Cry9C Protein Research Articles

Transgenic Bt cotton driven by the green tissue-specific promoter shows strong toxicity to lepidopteran pests and lower Bt toxin accumulation in seeds.

A promoter of the PNZIP (Pharbitis nil leucine zipper) gene (1.459 kb) was cloned from Pharbitis nil and fused to the GUS (β-glucuronidase) and Bacillus thuringiensis endotoxin (Cry9C) genes. Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation. Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants. In contrast, GUS staining in the reproductive structures such as petals, anther, and immature seeds of PNZIP::GUS cotton was very faint. Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus (CaMV) 35S::Cry9C line were selected for enzyme-linked immunosorbent assay (ELISA) and insect bioassays. Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from 24.6 to 45.5 μg g(-1) fresh weight. In green tissues such as the leaves, boll rinds, and bracts of the PNZIP::Cry9C line, the Cry9C protein accumulated up to 50.2, 39.7, and 48.3 μg g(-1) fresh weight respectively. In contrast, seeds of the PNZIP::Cry9C line (PZ1.3) accumulated only 0.26 μg g(-1) fresh weight of the Cry9C protein, which was 100 times lower than that recorded for the seeds of the CaMV 35S::Cry9C line. The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm. The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds. These features should allay public concerns about the safety of transgenic foods. We propose the future utility of PNZIP as an economical, environmentally friendly promoter in cotton biotechnology.

Pyramiding Bt genes for increasing resistance of cotton to two major lepidopteran pests: Spodoptera litura and Heliothis armigera

To more effectively control two major cotton insects (cotton bollworm and Spodoptera litura) and improve the efficacy of the pest resistance management, novel transgenic plants expressing Bacillus thuringiensis Cry9C gene were generated, and gene stacking strategy was incorporated. Initially, a binary plasmid vector harboring Cry9C gene was introduced into an elite cotton cultivar Simian-3 by Agrobacterium-mediated transformation. Integration and expression of the Cry9C genes in three transgenic lines were confirmed by PCR and RT-PCR. Among these transgenic lines, T0 generation of line 16 (L-16) with normal phenotypes were selected by ELISA assays for its highest expression level of Cry9C. In T1 population of L-16, the expression level of Cry9C ranged from 29 to 45 μg/g fresh leaf. The following insect bioassays demonstrated that transgenic S3-35S::Cry9C cotton plants exhibited moderate toxicity to Heliothis armigera but strong toxicity to S. litura compared with the transgenic plants expressing Cry 1Ac gene. For incorporation of gene staking strategy, Cry9C gene and Cry 2A or Cry 1Ac were pyramided, respectively by sexual crossing. The expression of Cry9C protein in all F1 progenies had a similar level as the parent plants indicating the high heritability of Bt genes in transgenic progenies. Progenies from both Cry9C × Cry 2A and Cry9C × Cry 1Ac exhibited higher resistance to S. litura compared with their parents. Together our data demonstrated that our newly generated transgenic plants represent a reservoir of novel insect-resistant materials in cotton breeding, and the successful incorporation of gene pyramiding technology can provide a new solution of developing multiple resistance management strategies.

Tevaluation of transgenic event CBH 351 (StarLink) corn in pig

This study examined the influence of transgenic event CBH (StarLink; SL)-derived hybrid corn on growth, health and physiological functions of pigs, as well as the possibility of transferring the cry9C gene or Cry9C protein to the blood, liver or muscles, in comparison with pigs fed a diet with non-transgenic (isogenic) corn (non-SL). The diet for the SL group was composed of 70% SL corn, and the diet for the non-SL group was composed of 70% non-SL corn. Forty pigs approximately 3 months in age were used in the current experiment. After the pigs were acclimatized to their environment for 7 days, they were fed piglet diets for 7 weeks, and afterwards fed growing-finishing diets until the end of the experiment. There were no significant differences in bodyweight gain, feed intake or feed conversion ratio between the pigs fed SL diet and those of non-SL diet. No abnormalities were observed in the health conditions of either the SL or the non-SL group. Moreover, no significant differences were observed between the two groups in hematological values, histopathological examination and necropsy findings. Although the serum biochemical values within each group were normal, the blood urea nitrogen values of the SL group showed a tendency to be slightly higher than those of the non-SL group. Also, the blood glucose values of the SL group were significantly lower than those of the non-SL group. However, the cause of the significant differences in the blood glucose values between the two groups is unknown. The PCR and ELISA did not detect the cry9C gene and Cry9C protein in the blood, liver or muscles of the pigs at the end of the experiment.

