Adsorption of transgenic insecticidal Cry1Ab protein to silica particles. Effects on transport and bioactivity

Bt crops are genetically modified to be resistant against insect pests by expressing insecticidal Cry proteins. The processes governing the fate and bioavailability of the expressed transgenic Cry proteins in soils are poorly understood. We studied adsorption of Cry1Ab to negatively charged silica (SiO(2)) particles, a major soil constituent and a model for negatively charged mineral surfaces, at pH 5 to 10 and ionic strengths I = 10 mM to 250 mM, both in solution depletion and saturated column transport experiments. Cry1Ab-SiO(2) interactions were dominated by patch-controlled electrostatic attraction (PCEA), as evident from increasing Cry1Ab attraction to SiO(2) with decreasing I at pH at which both Cry1Ab and SiO(2) were net negatively charged. Experimental and modeling evidence is provided that the surface heterogeneity of SiO(2) particles modulated PCEA, leading to a fraction of adsorption sites with slow Cry1Ab desorption kinetics. Desorption rates from these sites increased upon increasing the solution pH. In toxicity bioassays, we demonstrated that Cry1Ab retained insecticidal activity when adsorbed to SiO(2), suggesting high protein conformational stability during adsorption-desorption cycles. Models predicting Cry1A protein adsorption in soils therefore need to account for combined effects of the nonuniform protein surface charge distribution and of sorbent surface heterogeneity.     

Adsorption of transgenic insecticidal Cry1Ab protein to SiO2. 1. Forces driving adsorption

Genetically modified Bt crops express insecticidal Cry proteins (Bt toxins) that may enter agricultural soils. A mechanistic understanding of Cry protein adsorption to soils is critical for risk assessment, as this process governs Cry protein fate and bioavailability. We used quartz crystal microbalance and optical waveguide lightmode spectroscopy to elucidate the driving forces of the adsorption of monomeric Cry1Ab to negatively charged quartz (SiO(2)) and positively charged poly-L-lysine (PLL) at pH 5-8 and constant ionic strength of 50 mM (NaCl). Bovine serum albumin and hen egg white lysozyme were used as reference proteins because of their known adsorption behavior. Electrostatics governed Cry1Ab adsorption; as pH increased above the isoelectric point of Cry1Ab, the initial rate and the extent of adsorption decreased on SiO(2) and increased on PLL. Reversible adsorption to SiO(2) suggested weak Cry1Ab-SiO(2) electrostatic interactions and no irreversible conformational changes of Cry1Ab at the surface. High conformational stability of Cry1Ab was further supported by supply rate-independent extent of adsorption of Cry1Ab to apolar gold. Some evidence is presented that the nonuniform surface charge distribution of Cry1Ab resulted in patch-controlled electrostatic attraction with sorbents that carried the same net charge as Cry1Ab. A more detailed discussion of this mechanism is given in a companion paper.     

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.     

抗-Cry2Aa单链抗体分子互作及荧光免疫检测研究

Cry2Aa毒素是一种新型生物农药,分析该毒素与特异性单链抗体分子互作,并建立快速检测毒素残留的方法对保障食品和生态安全有重要意义。本研究以同源蛋白为模板对单链抗体进行建模,并进行了Cry2Aa毒素与单链抗体的分子对接模拟,确定关键结合位点,以此为基础将单链抗体作为检测抗体,建立了间接竞争时间分辨荧光免疫分析方法,对大米样品中Cry2Aa毒素进行了检测。利用生物信息学工具模拟获得单链抗体三维结构模型,分子对接结果显示重链可变区81NY82和121SGNY124区域及轻链可变区的245YSSN248氨基酸残基在与毒素结构Ⅱ区识别过程中起关键作用,为建立高效检测方法奠定基础,进一步基于该单链抗体建立的时间分辨检测方法灵敏度(IC10)为0.08 ng/mL,中抑制浓度(IC50)为7.99 ng/mL,线性检测范围(IC20~IC80)为0.24~263.77 ng/mL,且与常规双抗夹心ELISA法检测呈良好线性关系。     

Adsorption of Insecticidal Cry1Ab Protein to Humic Substances. 2. Influence of Humic and Fulvic Acid Charge and Polarity Characteristics

