human or animal consumption can be evaluated in the context of the safety of its donor proteins.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/efeitos adversos , Sequência de Aminoácidos , Animais , Endotoxinas/efeitos adversos , Feminino , Humanos , Inseticidas/efeitos adversos , Camundongos , Proteínas Recombinantes de Fusão/efeitos adversosRESUMO
The need for sustainable insect pest control is driving the investigation and discovery of insecticidal proteins outside of the typical 3-domain Cry protein family from Bacillus thuringiensis (Bt). Examples include Cry35 and Cry51 that belong to protein families (Toxin_10, ETX_MTX2) sharing a common ß-pore forming structure and function with known mammalian toxins such as epsilon toxin (ETX). Although ß-pore forming proteins are related to mammalian toxins, there are key differences in sequence and structure that lead to organism specificity that is useful in the weight-of-evidence approach for safety assessment. Despite low overall amino acid sequence identity among ETX_MTX2 proteins, sequence and structural similarities are found in the tail region responsible for the shared oligomerization and pore formation functions (causing the "relatedness"). Conversely, most of the sequence and structural diversity is located in the head region that is likely responsible for differential receptor binding and target species specificity (e.g., insecticidal vs. mammalian). Therefore, inclusion of a domain-based protein characterization approach that includes bioinformatic and functional comparisons of conserved and diverse domains will enhance the overall weight of evidence safety assessment of proteins including recently reported Cry51 protein variants (Cry51Aa1, Cry51Aa2, and Cry51Aa2.834_16).
Assuntos
Biologia Computacional/métodos , Endotoxinas/classificação , Inseticidas/classificação , Modelos Moleculares , Controle Biológico de Vetores/métodos , Sequência de Aminoácidos , Animais , Endotoxinas/química , Endotoxinas/genética , Inseticidas/química , Inseticidas/metabolismo , Relação Estrutura-AtividadeRESUMO
Dicamba tolerant (DT) soybean, cotton and maize were developed through constitutive expression of dicamba mono-oxygenase (DMO) in chloroplasts. DMO expressed in three DT crops exhibit 91.6-97.1% amino acid sequence identity to wild type DMO. All DMO forms maintain the characteristics of Rieske oxygenases that have a history of safe use. Additionally, they are all functionally similar in vivo since the three DT crops are all tolerant to dicamba treatment. None of these DMO sequences were found to have similarity to any known allergens or toxins. Herein, to further understand the safety of these DMO variants, a weight of evidence approach was employed. Each purified DMO protein was found to be completely deactivated in vitro by heating at temperatures 55 °C and above, and all were completely digested within 30 s or 5 min by pepsin and pancreatin, respectively. Mice orally dosed with each of these DMO proteins showed no adverse effects as evidenced by analysis of body weight gain, food consumption and clinical observations. Therefore, the weight of evidence from all these protein safety studies support the conclusion that the various forms of DMO proteins introduced into DT soybean, cotton and maize are safe for food and feed consumption, and the small amino acid sequence differences outside the active site of DMO do not raise any additional safety concerns.