Evaluation of Sampling Plans to Detect Cry9C Protein in Corn Flour and Meal

StarLink is a genetically modified corn that produces an insecticidal protein, Cry9C. Studies were conducted to determine the variability and Cry9C distribution among sample test results when Cry9C protein was estimated in a bulk lot of corn flour and meal. Emphasis was placed on measuring sampling and analytical variances associated with each step of the test procedure used to measure Cry9C in corn flour and meal. Two commercially available enzyme-linked immunosorbent assay kits were used: one for the determination of Cry9C protein concentration and the other for % StarLink seed. The sampling and analytical variances associated with each step of the Cry9C test procedures were determined for flour and meal. Variances were found to be functions of Cry9C concentration, and regression equations were developed to describe the relationships. Because of the larger particle size, sampling variability associated with cornmeal was about double that for corn flour. For cornmeal, the sampling variance accounted for 92.6% of the total testing variability. The observed sampling and analytical distributions were compared with the Normal distribution. In almost all comparisons, the null hypothesis that the Cry9C protein values were sampled from a Normal distribution could not be rejected at 95% confidence limits. The Normal distribution and the variance estimates were used to evaluate the performance of several Cry9C protein sampling plans for corn flour and meal. Operating characteristic curves were developed and used to demonstrate the effect of increasing sample size on reducing false positives (seller's risk) and false negatives (buyer's risk).

Suitability of Real-Time Quantitative Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay for cry9C Detection in Mexican Corn Tortillas: Fate of DNA and Protein After Alkaline Cooking

Alkaline-cooked corn, called nixtamal, is the basis for many traditional corn products such as tortillas, chips, and taco shells that are used widely in Mexico and Central America and in the preparation of snack foods that are consumed globally. To assess the effects of alkaline and thermal treatments on the detectability of DNA and protein for the presence of genetically modified sequences, various nixtamalized products were prepared from blends of conventional white corn containing 0.1, 1.0, and 10% transgenic corn (event CBH 351, StarLink). Real-time quantitative polymerase chain reactions (RTQ-PCR) and immunoassays were used to determine the cry9C gene and protein, respectively, in unprocessed corn kernels, freshly prepared alkaline-cooked and ground corn (masa), masa flour, tortillas prepared from masa by heat treatment, chips prepared from damp masa dough by deep frying, and from tortillas processed at high (200 degrees C) and low temperatures (70 degrees C). In spite of progressive degradation of genomic DNA during processing, RTQ-PCR genetic analysis allowed detection and quantification of the cry9C gene in all products prepared from 10, 1, and 0.1% StarLink corn, except deep-fried chips containing 0.1% StarLink. Enzyme-linked immunosorbent assays readily detected <1 ppm cry9C protein in all blends of unprocessed corn (10, 1, and 0.1% StarLink) as well as in nonfried tortilla and masa products. This technique was not suitable for thermally treated nixtamalized products containing <1% transgenic corn.

Influence of transgenic corn (CBH 351, named Starlink) on health condition of dairy cows and transfer of Cry9C protein and cry9C gene to milk, blood, liver and muscle

AbstractThe influence of transgenic event CBH 351 (Starlink)‐derived hybrid corn (SL) on the health condition, physiological function and lactational performance of dairy cows as well as the transfer of the cry9C gene and Cry9C protein present in SL to milk, blood, liver and muscles was examined, and compared with a diet containing non‐transgenic (isogenic) control corn (non‐SL). After adapting to a diet containing non‐SL for 2 weeks, four Holstein cows were assigned to each of the non‐SL and SL groups and were fed diets containing non‐SL or SL, respectively, for 5 weeks. There were no significant influences on the physiological condition, milk yield or serum biochemical and hematological values after feeding with SL. There was also no influence on pH value, cell density of protozoa, or volatile fatty acid concentration and composition of rumen fluids. In addition, no significant differences were observed on histopathological examination of the major organs and tissues between the SL and non‐SL groups. Moreover, the cry9C gene and Cry9C protein were not detected by the polymerase chain reaction method and ELISA in the milk, blood, liver and muscles of the cows at the end of the experiment.

