Fate of Cry1Ab protein in agricultural systems under slurry management of cows fed genetically modified maize (Zea mays L.) MON810: a quantitative assessment

The objective of the study was to track the fate of recombinant Cry1Ab protein in a liquid manure field trial when feeding GM maize MON810 to dairy cows. A validated ELISA was applied for quantification of Cry1Ab in the agricultural chain from GM maize plants, feed, liquid manure and soil to crops grown on manured fields. Starting with 23.7 μg of Cry1Ab g(-1) dry weight GM maize material, a rapid decline of Cry1Ab levels was observed as 2.6% and 0.9% of Cry1Ab from the GM plant were detected in feed and liquid manure, respectively. Half of this residual Cry1Ab persisted during slurry storage for 25 weeks. After application to experimental fields, final degradation of Cry1Ab to below detectable levels in soil was reported. Cry1Ab exhibited a higher rate of degradation compared to total protein in the agricultural processes. Immunoblotting revealed a degradation of the 65 kDa Cry1Ab into immunoreactive fragments of lower size in all analyzed materials.     

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.     

Rapid digestion of Cry34Ab1 and Cry35Ab1 in simulated gastric fluid

Two genes were identified in Bacillus thuringiensis Berliner (Bt) that code for the proteins that comprise a Cry34Ab1/Cry35Ab1 binary insecticidal crystal protein. Maize, Zea mays L., plants have been transformed to express the Cry34Ab1/Cry35Ab1 proteins, and as a result, these plants are resistant to attack by western corn rootworm, Diabrotica virgifera virgifera LeConte, a major pest in the Midwestern corn-growing area of the U.S.A. As part of the safety assessment for the proteins, digestibility studies were conducted. Digestion experiments with both proteins demonstrated rapid degradation in simulated gastric fluid, comparable to other registered plant-incorporated protectants. Quantitative and qualitative approaches for determining digestibility are illustrated.     

Biodegradation of Cry1Ab protein from Bt transgenic rice in aerobic and flooded paddy soils

Degradation of Cry1Ab protein from Bt transgenic rice was examined under both aerobic and flooded conditions in five paddy soils and in aqueous solutions. The hydrolysis rate of Cry1Ab protein in aqueous solutions was correlated inversely with the solution pH in the range of 4.0 to 8.0, and positively with the initial concentration of Cry1Ab protein. Rapid degradation of Cry1Ab protein occurred in paddy soils under aerobic conditions, with half-lives ranging from 19.6 to 41.3 d. The degradation was mostly biotic and not related to any specific soil property. Degradation of the Cry1Ab protein was significantly prolonged under flooded conditions compared with aerobic conditions, with half-lives extended to 45.9 to 141 d. These results suggest that the toxin protein, when introduced into a paddy field upon harvest, will probably undergo rapid removal after the field is drained and exposed to aerobic conditions.     

Effects of physical and chemical properties of soils on adsorption of the insecticidal protein (Cry1Ab) from Bacillus thuringiensis at Cry1Ab protein concentrations relevant for experimental field sites

The adsorption of the insecticidal Cry1Ab protein of Bacillus thuringiensis (Bt) on Na-montmorillonite (M-Na) and soil clay fractions was studied. The aim of this study was not to find the adsorption capacity of the soils from the experimental field site, where Bt corn (MON810) was cultivated, but rather to characterize the adsorption behavior of the Cry1Ab protein at concentrations typically found at experimental field sites. In kinetic experiments, the Cry1Ab protein adsorbed rapidly (<60 min) on M-Na. As the concentration of M-Na was varied and the added Cry1Ab protein concentration was kept constant (20 and 45 ng ml⁻¹), the adsorption per unit weight of Cry1Ab protein decreased with increasing concentrations of M-Na. Adsorption of Cry1Ab protein on M-Na decreased as the pH value of the suspension increased. All adsorption isotherms could be described mathematically by a linear regression with the parameter k, the distribution coefficient, being the slope of the regression line. Although their mineralogical composition was nearly identical, the soil clay fractions showed different k values. The different k values were correlated with the physical and chemical properties of the soil clay fractions, such as the organic carbon content, the specific external surface area, and the electrokinetic charge of the external surfaces of the clays, as well as with the external surface charge density. An increase in the amount of soil organic matter, as well as an increase in the electrokinetic external surface charge of the soil clays, decreased the distribution coefficient k. An increase of the specific external surface areas of the soil clays resulted in a higher distribution coefficient k.Less than 10% of adsorbed Cry1Ab protein was reversibly adsorbed on the soil clays and, thus, desorbed. The desorption efficiency of distilled water was higher than that of a solution of CaCl₂ (2.25 mmol) and of dissolved organic carbon (50 mg C l⁻¹).     