Assessing the fate and potential risks of transgenic Cry proteins in soils requires understanding of Cry protein adsorption to soil particles. The companion paper provided evidence that patch-controlled electrostatic attraction (PCEA) and the hydrophobic effect contributed to Cry1Ab protein adsorption to an apolar humic acid (HA). Here, we further assess the relative importance of these contributions by comparing Cry1Ab adsorption to seven humic substances varying in polarity and charge, at different solution pH and ionic strength, I. Cry1Ab adsorption to relatively apolar HAs at I = 50 mM exhibited rapid initial rates, was extensive, and was only partially reversible at pH 5-8, whereas adsorption to more polar fulvic acids was weak and reversible or absent at pH >6. The decrease in adsorption with increasing HS polarity at all tested pH strongly supports a large contribution from the hydrophobic effect to adsorption, particularly at I = 50 mM when PCEA was effectively screened. Using insect bioassays, we further show that Cry1Ab adsorbed to a selected HA retained full insecticidal activity. Our results highlight the need to consider adsorption to soil organic matter in models that assess the fate of Cry proteins in soils.     

Development of nanocolloidal gold based immunochromatographic assay for rapid detection of transgenic vegetative insecticidal protein in genetically modified crops

Vegetative insecticidal protein (Vip) is now being used for transgenic expression in several crops; conferring resistance against lepidopteron pests. A rapid, single step, sensitive and specific immunochromatographic (IC) strip test for the detection of recombinant Vip-S protein in the transgenic samples was developed. Polyclonal rabbit anti-Vip-S IgG conjugated to nanocolloidal gold served as a probe to detect Vip protein in test samples. The detection limit for the developed IC strip was 100 ng/ml (100 ppb) and on addition of gold enhancer the sensitivity increased to 1 ng/ml (1 ppb) of Vip-S protein. The assay was validated with transgenic brinjal samples. The assay time was less than 10 min, suitable for rapid on-site testing. No cross-reactivity was observed with other transgenic plant proteins employed for pest and weed management, i.e. Cry1Ac, Cry1Ab, and CP4-EPSPS. This on-site test offers rapid screening for a genetically modified crops having relatively new transgene (vip) entering the global market.     

Adsorption of Insecticidal Cry1Ab Protein to Humic Substances. 1. Experimental Approach and Mechanistic Aspects

Adsorption is a key process affecting the fate of insecticidal Cry proteins (Bt toxins), produced by genetically modified Bt crops, in soils. However, the mechanisms of adsorption to soil organic matter (SOM) remain poorly understood. This work assesses the forces driving the adsorption of Cry1Ab to Leonardite humic acid (LHA), used as a model for SOM. We studied the effects of solution pH and ionic strength (I) on adsorption using a quartz crystal microbalance with dissipation monitoring and optical waveguide lightmode spectroscopy. Initial Cry1Ab adsorption rates were close to diffusion-limited and resulted in extensive adsorption, even at pH >6, at which LHA and Cry1Ab carry negative net charges. Adsorption increased with decreasing I at pH >6, indicating Cry1Ab-LHA patch-controlled electrostatic attraction via positively charged domains of Cry1Ab. Upon rinsing, only a fraction of Cry1Ab desorbed, suggesting a range of interaction energies of Cry1Ab with LHA. Different interaction energies likely resulted from nonuniformity in the LHA surface polarity, with higher Cry1Ab affinities to more apolar LHA regions due to the hydrophobic effect. Contributions from the hydrophobic effect were substantiated by comparison of the adsorption of Cry1Ab and the reference proteins albumin and lysozyme to LHA and to apolar and polar model surfaces.     

A real-time immuno-PCR assay for the detection of transgenic Cry1Ab protein

The adaptation and production of transgenic crops harboring Cry1Ab protein is increasing every year globally due to their potent insecticidal activity. The Cry1Ab protein produced by Bacillus thuringiensis confers resistance against several lepidopteran insect pests. The release of transgenic crops/produce in the market worldwide has increased the need of regulatory affairs to monitor and verify the presence of transgenic protein in crops/produce. In this regard, the real-time immuno-PCR (IPCR) assay was developed for the detection and quantification of Cry1Ab protein. The IPCR assay showed high sensitivity with minimum detection limit of 100 pg/mL (0.1 ppb) and found to be 10 times more sensitive than sandwich ELISA. Under the optimized assay conditions, Cry1Ab protein can be determined in the concentration ranged from 100 pg/mL to 100 ng/mL. As results suggest, this assay could be a powerful tool for the detection of even trace amounts of Cry1Ab protein in transgenic crops.     

