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.     

杂交狼尾草厌氧发酵的物质与能量转化率分析

为了解厌氧发酵过程中的C、N元素流动状况以及物质和能量的转化利用效率,以杂交狼尾草为原料,采用中温(35±1)℃批式厌氧发酵工艺,研究了杂交狼尾草厌氧发酵制备沼气的产气性能,在此基础上结合物质流分析方法分析了发酵过程中C、N元素的分布情况以及物质与能量的转化率.研究结果表明:C/N 167和25下,杂交狼尾草鲜料的VS(挥发性固体)产气量分别为280.02和242.33 mL/g,其中CH4体积分数分别为59.96%和61.46%;C/N 137和25下,青贮料的VS产气量分别为327.02和295.43 mL/g,其中CH4体积分数分别为58.49%和61.05%;C元素的流向分布:沼气33.1%,发酵液8.0%,发酵渣58.9%;N元素主要流入剩余物中:发酵液69.4%,发酵渣30.7%.杂交狼尾草厌氧发酵制备沼气的物质转化率和能量转化率分别为42.1%和33.1%.该研究为能源草本植物的资源管理和厌氧能源化利用提供了理论依据.     

Bt水稻杀虫蛋白时空变化及秸秆还田后在土壤中的持留规律

以Bt抗虫水稻华池B6、TT51及其非转基因水稻亲本嘉早935、明恢63,以及与它们亲缘较远但农艺性状相近的水稻品种中九B、R9311为试验材料,研究了田间种植条件下Bt抗虫水稻杀虫蛋白的时空变化及其在根际土中的持留规律,同时,还研究了秸秆还田后Bt蛋白在土壤中的持留规律。结果表明：1）Bt抗虫水稻华池B6植株各个部位...     

Are survival and reproduction of Enchytraeus albidus (Annelida: Enchytraeidae) at risk by feeding on Bt-maize litter?

Enchytraeids are saprophagous soil organisms, appearing in high abundances and contributing to ecological processes within the soil. For decades they have been used as model species for biological research. In the framework of research on genetically modified plants, however, they have not been considered to date. Following the ISO/DIS guideline, survival and reproduction of Enchytraeus albidus, fed with diets containing Bt-maize (N4640Bt Cry1Ab, DKC5143Bt Cry3Bb1) leaf material were analysed. For comparison, diets with the corresponding untransformed near-isolines (N4640, DKC5143) were examined. Additionally a high quality control diet (oat flakes) was included. Survival and reproduction showed no significant differences between the Cry3Bb1 treatment and the treatment with the untransformed counterpart. For the Cry1Ab treatment survival was significantly higher than for the treatment with the corresponding near-isoline. In contrast, reproduction was significantly lower for the Cry1Ab treatment compared to that for the isoline. For the Cry3Bb1 treatment, no effect was shown on survival or reproduction. For the Cry1Ab variety and its untransformed counterpart, a contrasting result was detected, which is unlikely to be caused by the Bt-protein but rather by differences in other plant components. Overall survival and reproduction were highest for the control.     

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.     

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.     

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.     

Composition of forage and grain from second-generation insect-protected corn MON 89034 is equivalent to that of conventional corn (Zea mays L.)

Insect-protected corn hybrids containing Cry insecticidal proteins derived from Bacillus thuringiensis have protection from target pests and provide effective management of insect resistance. MON 89034 hybrids have been developed that produce both the Cry1A.105 and Cry2Ab2 proteins, which provide two independent modes of insecticidal action against the European corn borer ( Ostrinia nubilalis ) and other lepidopteran insect pests of corn. The composition of MON 89034 corn was compared to conventional corn by measuring proximates, fiber, and minerals in forage and by measuring proximates, fiber, amino acids, fatty acids, vitamins, minerals, antinutrients, and secondary metabolites in grain collected from 10 replicated field sites across the United States and Argentina during the 2004-2005 growing seasons. Analyses established that the forage and grain from MON 89034 are compositionally comparable to the control corn hybrid and conventional corn reference hybrids. These findings support the conclusion that MON 89034 is compositionally equivalent to conventional corn hybrids.     