Assuntos
Produtos Agrícolas/toxicidade , Dicamba/farmacologia , Resistência a Medicamentos , Alimentos Geneticamente Modificados/toxicidade , Glycine max/toxicidade , Gossypium/toxicidade , Herbicidas/farmacologia , Oxigenases de Função Mista/toxicidade , Oxirredutases O-Desmetilantes/toxicidade , Plantas Geneticamente Modificadas/toxicidade , Zea mays/toxicidade , Administração Oral , Sequência de Aminoácidos , Animais , Biologia Computacional , Qualidade de Produtos para o Consumidor , Produtos Agrícolas/enzimologia , Produtos Agrícolas/genética , Bases de Dados de Proteínas , Resistência a Medicamentos/genética , Estabilidade Enzimática , Feminino , Inocuidade dos Alimentos , Alimentos Geneticamente Modificados/parasitologia , Regulação da Expressão Gênica de Plantas , Gossypium/enzimologia , Gossypium/genética , Humanos , Masculino , Camundongos , Oxigenases de Função Mista/administração & dosagem , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Pancreatina/metabolismo , Pepsina A/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Desnaturação Proteica , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Medição de Risco , Glycine max/enzimologia , Glycine max/genética , Stenotrophomonas maltophilia/enzimologia , Stenotrophomonas maltophilia/genética , Temperatura , Testes de Toxicidade Aguda , Zea mays/enzimologia , Zea mays/genéticaAssuntos
Alérgenos/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Produtos Agrícolas/imunologia , Hipersensibilidade Alimentar/diagnóstico , Alimentos Geneticamente Modificados/efeitos adversos , Plantas Geneticamente Modificadas/imunologia , Alérgenos/biossíntese , Proteínas de Bactérias/biossíntese , Produtos Agrícolas/química , Produtos Agrícolas/genética , Hipersensibilidade Alimentar/etiologia , Hipersensibilidade Alimentar/genética , Hipersensibilidade Alimentar/imunologia , Humanos , Soros Imunes/química , Imunoglobulina E/sangue , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Medição de Risco , Transgenes , IncertezaRESUMO
The number of evaluations of the nutrient composition of food and feed crops has increased over the past 15years due to the introduction of new crops using the tools of modern biotechnology. The composition of these crops has been extensively compared with conventional (non-transgenic) controls as an integral part of the comparative safety assessment process. Following guidelines outlined in the Organization of Economic Co-operation and Development (OECD) Consensus Documents, most of these studies have incorporated field trials at multiple geographies and a diverse range of commercially available varieties/hybrids that are analyzed to understand natural variability in composition due to genetic and environmental influences. Using studies conducted in the US, Argentina and Brazil over multiple growing seasons, this report documents the effect of geography, growing season, and genetic background on soybean composition where fatty acids and isoflavones were shown to be particularly variable. A separate investigation of 96 different maize hybrids grown at three locations in the US demonstrated that levels of free amino acids, sugars/polyols, and molecules associated with stress response can vary to a greater degree than that observed for more abundant components. The International Life Sciences Institute (ILSI) crop composition database has proven to be an important resource for collecting and disseminating nutrient composition data to promote a further understanding of the variability that occurs naturally in crops used for food and feed.
Assuntos
Ração Animal/análise , Produtos Agrícolas/química , Análise de Alimentos/métodos , Animais , Biotecnologia/métodos , Produtos Agrícolas/genética , Bases de Dados Factuais , Guias como Assunto , Humanos , Valor Nutritivo , Glycine max/química , Glycine max/genética , Zea mays/química , Zea mays/genéticaRESUMO
The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysine revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.
Assuntos
Corynebacterium glutamicum/enzimologia , Hidroliases/química , Lisina/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X/métodos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Humanos , Concentração Inibidora 50 , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The Organization for Economic Co-operation and Development (OECD) recommends the measurement of specific plant components for compositional assessments of new biotechnology-derived crops. These components include proximates, nutrients, antinutrients, and certain crop-specific secondary metabolites. A considerable literature on the natural variability of these components in conventional and biotechnology-derived crops now exists. Yet the OECD consensus also suggests measurements of any metabolites that may be directly associated with a newly introduced trait. Therefore, steps have been initiated to assess natural variation in metabolites not typically included in the OECD consensus but which might reasonably be expected to be affected by new traits addressing, for example, nutritional enhancement or improved stress tolerance. The compositional study reported here extended across a diverse genetic range of maize hybrids derived from 48 inbreds crossed against two different testers. These were grown at three different, but geographically similar, locations in the United States. In addition to OECD analytes such as proximates, total amino acids and free fatty acids, the levels of free amino acids, sugars, organic acids, and selected stress metabolites in harvested grain were assessed. The major free amino acids identified were asparagine, aspartate, glutamate, and proline. The major sugars were sucrose, glucose, and fructose. The most predominant organic acid was citric acid, with only minor amounts of other organic acids detected. The impact of genetic background and location was assessed for all components. Overall, natural variation in free amino acids, sugars, and organic acids appeared to be markedly higher than that observed for the OECD analytes.
