CP4 EPSPS Protein Research Articles

The Combination of Quantitative PCR and Western Blot Detecting CP4‐EPSPS Component in Roundup Ready Soy Plant Tissues and Commercial Soy‐Related Foodstuffs

With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods would supplement each other for genetically modified detection from nucleic acid and protein levels. Accordingly, qPCR and western blot could be used in CP4-EPSPS detection in a wide variety of soy-related foodstuffs.

Development of antibiotic marker-free creeping bentgrass resistance against herbicides

Herbicide-resistant creeping bentgrass plants (Agrostis stolonifera L.) without antibiotic-resistant markers were produced by Agrobacterium-mediated transformation. Embryogenic callus tissues were infected with Agrobacterium tumefaciens EHA105, harboring the bar and the CP4-EPSPS genes for bialaphos and glyphosate resistance. Phosphinothricin-resistant calli and plants were selected. Soil-grown plants were obtained at 14-16 weeks after transformation. Genetic transformation of the selected, regenerated plants was validated by PCR. Southern blot analysis revealed that at least one copy of the transgene was integrated into the genome of the transgenic plants. Transgene expression was confirmed by Northern blot. CP4-EPSPS protein was detected by ELISA. Transgenic plants remained green and healthy when sprayed with Basta, containing 0.5% glufosinate ammonium or glyphosate. The optimized Agrobacterium-mediated transformation method resulted in an average of 9.4% transgenic plants. The results of the present study suggest that the optimized marker-free technique could be used as an effective and reliable method for routine transformation, which may facilitate the development of varieties of new antibiotic-free grass species.

Scientific Opinion on application (EFSA‐GMO‐NL‐2007‐38) for the placing on the market of insect resistant and/or herbicide tolerant genetically modified maize MON89034 × NK603 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto

This opinion reports on an evaluation of a risk assessment for placing on the market the genetically modified herbicide tolerant and insect resistant maize MON89034 × NK603 for food and feed uses, import and processing. Conventional breeding methods were used in the production of maize MON89034 × NK603 from inbred lines of the respective parental events. The structural integrity of the inserts in the single events as well as the phenotypes were retained in the hybrid. The expression levels of the Cry1A.105, Cry2Ab2 and CP4 EPSPS proteins in maize MON89034 × NK603 were demonstrated to be comparable with those of the single events. The comparative analysis of phenotypic, agronomic and compositional characteristics of this GM maize indicated equivalence with its non-GM counterpart and conventional maize except for the expression of the target proteins, providing resistance to certain lepidopteran pests and tolerance to glyphosate herbicide. The safety assessment identified no concerns regarding potential toxicity and allergenicity of maize MON89034 × NK603. A feeding study on broiler chickens confirmed the nutritional equivalence of this GM maize to non-GM counterpart and conventional maize. Considering the intended uses of maize MON89034 × NK603, which excludes cultivation within the European Union, no scientific assessment of potential environmental effects associated with cultivation of maize MON89034 × NK603 was required. In case of accidental release of viable maize grain of MON89034 × NK603 into the environment during transportation and processing, there are no indications of increased likelihood of establishment or survival of feral maize plants except in the presence of the herbicide. The GMO Panel concludes that maize MON89034 × NK603 is as safe as its non-GM counterpart with respect to effects on human and animal health and the environment.

Application (Reference EFSA-GMO-CZ-2006-33) for the placing on themarket of the insect-resistant and glyphosate-tolerant genetically modifiedmaize MON 88017 x MON 810, for food and feed uses, import andprocessing under Regulation (EC) No 1829/2003 from Monsanto

Application (Reference EFSA-GMO-CZ-2006-33) for the placing on themarket of the insect-resistant and glyphosate-tolerant genetically modifiedmaize MON 88017 x MON 810, for food and feed uses, import andprocessing under Regulation (EC) No 1829/2003 from Monsanto

Serum testing of genetically modified soybeans with special emphasis on potential allergenicity of the heterologous protein CP4 EPSPS