Effects of Cry1F and Cry34Ab1/35Ab1 on storage pests

Two crystalline protoxins from Bacillus thuringiensis (Bt), Cry1Fa1 and Cry34Ab1/Cry35Ab1 (Cry1F, Cry34/35Ab1), were evaluated for efficacy against lepidopteran and coleopteran storage pests. Cry1F was tested against the lepidopterans Sitotroga cerealella (Angoumois grain moth) and colonies of Plodia interpunctella (Indian mealmoth) that are susceptible or resistant to Bt Cry1Ab and Cry1Ac toxins, Bt subspecies entomocidus, and the commercial formulation Dipel^®. Cry1F was also tested against the coleopterans Cryptolestes pusillus (flat grain beetle) and Tribolium castaneum (red flour beetle). Cry34/35Ab1 was tested against S. cerealella, C. pusillus, and T. castaneum, and against additional coleopteran storage pests, including Tenebrio molitor (yellow mealworm), Trogoderma variabile (warehouse beetle), Oryzaephilus surinamensis (sawtoothed grain beetle), Rhyzopertha dominica (lesser grain borer), and Sitophilus oryzae (rice weevil). Strains of Bt-susceptible or -resistant P. interpunctella generally were more sensitive to Cry1A protoxin or toxin than either Cry1F protoxin or Dipel. Despite difficulties with the bioassay of S. cerealella larvae, the data suggest that Cry1F and Cry34/35Ab1 caused increased larval mortality, and a developmental delay was observed and no pupae emerged with 0.9% Cry1F. Neither Cry1F nor the corn rootworm-active toxin Cry34/35Ab1 significantly affected the biological parameters of the coleopteran species evaluated.     

Increased digestibility of two products in genetically modified food (CP4-EPSPS and Cry1Ab) after preheating

We performed experiments on in vitro digestion of newly expressed proteins by SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) to assess the allergenicity of food components derived from biotechnological modification. For newly expressed proteins, we chose CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4) and Cry1Ab derived from Bacillus thuringiensis subsp. kurstaki strain HD-1. The former is expressed in GM-soybeans and the latter is expressed in GM-corns. Firstly, we examined the digestibility of purified CP4-EPSPS and Cry1Ab by SGF. Both proteins were rapidly digested within 60 sec. After preheating, the digestibility by SGF was slightly increased. Secondly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SGF. The digestion time of both proteins by SGF was almost the same as that of the purified proteins. Thirdly, the digestibility of CP4-EPSPS and Cry1Ab by SIF was examined. The digestion time of these proteins was 240 min or more. However, digestibility of these proteins by SIF was dramatically increased by preheating, and the digestion time was less than 5 sec. Fourthly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SIF. Digestion time of both proteins by SIF was almost the same as that of the purified proteins. From these results, we concluded that the digestibility of both CP4-EPSPS and Cry1Ab by SGF and SIF was increased by preheating. Therefore, we suggest that the allergenicity of both proteins should be extremely low because of the easy digestibility of these proteins by SGF and also by SIF with preheating.     

大肠杆菌中表达Cry1C基因及其重组表达产物的纯化及复性

通过PCR方法将苏云金芽孢杆菌Cry1C基因从原始质粒中克隆出来。由于Cry1C基因携带了大量大肠杆菌稀有密码子,因此用PCR方法对其前86个密码子进行修改,以提高其在大肠杆菌BL21(DE3)中的表达量。Cry1C蛋白在大肠杆菌BL21(DE3)中以包涵体形式大量表达,将包涵体用8mol/L尿素溶解并用His TrapTM FF凝胶柱纯化。所得纯化蛋白在复性缓冲液中复性折叠,最终得到可溶并有生物活性(由三化螟活性实验验证)的蛋白。Cry1C蛋白纯度可达99.2%。     

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.     