玉米秸秆与废弃白菜的混合青贮品质及产沼气能力分析

为实现干玉米秸秆(dried maize straw,DMS)的长时间保质贮存,利用青贮原理将DMS与废弃白菜(cabbage waste,CW)连续混贮90 d,设置DMS单贮组和6个不同质量比例(DMS:CW=29:19、27:21、25:23、23:25、21:27和19:29)混贮组,间隔30 d分析其贮存品质,筛选适宜贮存条件,并考察秸秆贮存前后的微观结构变化和产沼气潜力。结果表明,与单贮组相比,6个混贮组的p H值均显著下降(P0.05),乳酸含量显著升高(P0.05),混贮品质优于单贮。6个混贮组贮存30 d时的p H值均为最低,乳酸含量最高,感官评价均为优级,30 d后p H值有所上升,乳酸含量急剧下降,60d时的木质素含量显著降低(P0.05),综纤维素含量显著升高(P0.05)。随着CW比例增加,混贮组p H值逐渐减小,乳酸含量逐渐增加,MEⅤ组(DMS:CW=21:27)p H值最低,乳酸含量最高,能使DMS连续贮存60 d不变质。扫描电镜结果显示,青贮发酵使干秸秆原先致密复杂的木质纤维结构开始瓦解,青贮秸秆表面有很多裂缝和孔洞,与微生物或酶的可接触面积增加。沼气发酵试验表明,MEⅤ组混贮秸秆的累积产气量略高于未贮存干秸秆,且累积甲烷产量提高了61.67%,是未贮存干秸秆的1.6倍。Modified Gompertz模型显示混贮秸秆的甲烷生成速率增加,产甲烷能力提高。总之,当DMS∶CW质量比为21∶27(含水率为73%)时DMS能连续保质贮存2月,且贮存后秸秆的甲烷产量明显提升。     

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.     

Purification and characterization of a chimeric Cry1F delta-endotoxin expressed in transgenic cotton plants

Cotton plants were genetically modified through the introduction of a synthetic gene that encodes a Bacillus thuringiensis insecticidal protoxin referred to as Cry1F(synpro). This protoxin is a chimeric, full-length delta-endotoxin of 130 kDa, comprised of the core toxin of Cry1Fa2 protein and parts of the nontoxic portions of Cry1Ca3 and Cry1Ab1 proteins, all of which originated from Bacillus thuringiensis. The Cry1F(synpro) expressed in cotton plants confers resistance to lepidopteran pests. The current study was conducted to characterize the Cry1F(synpro) protein expressed in the transgenic cotton event 281-24-236. Results showed that the full-length Cry1F(synpro) produced in the transgenic cotton plants was sensitive to the host cell protease cleavage, resulting in a truncated, biologically active form (core toxin) with an apparent molecular mass of 65 kDa. This truncated toxin was purified by immunoaffinity chromatography from the cotton leaf extract. N-terminal sequencing, peptide mass fingerprinting by MALDI-TOF MS, and internal peptide sequencing by MS/MS confirmed the identity of the truncated core toxin of Cry1F. The mechanism of truncation was explored with Cry1F(synpro) derived from a recombinant Pseudomonas fluorescens. The transgenic cotton-produced Cry1F showed equivalent insecticidal activity to that of Pseudomonas fluorescens-derived Cry1F.     

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.     

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.     

Cry1Ab蛋白在不同土壤中的吸附与解吸

以转cry1Ab基因高粱为材料提取Cry1Ab蛋白，测定了Cry1Ab蛋白在6种土壤中的吸附与解吸，分析了Cry1Ab蛋白溶液浓度、土壤理化性质对Cry1Ab蛋白吸附与解吸的影响。结果表明，不同类型的土壤对Cry1Ab的吸附与解吸存在明显的差异，吸附量表现为红泥土〉灰化土〉青紫泥田〉黄筋泥〉黄松田〉红砂土，解吸量表现为灰化土〉青紫泥田〉红砂土〉黄筋泥〉红泥土〉黄松田；Cry1Ab蛋白溶液的浓度与吸附量和解吸量呈显著正相关，相关系数分别为0．86和0．99；土壤有机质、pH值与土壤吸附Cry1Ab蛋白的能力呈显著正相关，相关系数分别为0．83和0．82；土壤全氮、有效磷的含量与土壤吸附Cry1Ab蛋白呈正相关，土壤全氮、有效磷和速效钾的含量与解吸均呈负相关。土壤吸附、解吸Cry1Ab蛋白是加入Cry1Ab蛋白溶液的浓度及不同土壤的理化性质共同作用的结果。     