Determination of Cry9C Protein in Processed Foods Made with StarLink™ Corn

StarLink (Aventis CropScience US) hybrid corn has been genetically modified to contain a pesticidal protein, Cry9C, which makes it more resistant than traditional varieties to certain types of corn insect pests. Unlike other varieties of genetically engineered corn, the U.S. Environmental Protection Agency authorized the use of StarLink corn for animal feed and industrial use only, not for human consumption. However, some Cry9C-containing corn was mistakenly or inadvertently comingled with yellow corn intended for human food use. Because corn containing the Cry9C construct was not approved for human use, the U.S. Food and Drug Administration considers food containing it to be adulterated. Consequently, this regulatory violation resulted in hundreds of recalls of corn-based products, such as taco shells, containing cry9C DNA. Detecting the novel protein in StarLink corn is an emerging issue; therefore, there is no standardized or established analytical method for detecting Cry9C protein in processed foods. We developed a procedure for quantitation of Cry9C protein, with validation data, in processed food matrixes with a limit of quantitation at 1.7 ng/g (ppb), using a commercial polyclonal antibody-based Cry9C kit that was intended for corn grain samples. Intra- and interassay coefficients of variation were 2.8 and 11.8%, respectively. Mean recoveries were 73 and 85% at 2 and 5 ng/g Cry9C fortifications, respectively, for 19 control non-StarLink corn-based matrixes. Our data demonstrate only 0-0.5% of Cry9C protein survived the processing of tortilla chips and soft tortillas made from 100% StarLink corn, resulting in levels from below the detection limit to 45 ppb.

Evaluation of transgenic event CBH 351 (StarLink) corn in broiler chicks

ABSTRACTThe influence of transgenic event CBH 351 (StarLink; SL)‐derived hybrid corn on the growth performance, health condition and physiological function in broiler chicks, as well as the possible transfer of the cry9C gene and Cry9C protein to blood, liver and muscle were examined in comparison with chicks fed on a diet with non‐transgenic corn (SL‐F). Bodyweight gain and feed conversion ratio in the chicks fed on a diet with SL were significantly greater than in chicks fed on a diet with SL‐F during the starter phase (0–3 weeks of age), but this significant difference disappeared during the finisher phase (4–7 weeks of age). No abnormalities in health condition in either SL or SL‐F groups were observed, and livability did not differ significantly between SL and SL‐F groups. Moreover, no significant differences in serum biochemical and hematological values, histopathological observation and necropsy findings were observed between SL and SL‐F groups at the end of the experiment. The cry9C gene and Cry9C protein were not detected in blood, liver and muscle of chicks at 3, 5 or 7 weeks of age. The results indicate that feeding SL does not influence growth performance, health condition or physiological function in broiler chicks, and the cry9C gene and Cry9C protein are not transferred to the blood, liver and muscles of broilers.

Analysis of Flour and Food Samples for cry9C from Bioengineered Corn

StarLink corn is a variety of yellow corn that has been genetically modified by the insertion of an altered cry9C gene into the plant genome, resulting in expression of the insecticidal Cry9C protein. The U.S. Environmental Protection Agency has approved StarLink corn for use in animal feed but not in food intended for human consumption. Therefore, under the U.S. Food, Drug, and Cosmetic Act, any food intended for human consumption in which the presence of StarLink corn is indicated by the presence of either the Cry9C protein or the cry9C gene would be considered adulterated. Extraction and PCR-based methods were used to detect the presence of the cry9C DNA initially in corn flour and corn meal, and then these methods were extended to the analysis of processed corn products, including taco shells, cereals, baby foods, party snacks, and chips, for the presence of this modified genetic material. In a survey of 63 products, the cry9C transgene was detected in 4 taco shells.

Effect of subchronic feeding of genetically modified corn (CBH351) on immune system in BN rats and B10A mice.