Characterization of Cry34Ab1 and Cry35Ab1 insecticidal crystal proteins expressed in transgenic corn plants and Pseudomonas fluorescens

Cry34Ab1 and Cry35Ab1 proteins, identified from Bacillus thuringiensis strain PS149B1, act together to control corn rootworms. Transgenic corn lines coexpressing the two proteins were developed to protect corn against rootworm damage. Large quantities of the two proteins were needed to conduct studies required for assessing the safety of this transgenic corn crop. Because it was technically infeasible to obtain sufficient quantities of high purity Cry34Ab1 and Cry35Ab1 proteins from the transgenic corn plants, the proteins were produced using a recombinant Pseudomonas fluorescens (Pf) production system. The two proteins from both the transgenic corn and the Pf were purified and characterized. The proteins from each host had the expected molecular mass and were immunoreactive to specific antibodies in enzyme-linked immunosorbent assay and Western blot analysis. Data from N-terminal sequencing, tryptic peptide mass fingerprinting, internal peptide sequencing, and biological activity provided direct evidence that the Cry34Ab1 and Cry35Ab1 proteins produced in Pf and transgenic corn were, respectively, comparable or equivalent molecules. In addition, neither protein had detectable glycosylation regardless of the host.     

苏云金杆菌Cry2Ab可溶蛋白的原核表达及多克隆抗体的制备

苏云金杆菌Cry2Ab蛋白是一类对鳞翅目昆虫有特异性毒性作用的毒素蛋白,已广泛应用于针对鳞翅目害虫的防治之中。依据苏云金杆菌cry2Ab基因序列设计一对全长引物,从苏云金杆菌(Bacillus thuringiensis)WB9菌株总DNA中克隆出cry2Ab基因全序列,构建Cry2Ab-PK表达载体,将获得的Cry2Ab-PK阳性克隆进行原核诱导表达,获得约90kD的Cry2Ab-GST融合蛋白,经批量纯化并切除GST标签后获得可溶Cry2Ab蛋白,约65kD。利用纯化Cry2Ab可溶蛋白免疫新西兰雄性大白兔(Oryctolagus cuniculus),制备Cry2Ab兔源多克隆抗血清,通过间接ELISA法测定Cry2Ab抗血清效价超过1:150000。Western blot测定结果表明,制备的Cry2Ab兔源抗血清能特异性识别Cry2Aa及Cry2Ab抗原蛋白,不能识别Cry1Ab及Cry3Aa抗原蛋白。研究结果对深入研究Cry2A毒素蛋白作用机理及毒素与受体间互作关系提供了技术支持。     

High sensitive detection of Cry1Ab protein using a quantum dot-based fluorescence-linked immunosorbent assay

Protein-based detection methods, enzyme-linked immunosorbent assay (ELISA) and lateral flow strip, have been widely used for rapid, spot, and sensitive detection of genetically modified organisms (GMOs). Herein, one novel quantum dot-based fluorescence-linked immunosorbent assay (QD-FLISA) was developed employing quantum dots (QDs) as the fluorescent marker for the detection of the Cry1Ab protein in MON810 maize. The end-point fluorescent detection system was carried out using QDs conjugated with goat anti-rabbit secondary antibody. The newly developed Cry1Ab QD-FLISA assay was highly specific to the Cry1Ab protein and had no cross-reactivity with other target proteins, such as Cry2Ab, Cry1F, and Cry3Bb. The quantified linearity was achieved in the value range of 0.05-5% (w/w). The limits of detection (LOD) and quantification (LOQ) of the QD-FLISA were 2.956 and 9.854 pg/mL, respectively, which were more sensitive than the conventional sandwich ELISA method. All of the results indicated that QD-FLISA was a highly specific and sensitive method for the monitoring of Cry1Ab in GMOs.     