北京大中型沼气工程冬季运行状况及发酵前后物料理化生物特性

为了解北京市大中型沼气工程冬季运行情况及发酵剩余物理化特性,该文实地调查了北京市29座沼气工程冬季运行情况,以调研问卷的形式了解沼气工程运行状况,并采样分析了发酵原料、沼渣和沼液样品的养分含量、粪大肠菌群数、铜、锌、砷、铅等指标。结果表明:北京29座冬季运行的沼气工程中主要以畜禽粪便为原料,其中有20座沼气工程采用猪粪进行厌氧发酵,厌氧消化反应器类型主要是全混式厌氧反应器和升流式厌氧固体反应器,沼气以供周边农户使用为主,沼渣和沼液以农田利用为主,从区域沼气工程运行状况来看,房山区的沼气工程运行状况相对较好,大兴区和顺义区的沼气工程运行状况参差不齐;所调研沼气工程沼渣和沼液的pH值适宜向农田施用,沼渣的EC值相对原料平均下降了56.97%,沼渣有机质和可挥发性固体的质量分数相对原料分别降低了20.99%和27.93%,沼渣的养分质量分数较高,沼液的养分质量浓度较低,沼渣中的粪大肠菌群数和沼液的化学需氧量、粪大肠菌群数整体偏高,有6座沼气工程的砷含量较高。调查结果可为北京市沼气工程冬季运行效果及沼渣沼液资源化利用提供基础数据,促进北京市沼气工程的可持续发展,提高生态和经济效益。     

Liquid chromatographic determination of fumonisins B1, B2, and B3 in corn silage

Corn silage was dried, ground, and then extracted with 0.1 M ethylenediaminetetraacetic acid. The filtrate was applied to a FumoniTest immunoaffinity column. Fumonisins were derivatized with naphthalene-2,3-dicarboxaldehyde, separated on a C(18) liquid chromatographic column, and detected by fluorescence. The detection limits for fumonisin B(1), fumonisin B(2), and fumonisin B(3) were 50, 25, and 25 ng/g of dried silage, respectively. Recoveries of fumonisin B(1), fumonisin B(2), and fumonisin B(3) from wet and dried corn silage spiked over the range of 100-5000 ng/g averaged 91-106%. The method was applied to corn silage samples collected from the midwestern area of the United States during 2001-2002. Of 89 corn silage samples, fumonisin B(1), fumonisin B(2), and fumonisin B(3) were found in 86 (97%), 64 (72%), and 51 (57%) of the samples. The mean positive levels of fumonisin B(1), fumonisin B(2), and fumonisin B(3) were 615, 93, and 51 ng/g, respectively, in dried silage. This suggests that fumonisins may be frequent low level contaminants in corn silage.     

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.     

A highly specific enzyme-linked immunosorbent assay for the detection of Cry1Ac insecticidal crystal protein in transgenic WideStrike cotton

A highly selective enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitative detection of the Cry1Ac protein expressed in transgenic cotton. Two Cry1Ac-specific monoclonal antibodies (MAb), Kbt and 158E6, were developed and selected to form a sandwich format ELISA. The MAb Kbt was used as a capture antibody, and 158E6 was conjugated with horseradish peroxidase and served as a detection antibody. The assay was optimized and validated with different cotton matrices. Tissues were extracted with phosphate-buffered saline containing 0.05% Tween 20 and 1% polyvinylpyrrolidone. The extract was then treated with trypsin to truncate full-length Cry1Ac into the core toxin for quantitation. The resulting assay has good accuracy and precision with a validated limit of quantitation ranging from 0.1 to 0.375 mug/g dry weight of cotton tissues. This assay is highly specific for Cry1Ac protein and has no cross-reactivity with the nontarget proteins tested such as Cry1Ab and Cry1F.     

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.     

华恢1号衍生系旱恢3T的选育及抗虫表现

水稻作为世界上主要的粮食作物之一,容易受到螟虫、稻飞虱、稻象甲等主要害虫为害造成减产.为改良节水抗旱稻的抗虫性,以华恢1号(TT51)为cry1Ab/Ac基因供体,与节水抗旱稻恢复系品种旱恢3号杂交,利用分子标记辅助选择技术对目标基因进行筛选,最终获得BC3F4纯合、稳定株系.通过对Cry1Ab/Ac蛋白的定性和定量检...