Subchronic animal feeding studies to examine the effect on the immune system of genetically modified corn CBH351, which contains the Cry9C protein derived from Bacillus thuringiensis subspecies tolworthi, were conducted in female BN rats and B10A mice. The studies were designed to compare the effect of a line of genetically modified corn CBH351 (GM corn) with that of isoline corn (non-GM corn). Heat-treated corn meal was incorporated into the diets of the rats and mice at a concentration of 50%. The study duration was 13 weeks. Growth, food intake, and organ weights of the thymus, spleen, and liver were compared between animals fed the non-GM and GM lines. The histological findings in thymus, spleen, mesenteric lymph nodes, Peyer's patches, small intestines, liver, kidney, and bone marrow, and the presence of Cry9C-specific IgE, IgG, IgG1 and IgA antibodies in serum were also compared. The results showed no significant differences in growth, feeding value, or the histological findings in immunity-related organs between the animals fed the GM and non-GM lines. Production of Cry9 C-specific IgE and IgA was not detected in the serum of either group. Production of Cry9C-specific IgG and IgG1 was slightly increased in the 50% GM groups of BN rats. No Cry9C-specific IgG or IgG1 was detected in the serum of BN rats fed the diet containing 5% GM-corn In conclusion, no immunotoxic activity was detected in the GM-corn-fed rats and mice in this subchronic dietary study.

Determination of Cry9C Protein in Corn-Based Foods by Enzyme-Linked Immunosorbent Assay: Interlaboratory Study

The performance of a commercially available enzyme-linked immunosorbent assay kit (Enviro-Logix) was assessed for the determination of Cry9C protein, which is produced by the genetically modified corn StarLink, in 8 types of corn-based foods (starch, refined oil, soft tortillas, tortilla chips, corn flakes, corn puffs, corn muffins, and corn bread) in an interlaboratory study involving 7 laboratories in the United States. The assay kit is a double antibody sandwich and is based on the specific interaction between antibody and antigen. The Cry9C protein analyte is sandwiched between 2 antibodies, one to capture the analyte and the other is conjugated to the enzyme, horseradish peroxidase. The enzyme uses tetramethylbenzidine/peroxide for color development. A strong acid stopping reagent is then used to change the color from blue to a stable yellow. The intensity of the color is proportional to the concentration of the Cry9C protein. In this study blind duplicates of control samples (blank material prepared from non- StarLink corn), spiked samples (blank material with the addition of Cry9C protein), and samples containing incurred analyte (products prepared with StarLink corn) were analyzed. Cry9C protein from 2 different sources was used to spike the food products. Cry9C protein produced and purified from a bacterial host was used to prepare spiked test samples at 2.72 and 6.8 ng/g. Cry9C protein from StarLink corn flour was used to prepare spiked samples at 1.97 ng/g. Average recoveries for samples spiked with corn flour Cry9C protein at 1.97 ng/g ranged from 73 to 122%, within-laboratory relative standard deviations (RSDr) ranged from 6 to 22%, and between-laboratories relative standard deviations (RSDR) ranged from 16 to 56%. Average recoveries for samples spiked with bacterial Cry9C protein at 2.72 and 6.8 ng/g ranged from 27 to 96% and from 32 to 113%, respectively; RSDr values ranged from 10 to 35% and from 7 to 38%, respectively; and the RSDR ranged from 28 to 84% and 15 to 75%, respectively. The incurred test samples were found to contain Cry9C protein at levels ranging from 0.8 to 3187 ng/g depending on the product, RSDr values ranged from 5 to 16% and RSDR values ranged from 11 to 71%. Results of the statistical analysis indicate that this method is applicable to the determination of Cry9C protein in the 8 types of collaboratively studied corn-based products containing Cry9C protein (from StarLink) at levels of > or =2 ng/g.

Establishment of Cry9C susceptibility baselines for European corn borer and southwestern corn borer (Lepidoptera: Crambidae).

In 1997 and 1998, Cry9C susceptibility baselines were established for field-collected populations of European corn borer, Osrinia nubilalis (Hubner), and southwestern corn borer, Diatraea grandiosella Dyar. Bioassay of neonate European corn borer larvae of 16 colonies collected from the midwestern United States indicated LC50 values ranging from 13.2 to 65.1 ng of Cry9C protein per square centimeter. Neonate European corn borer LC50 values ranged from 46.5 to 214 ng/cm2. Neonate larvae of three colonies of southwestern corn borer collected from the southern and southwestern United States exhibited LC50 values from 16.9 to 39.9 ng of Cry9C protein per square centimeter. Southwestern corn borer neonate LC90 confidence limit values ranged from 40.3 to 157 ng of Cry9C protein per centimeter. The most sensitive southwestern corn borer colony was collected from the Mississippi delta exhibiting an LC50 value of 22.6 ng of Cry9C per cm2 and also displayed the widest LC0 confidence limits of 40.3-94.8 ng of Cry9C per cm2. Geographic baseline susceptibility data establishes the natural genetic variation and provides the foundation for future testing of insect populations exposed to increased use of Bacillus thuringiensis-based crops. Insect resistance management and stewardship of Cry9C will rely upon baseline data for the validation of discriminating dose assays for European corn borer and southwestern corn borer.