Roundup Ready soy contains the CP4-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein. Serum IgE from two distinct populations of soy-allergic patients were recruited to determine their IgE-binding specificity. One population consisted of 10 adult patients from Europe, whose primary diagnosis was soy food allergy with some also having mite allergy. In addition, 6 primarily mite-allergic, 6 food-allergic (celery, carrot, milk, shrimp, walnut, and apple), and 5 non-allergic patients were tested. Another population consisted of 13 children from Korea, whose primary diagnosis was atopic dermatitis and secondarily soy and egg sensitization. In addition, 11 non-allergic patients were tested. Each patient population was extensively characterized with respect to clinical symptoms, specific IgE (CAP) scores, and total IgE. Immunoblots and ELISA assays were developed using serum IgE from these patients and soy extracts, CP4 EPSPS, rice extract, ovalbumin, rubisco, purified major peanut allergen Ara h 2, the putative soy allergen Gly m Bd 30k and mite allergen Der f 2 proteins as the intended targets. Immunoblot results indicated that soy-allergic patients bound soy extracts but did not specifically bind rubisco or CP4 EPSPS. ELISA results were in general agreement with the immunoblot results except that rubisco bound significant quantities of serum IgE from some patients. These results indicate that the CP4 EPSPS protein does not bind significant quantities of IgE from two geographically distinct sensitive populations and there is no evidence for an increased allergenic potential of this biotech protein.

Establishment of transgenic herbicide‐resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats

Concerns about genetically modified (GM) crops include transgene flow to compatible wild species and unintended ecological consequences of potential transgene introgression. However, there has been little empirical documentation of establishment and distribution of transgenic plants in wild populations. We present herein the first evidence for escape of transgenes into wild plant populations within the USA; glyphosate-resistant creeping bentgrass (Agrostis stolonifera L.) plants expressing CP4 EPSPS transgenes were found outside of cultivation area in central Oregon. Resident populations of three compatible Agrostis species were sampled in nonagronomic habitats outside the Oregon Department of Agriculture control area designated for test production of glyphosate-resistant creeping bentgrass. CP4 EPSPS protein and the corresponding transgene were found in nine A. stolonifera plants screened from 20,400 samples (0.04 +/- 0.01% SE). CP4 EPSPS-positive plants were located predominantly in mesic habitats downwind and up to 3.8 km beyond the control area perimeter; two plants were found within the USDA Crooked River National Grassland. Spatial distribution and parentage of transgenic plants (as confirmed by analyses of nuclear ITS and chloroplast matK gene trees) suggest that establishment resulted from both pollen-mediated intraspecific hybridizations and from crop seed dispersal. These results demonstrate that transgene flow from short-term production can result in establishment of transgenic plants at multi-kilometre distances from GM source fields or plants. Selective pressure from direct application or drift of glyphosate herbicide could enhance introgression of CP4 EPSPS transgenes and additional establishment. Obligatory outcrossing and vegetative spread could further contribute to persistence of CP4 EPSPS transgenes in wild Agrostis populations, both in the presence or absence of herbicide selection.

Determining whether transgenic and endogenous plant DNA and transgenic protein are detectable in muscle from swine fed Roundup Ready soybean meal.