A novel cell-free translation/glycosylation system prepared from insect cells

A cell-free translation/glycosylation system derived from lepidopteran (Sf21) cells, which are widely used to express high yields of foreign active proteins that have post-translational modifications, was constructed. The insect cell extract was prepared using a Mini-Bomb cell disruption chamber by nitrogen pressure treatment, which stably retains translational and post-translational components. The gp120 mRNA was transcribed from the human immunodeficiency virus type-1 envelope glycoprotein gp120 gene with T7 RNA polymerase. When the gp120 mRNA was translated in the insect cell-free system, gp120 having a molecular mass of 100 kDa was detected by Western blot analysis. Synthesized gp120 and gp120 expressed in the intracellular fraction of recombinant-baculovirus-infected Sf21 cells had the same molecular mass, and they both had reduced mobility compared with gp120 secreted by recombinant baculovirus-infected Sf21 cells. In contrast, the 56-kDa gp120 protein, which corresponds to the polypeptide backbone of gp120, was synthesized in wheat germ and rabbit reticulocyte systems. The molecular mass of synthesized gp120 decreased from 100 kDa to 61 kDa after endoglycosidase H treatment, indicating that synthesized gp120 had been glycosylated with N-linked oligosaccharides. Furthermore, glycosylated gp120 was bound to human CD4 molecules expressed on the surface of quail cells. These results revealed that the insect cell-free system can synthesize gp120 that is folded in the proper conformation to provide a CD4-binding domain.     

Detection of genetically modified plant products by protein strip testing: an evaluation of real-life samples

The determination of the presence of genetically modified plant material by the detection of expressed genetically engineered proteins using lateral flow protein strip tests has been evaluated in different matrices. The presence of five major genetically engineered proteins (CP4-EPSPS, CryIAb, Cry9C, PAT/pat and PAT/bar protein) was detected at low levels in seeds, seed/leaf powder and leaf tissue from genetically modified soy, maize or oilseed rape. A comparison between “protein strip test” (PST) and “polymerase chain reaction” (PCR) analysis of genetically modified food/feed samples demonstrates complementarities of both techniques.     

Immunochemical detection of Cry1A(b) protein in model processed foods made with transgenic maize

Immunoassays are used to screen for the presence of genetically modified organisms in raw materials. However, processing may condition the usefulness of immunoassays to analyse genetically modified foods because it leads to protein denaturation that affects recognition by antibodies. We studied the effect of processing on the detection of Cry1A(b) protein in model foods prepared with transgenic maize using a sandwich ELISA. Nixtamalization at 100 °C for 5 min and at 85 °C for 60 min gave 40 and 70% loss of Cry1A(b) protein. In the preparation of porridge, the concentration of Cry1A(b) protein did not change until the mixture reached 75 °C, but it decreased by 90% after 3 min at that temperature. Concentration of Cry1A(b) protein decreased by 90% in tortillas griddled at 180 °C for 20 s, but no protein was detected in fried tortillas after 10 s at 190 °C. Cry1A(b) protein is rapidly denatured by heat treatment resulting in a marked decline in concentration and decreased detection in processed foods.     

Adsorption of transgenic insecticidal Cry1Ab protein to SiO2. 2. Patch-controlled electrostatic attraction

Adsorption governs the fate of Cry proteins from genetically modified Bt crops in soils. The effect of ionic strength (I) on the adsorption of Cry1Ab (isoelectric point IEP(Cry1Ab) ≈ 6) to negatively charged quartz (SiO(2)) and positively charged poly-L-lysine (PLL) was investigated at pH 5 to 8, using quartz crystal microbalance with dissipation monitoring and optical waveguide lightmode spectroscopy. Cry1Ab adsorbed via positively and negatively charged surface patches to SiO(2) and PLL, respectively. This patch controlled electrostatic attraction (PCEA) explains the observed increase in Cry1Ab adsorption to sorbents that carried the same net charge as the protein (SiO(2) at pH > IEP(Cry1Ab) and PLL at pH < IEP(Cry1Ab)) with decreasing I. In contrast, the adsorption of two reference proteins, BSA and HEWL, with different adsorption mechanism, were little affected by similar changes of I. Consistent with PCEA, Cry1Ab desorption from SiO(2) at pH > IEP(Cry1Ab) increased with increasing I and pH. Weak Cry1Ab-SiO(2) PCEA above pH 7 resulted in reversible, concentration dependent adsorption. Solution depletion experiments showed that PCEA also governed Cry1Ab adsorption to SiO(2) particles at environmentally relevant concentrations (a few ng mL(-1)). These results imply that models describing Cry1Ab adsorption to charged surfaces in soils need to account for the nonuniform surface charge distribution of the protein.     