Farmers Sue Aventis Over StarLink Corn

A class-action lawsuit has been filed against Aventis CropScience over the disruption its genetically engineered corn has caused in this year's corn harvest. Designed to be insect resistant, StarLink corn has been approved for animal feed but not for human consumption because it contains the insecticidal Cry9C protein, a potential human allergen. Although StarLink was planted on less than 1% of U.S. corn acreage this past year, the widespread discovery of it in food products and grain supplies has led to product recalls and grain inspections, caused export problems, and raised health concerns (see page 42). Filed on behalf of U.S. farmers who did not plant StarLink, the lawsuit contends that Aventis was negligent in informing farmers about restrictions outlined in its Environmental Protection Agency registration and failed to prevent StarLink from entering the food supply chain. These omissions allegedly resulted in a loss of export and domestic markets for ...

Effects of transgenic Bacillus thuringiensis maize grain on B. thuringiensis-susceptible Plodia interpunctella (Lepidoptera: Pyralidae).

Percentage survivorship, developmental time, adult body length, and sex ratio of Plodia interpunctella (Hübner) reared on field-produced grain from sixteen cultivars of maize, Zea mays L., including several transgenic Bacillus thuringiensis (Bt) Berliner hybrids and selected non-Bt isolines, were evaluated under laboratory conditions. Compared with isolines, development was delayed and survivorship reduced for P. interpunctella reared on grain from transgenic hybrids with the CaMV/35s promoter that express Cry1Ab protein. Similarly, compared with non-Bt hybrids, a transgenic hybrid with the CaMV/35s promoter that expresses Cry9C protein delayed development, decreased survivorship, and caused reductions in adult body length of P. interpunctella. In contrast, no significant differences in P. interpunctella developmental times or survivorship were observed between transgenic hybrids with the PEPC promoter expressing Cry1Ab and their isolines. Additionally, developmental time, survivorship, and adult body length were similar between P. interpunctella reared on a transgenic hybrid with the CaMV/35s promoter expressing Cry1Ac and non-Bt hybrids. Our data demonstrate that transgenic Bt maize grain, especially grain from hybrids with the CaMV/35s promoter expressing Cry1Ab or Cry9C, can significantly affect B. thuringiensis-susceptible P. interpunctella populations up to 4 or 5 mo after harvest.

Transgenic Corn Expressing a Cry9C Insecticidal Protein from Bacillus thuringiensis Protected from European Corn Borer Damage

The European corn borer, Ostrinia nubilalis (Hübner), is devastating insect pest in the corn (Zea mays L.) growing regions of North America and Europe. Field evaluations in the USA and Belgium showed that transgenic corn events expressing Cry9C, an insecticidal crystal protein from Bacillus thuringiensis subsp, tolworthi, very effectively control both generations of the European corn borer. Second to fourth instar larvae fed with leaf material of event CBH351 expressing the Cry9C protein all died within 4 d. Cry9C events, expressing high levels of the insecticidal protein, showed minimal stalk tunneling after heavy artificial infestations. Event CBH351 tested in plots containing only Cry9C transgenic plants had 0.14‐ and 0.09‐cm tunneling per stalk compared with more than 30‐ and 23‐cm tunneling per stalk for the negative controls, in the Belgium and Iowa field trial, respectively. In plots containing 30% non‐transgenic plants the event CBH351, showed only 1.45‐cm tunneling per stalk. Leaf, tassel, and pith tissue contained 39.0, 17.4, and 84.8 μg Cry9C protein mg−1 soluble protein, respectively, in analyses conducted at harvest of the Belgium trial. The implications of Cry9C use for resistance management strategies are discussed.