转基因高粱Cry1Ab蛋白含量的比较研究

通过农杆菌介导法将杀虫晶体蛋白基因cry1Ab导入高粱恢复系115中,共获得13个独立的转基因株系,26株转基因植株,转化率为5.1%;GUS活性、PCR、Southern和Western杂交分析表明,此基因已整合进高粱基因组并得到正确表达。利用ELISA试剂盒测定Cry1Ab蛋白含量,结果表明,不同转基因植株的Cry1Ab蛋白含量有明显差异,最高可达0.850 μg/g,占可溶性蛋白的0.016%,还有一些转基因植株中不能表达;同一转基因植株的不同组织中表达量有明显差异,其顺序为：叶>颖壳>籽粒>根>茎;不同部位的叶片也有差异,其顺序为：中部叶>基部叶>新叶。     

Fate of fatty acids derived from biogas residues in arable soil

Biogas residues are rich in microbial biomass and contribute to organic matter formation when applied to soils. Here we present a detailed analysis of the fatty acids derived from ¹³C-labelled biogas residues applied to arable soil and incubated for 378 days. We applied a differential approach using phospholipid fatty acids and total fatty acids to evaluate the carbon dynamics in living biomass and non-living soil organic matter. Biogas residue addition increased the microbial biomass in soil. The sum of ¹³C-labelled phospholipid fatty acids decreased to ∼60% during incubation whereas the decrease of t-FA was higher (to 33%). Compound-specific fatty acid analysis showed fatty acid specific incorporation or loss of ¹³C, indicating hints for the carbon flow within the microbial food web. Overall, microbial biomass in biogas residues may be a significant contributor to soil organic matter formation.     

Effects of temperature, water content and pH on degradation of Cry1Ab protein released from Bt corn straw in soil

We investigated the effects of soil temperature (15 °C, 25 °C, 35 °C), water content (20%, 33%, 50%) and pH (4.5, 7.0, 9.0) on the degradation of Cry1Ab protein released from the straw of Bt corn varieties 34B24 and 1246 × 1482 both expressing Cry1Ab protein. Our results showed that Cry1Ab protein released from both 34B24 and 1246 × 1482 straw was degraded in a similar way in all treatments, which demonstrated a rapid decline in the early stage but a slow decline in the middle and late stages. In the late stage (180 days after the experiment commenced) 0.03%-1.51% and 0.02%-0.91% of initial Cry1Ab protein released from 34B24 and 1246 × 1482 straw was detected in soil. In addition, degradation dynamics of Cry1Ab protein under different environmental conditions was well described by the shift-log model. DT₅₀ of Cry1Ab protein released from 34B24 and 1246 × 1482 straw was 0.97-9.97 d and 0.75-10.89 d, respectively, and DT₉₀ was 4.66-162.45 d and 6.44-57.46 d, respectively. The results suggested that soil temperature had significant effects on the degradation of Cry1Ab protein, with a higher degradation rate at higher temperature, but soil water content and pH had no obvious effects on the degradation of Cry1Ab protein.     