Kinetic and Thermodynamic Parameters for Heat Denaturation of Cry1A(b) Protein from Transgenic Maize (Zea mays)

ABSTRACT:  The effect of heat treatment on the denaturation of Cry1A(b) protein expressed in transgenic maize was studied over a temperature range of 69 to 77 °C. Denaturation of Cry1A(b) protein was measured by the loss of reactivity with its specific antibodies using a sandwich ELISA. The process of denaturation was studied by analyzing the values of inmunoreactive protein after each heat treatment by kinetic analysis. Denaturation of Cry1A(b) protein was best described assuming a reaction order of 1.5. D -values calculated were 4338, 2350, 1272, 734, and 601 s at 69, 71, 73, 75, and 77 °C, respectively. Z -value was estimated to be 9.0 °C and the activation energy value was 266.15 kJ/mol. Thermodynamic parameters for the process of denaturation of Cry1A(b) protein were also calculated. The high values of the enthalpy of activation and the positive values of the entropy of activation obtained for Cry1A(b) protein are typical of a reaction in which the denaturation of the protein is the rate-determining process that predominates over an aggregation process during heating.     

基于同源建模与分子对接技术构建抗Bt Cry1类毒素单链抗体定点饱和突变库

转基因食品中苏云金芽胞杆菌(Bt)Cry毒素种类较多,对生态环境和食品安全等造成的潜在风险日益受到关注,制备针对多种Cry毒素的高效广谱抗体,进而建立广谱性快速检测方法,是对转Bt基因作物进行监管并确保生态环境和食品安全的基础。本研究利用基因工程抗体易于定向修饰及Cry毒素具有相似结构的特点,以实验室前期获得的人源化抗苏云金芽胞杆菌(Bt)Cry1Ac毒素单链抗体为材料,通过同源建模模拟单链抗体及五种Cry1类毒素的三维结构并利用分子对接技术分析单链抗体与Cry1类毒素结合的关键氨基酸位点;在此基础上利用重叠延伸PCR技术对关键氨基酸位点及其邻近位点进行定向改造,以构建定点饱和突变库,为筛选检测多种Cry1类毒素的广谱特异性抗体提供实验材料。     

Microbial control using bacteria of the almond moth,Cadra (Ephestia) cautella Walker (Lepidoptera: Pyralidae)

Almond moth, Cadra (Ephestia) cautella (Lepidoptera: Pyralidae) is one of the most serious pests of dried fruits and other stored products. Almond moth is polyphagous pest that is widespread in Turkey and all over the world. Almond moth larvae cause serious damages on trees, warehouse and threshing floor of drying fig. Though various cultural, chemical and biological methods are used to control this pest, it is still effective in Turkey and various part of the world. In order to find a significant microbial control agent against this pest, we isolated 13 bacterial isolates and identified these isolates based on morphological, physiological, biochemical and molecular characteristics. According to these characteristics, isolates were identified as Serratia marcescens (Eca1 and Eca3), Serratia sp. (Eca11), Bacillus thuringiensis (Eca2, Eca4, Eca6, Eca7, Eca8, Eca9, Eca10, Eca12, Eca13) and Bacillus axarquiensis (Eca5). The insecticidal activities of these isolates were performed against three insect species from Lepidoptera group which cause serious damage in warehouses. The highest insecticidal activity is 57% for Eca9 isolate on the 3rd instar larvae of C. cautella, 100% for Eca9 isolate on the 3rd instar larvae of Plodia interpunctella and 100% for Eca10 and Eca3 isolate on the 3rd instar larvae of E. kuehniella. Results indicate that Eca9, Eca3 and Eca10 isolates may be valuable as potential biological control agents for the control of warehouse pests.     

转Cry1Ab/Cry1Ac基因大米粉饲喂印度谷螟后的比较转录组分析（网络首发、推荐阅读）

转Cry1Ab/Cry1Ac基因作物可以有效防治害虫。以世界性的储粮害虫印度谷螟Plodia interpunctella (Hübener)为研究对象,借助高通量测序法研究试虫中与Bt毒蛋白密切相关的基因,以期初步揭示转Bt稻谷的杀虫作用机制。结果表明,在对印度谷螟非胁迫种群和转Bt基因大米粉胁迫种群分别进行转录组测序,经De novo组装后共得到37 246个Unigene,其中有23 310个Unigene获得注释。随后,通过比较分析上述两种品系试虫基因表达量,共筛选出34 466条显著差异表达的基因,其中在胁迫品系中有15 741条基因表达量显著上调,另有18 725条基因表达量显著下调。一方面大大扩充了针对该物种的基因信息,另一方面为转基因稻谷抗印度谷螟的相关机理研究提供了新思路。