Assuntos
Meio Ambiente , Valor Nutritivo , Sementes/química , Zea mays/química , Zea mays/genética , Aminoácidos/análise , Carboidratos/análise , Ácidos Graxos/análise , Hibridização GenéticaRESUMO
During the last decade, the area of biotech crops modified for agronomic input traits (e.g., herbicide tolerance and insect protection) has increased to 90 million halyear, grown by over 8 million farmers in a total of 17 countries. As adoption of these improved agronomic trait biotech crops has grown, so has interest in biotech crops that have improved nutritional characteristics for use as feed and food. A previous publication by the International Life Sciences Institute (ILSI) reported on the principles and concepts proposed for the nutritional and safety assessments of foods and feeds nutritionally improved through biotechnology. In this paper, the guidelines and principles recommended in the earlier publication are discussed relative to a specific case study, Lysine maize. Lysine maize is a feed ingredient with enhanced nutritional characteristics for poultry and swine and provides an alternative to the need for addition of supplemental lysine to some diets for these animals. The 2004 Task Force of the ILSI has also applied the concepts from that report to 4 other case studies: sweet potato enriched in provitamin A (2 examples, one using biotechnology and one using conventional breeding); Golden Rice 2; double-embryo maize; and ASP-1 enhanced protein sweet potato.
Assuntos
Biotecnologia/métodos , Lisina/química , Ciências da Nutrição , Zea mays/genética , Ração Animal , Animais , Qualidade de Produtos para o Consumidor , Produtos Agrícolas/genética , Análise de Alimentos , Alimentos Geneticamente Modificados , Humanos , Modelos Biológicos , Plantas Geneticamente Modificadas , Aves Domésticas , SuínosRESUMO
The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety of proteins.
Assuntos
Proteínas Alimentares/química , Inocuidade dos Alimentos , Pepsina A/química , Proteínas Alimentares/imunologia , Concentração de Íons de Hidrogênio , Fatores de TempoRESUMO
Lipid transfer protein (LTP) is the main causative agent for rare food allergic reactions to maize. This paper describes a new, validated ELISA that accurately measures maize LTP concentrations from 0.2 to 6.4 ng/mL. The levels of LTP ranged from 171 to 865 µg/g of grain, a 5.1-fold difference, across a set of 49 samples of maize B73 hybrids derived from the Nested Association Mapping (NAM) founder lines and a diverse collection of landrace accessions from North and South America. A second set of 107 unique samples from 18 commercial hybrids grown over two years across 10 U.S. states showed a comparable range of LTP level (212-751 µg/g of grain). Statistical analysis showed that genetic and environmental factors contributed 63 and 6%, respectively, to the variance in LTP levels. Therefore, the natural variation of maize LTP is up to 5-fold different across a diverse collection of varieties that have a history of safe cultivation and consumption.
Assuntos
Proteínas de Transporte/análise , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas de Plantas/análise , Zea mays/química , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Zea mays/genética , Zea mays/imunologiaRESUMO
Cre recombinase, when used as a tool in agricultural biotechnology, can precisely excise DNA sequences that may be useful in the introduction of a new trait but are not needed in the commercial product. Although the cre genetic material would not be present in the final product, the present studies were performed to assess the safety of Cre recombinase to provide confirmatory evidence of the safe use of Cre-lox technology in agricultural biotechnology. Cre recombinase shares no relevant sequence similarity to known allergens or toxins. When Cre recombinase was exposed to a pH 1.2 solution of simulated gastric fluid lacking pepsin, CD spectroscopy showed that there was a loss of secondary structure and that the protein was no longer active in a functional assay. Cre recombinase was degraded rapidly when exposed to pepsin in a standardized gastric digestion model; therefore, Cre recombinase would not survive the harsh gastric environment. When orally administered to mice as an acute dosage of 53 mg/kg of body weight, no treatment-related adverse findings were observed. These data support the conclusion that human and animal dietary exposure to Cre recombinase pose no known safety concerns; consistent with the fact that bacteriophage P1, the source of the cre gene and expressed protein, is commonly encountered in the environment and in normal enteric bacteria without reports of adverse consequences.