Adsorption of Cry1Ab protein isolated from Bt transgenic rice on bentone, kaolin, humic acids, and soils

Adsorption on a soil matrix of the insecticidal protein from Bacillus thuringiensis ( Bt) transgenic plants affects their accumulation and release and, hence, bioavailability in soil. Cry1Ab protein isolated from Bt transgenic rice was used to evaluate the adsorption and desorption on bentone, kaolin, and humic acids (HAs). The adsorption equilibrium of Cry1Ab protein was reached within 1-2 h for bentone and kaolin and within 4-8 h for HAs. The adsorption isotherms were better described by linear expressions ( R (2) >/= 0.973) rather than by the Freundlich model. No saturation was observed, even at the maximum concentration used (3.71 microg mL (-1)). The adsorbed protein was not easily desorbed at the used protein concentrations (0.18-3.71 microg mL (-1)); more than 50-70%, 70-80%, and 90% of the adsorbed protein remained on HAs, kaolin, and bentone, respectively, after washing with water. Adsorption and desorption of the Cry1Ab protein were further studied using five soils, and the isotherms were also well-described by linear equations ( p < 0.05). Adsorption of the Cry1Ab protein on soils was positively related to the soil organic matter content.     

南非冬闲期表施和混施入Bt层玉米落叶和Cry1Ab蛋白的分解研究

Unintended effects of genetic modification on chemical composition of Bt maize leaf litter may have impacts on its decomposition. In most agricultural systems in South Africa, maize litter is either left on the soil surface or incorporated into the soil during tillage. A litterbag experiment, using leaf litter of three maize hybrids （DKC80-12B, DKC80-10 and DKC6-125）, was carried out at the University of Fort Hare Research Farm, South Africa, to determine the effects of genetic modification on decomposition of maize leaf litter when left on the soil surface under field conditions between July and November, the normal fallow period, in 2008. Another litterbag experiment was conducted at the University of Fort Hare Research Farm and Zanyokwe Irrigation Scheme, South Africa, using leaf ~itter of two maize hybrids genetically modified with the erylAb gene （MONS10）, DKC75-15B and PAN6Q-3OSB, and their corresponding near isolines, CRN3505 and PAN6Q-121. The degradation of CrylAb protein in the litter, both surface-applied and soil-incorporated, was also investigated. Decomposition of Bt maize litter was similar to that of non-Bt maize litter both when applied on the surface and when incorporated into soil. Soil-incorporated litter, as well as its CrylAb protein, decomposed faster than that applied on the surface. The leaf litter C：N ratios of PAN6Q-308B and PAN6Q-121 were similar throughout the study, whereas those of DKC75-15B and CRN3505 declined by similar amounts during a 12-week period. These findings suggested that decomposition of leaf litter of Bt maize, with the MON810 event, was not affected by maize genetic modification, and that the CrylAb protein broke down together with plant leaf litter during the winter fallow regardless of whether the litter was applied on the soil surface or incorporated into soil.     

Cry1Ab Adsorption and Transport in Humic Acid-Coated Geological Formation of Alumino-Silica Clays

Genetically modified agricultural products have been introduced to increase food supply by enhancing their resistance to pests and diseases, along with easily adapting to environmental conditions. Due to the modification of DNA, public objections are prevalent, including concerns on the impact on the ecosystem. In this research, adsorption and transport of Cry1Ab, a toxin exuded by the transgenic Bt maize in alumino-silica clays, were evaluated in laboratory columns under steady-state flow conditions. Since Cry1Ab fate and transport were very responsive to animal waste field applications, during which humic acids were released, Cry1Ab adsorption and transport in humic acid-coated alumino-silica clays were also investigated. Cry1Ab breakthrough curves were simulated using the convection-dispersion transport models. It was discovered that the humic acid coating increased Cry1Ab deposition during the transport. Based on analysis of the breakthrough curves, adsorption isotherms of Cry1Ab in alumino-silica clays were obtained and compared with those of batch experiments. The humic acid coating changed the bonding energy between Cry1Ab and the adsorption receptor sites on alumino-silica clay surfaces, thereby changing Cry1Ab partition between the aqueous phase and the solid phase.     