Questions regarding the digestive fate of DNA and protein from transgenic feed have been raised in regard to human consumption and commercial trade of animal products (e.g., meat, milk, and eggs) from farm animals fed transgenic crops. Using highly sensitive, well-characterized analytical methods, pork loin samples were analyzed for the presence of fragments of transgenic and endogenous plant DNA and transgenic protein from animals fed meal prepared from conventional or glyphosate-tolerant Roundup Ready (RR) soybeans. Pigs were fed diets containing 24, 19, and 14% RR or conventional soybean meal during grower, early-finisher, and late-finisher phases of growth, respectively, and longissimus muscle samples were collected (12 per treatment) after slaughter. Total DNA was extracted from the samples and analyzed by PCR, followed by Southern blot hybridization for the presence of a 272-bp fragment of the cp4 epsps coding region (encoding the synthetic enzyme 5-enolpyruvylshikimate-3-phosphate synthase derived from Agrobacterium sp. strain CP4) and a 198-bp fragment of the endogenous soybean gene le1 (encoding soy lectin). Using 1 microgram of input DNA per reaction, none of the extracted samples was positive for cp4 epsps or le1 at the limit of detection (LOD) for these PCR/Southern blot assays. The LOD for these assays was shown to be approximately one diploid genome equivalent of RR soybean DNA, even in the presence of 10 micrograms of pork genomic DNA. A 185-bp fragment of the porcine preprolactin (prl) gene, used as a positive control, was amplified from all samples showing that the DNA preparations were amenable to PCR amplification. Using a competitive immunoassay with an LOD of approximately 94 ng of CP4 EPSPS protein/g of pork muscle, neither the CP4 EPSPS protein nor the immunoreactive peptide fragments were detected in loin muscle homogenates from pigs fed RR soybean meal. Taken together, these results show that neither small fragments of transgenic DNA nor immunoreactive fragments of transgenic protein are detectable in loin muscle samples from pigs fed a diet containing RR soybean meal.

Analysis of Expressed Proteins in Fiber Fractions from Insect‐Protected and Glyphosate‐Tolerant Cotton Varieties

The objective of this study was to determine the concentration of introduced proteins in the fiber fractions of transgenic cotton (Gossypium hirsutum L.) before and after processing. Two novel cotton varieties were developed by genetic transformation technology. One variety expressed the CryIA(c) insecticidal protein derived from Bacillus thuringiensis var. kurstaki. The second cotton variety expressed the CP4 EPSPS (5‐enolpyruvyl shikimate‐3‐phosphate synthase) protein, originally found in Agrobacterium sp., which confers tolerance to glyphosate herbicide. Levels of the CryIA(c) and CP4 EPSPS proteins in raw lint, combed lint, raw linters, and processed linter brown stock from these cotton varieties were analyzed by means of western blot, insect bioassay [for CryIA(c) protein only], or both. The CryIA(c) protein was present in raw linters (0.17 μg g−1), but it was not detected in either raw or combed lint. The CP4 EPSPS protein was detected at low levels (<0.5 μg g−1) in combed lint, but not in processed linter brown stock. Inactivation of the CryIA(c) and CP4 EPSPS proteins in the first processing step for linters indicates that these proteins will not be present in cotton linter products. Similarly, the typical heat and chemical processing of cotton lint should also denature any transgenic protein(s) present.

The Expressed Protein in Glyphosate-Tolerant Soybean, 5-Enolypyruvylshikimate-3-Phosphate Synthase from Agrobacterium sp. Strain CP4, Is Rapidly Digested In Vitro and Is not Toxic to Acutely Gavaged Mice

The safety of 5-enolpyruvylshikimate-3-phosphate synthase enzyme derived from Agrobacterium sp. strain CP4 (CP4 EPSPS) was assessed. CP4 EPSPS is the only protein introduced by genetic manipulation that is expressed in glyphosate-tolerant soybeans, which are being developed to provide new weed-control options for farmers. Expression of this protein in plants imparts high levels of glyphosate tolerance. The safety of CP4 EPSPS was ascertained by evaluating both physical and functional characteristics. CP4 EPSPS degrades readily in simulated gastric and intestinal fluids, suggesting that this protein will be degraded in the mammalian digestive tract upon ingestion as a component of food or feed. There were no deleterious effects due to the acute administration of CP4 EPSPS to mice by gavage at a high dosage of 572 mg/kg body wt, which exceeds 1000-fold the anticipated consumption level of food products potentially containing CP4 EPSPS protein. CP4 EPSPS does not pose any important allergen concerns because this protein does not possess characteristics typical of allergenic proteins. These data, in combination with seed compositional analysis and animal feeding studies, support the conclusion that glyphosate-tolerant soybeans are as safe and nutritious as traditional soybeans currently being marketed.