Assuntos
Biotecnologia/normas , Alimentos Geneticamente Modificados/normas , Integrases/administração & dosagem , Integrases/efeitos adversos , Ácidos , Administração Oral , Sequência de Aminoácidos , Animais , Peso Corporal/efeitos dos fármacos , Dicroísmo Circular , Estabilidade Enzimática , Alimentos Geneticamente Modificados/efeitos adversos , Integrases/genética , Integrases/metabolismo , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Segurança , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings.
Assuntos
DNA de Plantas/genética , Glycine max/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Sementes/genética , Análise de Sequência de DNA/métodos , Marcadores Genéticos/genética , Testes Genéticos , Plantas Geneticamente Modificadas/genética , Sementes/classificação , Glycine max/classificação , Fatores de TempoRESUMO
In order to provide farmers with better and more customized alternatives to improve yields, combining multiple genetically modified (GM) traits into a single product (called stacked trait crops) is becoming prevalent. Trait protein expression levels are used to characterize new GM products and establish exposure limits, two important components of safety assessment. Developing a multiplexed immunoassay capable of measuring all trait proteins in the same sample allows for higher sample throughput and savings in both time and expense. Fluorescent (bead-based) multiplexed immunoassays (FMI) have gained wide acceptance in mammalian research and in clinical applications. In order to facilitate the measurement of stacked GM traits, we have developed and validated an FMI assay that can measure five different proteins (ß-glucuronidase, neomycin phosphotransferase II, Cry1Ac, Cry2Ab2, and CP4 5-enolpyruvyl-shikimate-3-phosphate synthase) present in cotton leaf from a stacked trait product. Expression levels of the five proteins determined by FMI in cotton leaf tissues have been evaluated relative to expression levels determined by enzyme-linked immunosorbent assays (ELISAs) of the individual proteins and shown to be comparable. The FMI met characterization requirements similar to those used for ELISA. Therefore, it is reasonable to conclude that FMI results are equivalent to those determined by conventional individual ELISAs to measure GM protein expression levels in stacked trait products but with significantly higher throughput, reduced time, and more efficient use of resources.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Gossypium/química , Plantas Geneticamente Modificadas/química , Proteínas Recombinantes/análise , Fluorescência , Gossypium/genética , Plantas Geneticamente Modificadas/genéticaRESUMO
Understanding the impact of genetic diversity on crop biochemical composition is a prerequisite to the interpretation and potential relevance of biochemical differences experimentally observed between genotypes. This is particularly important in the context of comparative safety assessments for crops developed by new technologies such as genetic engineering. To interrogate the natural variability of biochemical composition, grain from seven maize hybrids grown at four geographically distinct sites in Europe was analyzed for levels of proximates (fat, protein, moisture, ash, and carbohydrates), fiber, amino acids, fatty acids, four vitamins, nine minerals, and secondary metabolites. Statistical evaluation of the compositional data at the p < 0.05 level compared each hybrid against every other hybrid (head-to-head) for all analytes at each site and then across all sites to understand the factors contributing to variability. Of the 4935 statistical comparisons made in this study, 40% (1986) were found to be significant. The magnitude of differences observed, as a percent, ranged between 0.84 and 149% when all individual sites and the combined sites were considered. The large number of statistically significant differences in the levels of these analytes between seven commercial hybrids emphasizes the importance of genetic background and environment as determinants of the biochemical composition of maize grain, reflects the inherent natural variability in those analytes across a representative sampling of maize hybrids, and provides a baseline of the natural range of these nutritional and antinutritional components in maize for comparative compositional assessments.