Earthworms of different functional groups affect the fate of the Bt-toxin Cry1Ab from transgenic maize in soil

The fate of the insecticidal Cry1Ab protein from crop residues (leaves and roots) of the transgenic maize variety MON810 was studied in the presence and absence of two earthworm species (Lumbricus terrestris, Aporrectodea caliginosa; separate incubations) in soil microcosms. The recombinant Cry1Ab protein was quantified using a highly sensitive ELISA. Control microcosms received corresponding non-transgenic plant material. All earthworms survived in the microcosms over a period of 5 weeks, irrespective of whether they received MON810 or non-transgenic plant material. Weight loss was observed for both earthworm species, independent of the plant material or transgenic modification. A strong decline of immunoreactive Cry1Ab in plant residues (mean initial concentration approx. 5000 ng g⁻¹) of MON810 was observed in all treatments, but in microcosms with earthworms this decline was significantly higher with less than 10% of the initial Cry1Ab concentration remaining after 5 weeks. Cry1Ab concentrations in casts were only 0.1% of those found in remaining plant material of the respective microcosms. No immunoreactive Cry1Ab proteins were found in earthworm tissues (threshold of detection: 0.58 ng g⁻¹ fresh weight). No further decline was found for Cry1Ab concentrations in casts of A. caliginosa during a subsequent period of 3 months of incubation in bulk soil (<0.1 ng g⁻¹) after removal of the earthworms from the microcosms, while in casts of L. terrestris the concentration decreased from 0.4 to below 0.1 ng g⁻¹. In conclusion, this study demonstrates that earthworms enhance the decline of immunoreactive Cry1Ab proteins from maize residues.     

Adsorption and Desorption of Cry1Ab Proteins on Differently Textured Paddy Soils

In recent years, selected cry genes from Bacillus thuringiensis (Bt) encoding the production of Cry proteins (Bt toxins) have been engineered into crop plants (Bt-crops). Through the cultivation of Bt crops and the application of Bt pesticides, Cry proteins could be introduced into arable soils. The interaction between the proteins and soils was analyzed in this study to investigate the affinity of Cry proteins in paddy soil ecosystems. Four Paddy soils were selected to represent different soil textures. Cry proteins were spiked in soils, and the amount of protein adsorbed was measured over 24 h. Desorption of Cry1Ab proteins from paddy soils was performed by washing with sterile Milli-Q water (H₂O_MQ), and subsequently extracted with an extraction buffer. The paddy soils had a strong affinity for Cry1Ab proteins. Most of the Cry1Ab proteins added (> 98%) were rapidly adsorbed on the paddy soils tested. More Cry1Ab proteins were adsorbed on non-sterile soils than on sterile soils. Less than 2% of the adsorbed Cry1Ab proteins were desorbed using H₂O_MQ, while a considerable proportion of the adsorbed proteins could be desorbed with the buffer, ranging from 20% to 40%. The amount of proteins desorbed increased with the increases in the initial amount of Cry1Ab proteins added to the paddy soils. The concentration of Cry1Ab proteins desorbed from the paddy soils was higher for sterile soils than non-sterile ones. Our results indicate that Bt toxins released via the cultivation of Bt crops, the application of Bt pesticides can be adsorbed on paddy soils, and soil texture could impose an impact on the adsorption capability.     

Release of the recombinant Cry3Bb1 protein of Bt maize MON88017 into field soil and detection of effects on the diversity of rhizosphere bacteria

A three-year experimental field study with a genetically engineered Bt maize (event MON88017) and three conventionally bred cultivars was conducted to quantify the recombinant Cry3Bb1 protein released into soil and detect effects on the diversity of soil bacteria. Protein extraction and an enzyme-linked immunosorbent assay (ELISA) allowed a threshold detection of 0.01 ng Cry3Bb1 g^?1 soil. The maximum amount found in field plots with Bt maize was 1.0 ng Cry3Bb1 g^?1 rhizosphere soil. Average concentrations during the growing seasons varied between years from 0.07 to 0.29 ng g^?1. No accumulation of Cry3Bb1 in soil occurred over the three growing seasons. Four weeks after harvest, the major Cry3Bb1 reservoirs on the field were the remaining root stubbles, but their Cry3Bb1 concentration declined by 98.30–99.99% in the following seven months. During the three consecutive years of study there were never significant differences between the rhizosphere bacterial community structure of the Bt maize and the other cultivars, as detected by cultivation independent profiling of PCR-amplified 16S rRNA genes. The low concentrations of soil extractable Cry3Bb1, its degradation in decaying roots, and the lack of effects on rhizosphere bacteria give no indications of adverse effects of MON88017 cultivation on soil ecology.     

用合成多肽为半抗原制备Bt Cry1Ab的单克隆抗体

由于Bt Cry1Aa、Cry1Ab和Cry1Ac晶体蛋白之间具有很高的同源性(82%~90%),采用常规的单抗制备方法很难制取特异性强的Bt Cry1Ab单抗,为了制备抗Bt Cry1Ab蛋白的特异性单克隆抗体(MAB),本研究从NCBI获得了Bt Cry1Ab蛋白的氨基酸序列,根据ANTHEPORT和DNAStar软件对其抗原性、亲水性和表位性分析结果选定BtCry1Ab特异性肽段进行人工合成,并将其偶联于匙孔血蓝蛋白(KLH)免疫动物,应用细胞融合技术制备了抗该肽段的杂交瘤细胞22株。通过ELISA试验从中筛选出与Bt Cry1Ab天然蛋白产生特异性反应的单克隆抗体杂交瘤细胞株一株(3A10)。经检测,其分泌的抗体亚类为IgG1型;轻链属κ型;杂交瘤细胞株染色体数目为89~108条;用其制作的腹水对Bt Cry1Ab合成肽的反应效价为1∶1×107;对Bt Cry1Ab天然蛋白的反应效价为1∶1×104;纯化后的抗体对Bt Cry1Ab合成肽的效价为1∶1×108;对Bt Cry1Ab天然蛋白的效价为1∶2×104。抗体的相对亲和力为0.5μg/mL,对Bt Cry1Ab蛋白的最低可检测值为10ng/mL。ELISA结果显示,3A10杂交瘤细胞株所分泌的MAB能特异性识别合成肽和Bt Cry1Ab蛋白,而对同源的Cry1Ac和Cry1Aa蛋白无交叉反应;本研究所制备的Bt Cry1Ab单克隆抗体能够对常规棉和抗虫棉(Gossypium hirsutumL.)进行有效的区分,并且能特异性的识别其中的Bt Cry1Ab蛋白。     

Rapid degradation of the Cry1F insecticidal crystal protein in soil

The gene for the core Cry1F insecticidal crystal protein (ICP) from Bacillus thuringiensis Berliner (Bt) has been incorporated into the genome of maize plants, Zea mays L. Plants expressing this ICP are protected from attack by various Lepidopteran pests including the European corn borer, Ostrinia nubilalis (Hübner). The stability of the Cry1F ICP in soil was assessed in a laboratory study designed to determine the persistence of the active protein residue in soil over time, using insect bioassay as the analytical quantification method. The GI(50) (concentration estimated to inhibit growth by 50%) rose at each consecutive incubation interval, indicating a consistent decline in Cry1F activity over time. The residue data were poorly described by a first-order model when fit to either the full data or a truncated data set where the last interval (28 days) was excluded. Data were well described by a shift-log model, and this model predicted DT(50) (time until 50% decay) and DT(90) (time until 90% decay) values of 0.6 and 6.9 days, respectively. This rapid degradation rate was consistent with other Bt proteins evaluated in our laboratory.     

不同发育时期转基因高粱不同株系Cry1Ab蛋白含量的比较

高粱的害虫种类众多,影响着产量与品质,培育抗虫品种是重要的育种目标,但目前高粱中缺乏有效的抗螟虫资源,难以通过常规育种培育出抗螟虫的品种。张明洲(2002)成功地将来自Bt密码子优化的抗虫基因cry1Ab转入高粱中,并经农业部批准进入中间试验阶段。为有效和合理地利用这一抗虫