苏云金杆菌4.0718菌株杀虫晶体蛋白的双向电泳飞行时间质谱分析

根据苏云金杆菌4.0718菌株杀虫晶体蛋白的特性,对裂解液组成、上样量、聚焦时间等相关技术进行了比较研究和条件优化,首次获得苏云金杆菌杀虫晶体蛋白双向电泳图谱,并对部分蛋白质点进行胰酶酶解,基质辅助激光解吸电离飞行时间质谱（matrixassisted laser desorption/ionization time of flight mass spectrometry,MALDITOFMS）测定肽质量指纹图谱,Mascot软件查询SwissProt数据库,最终鉴定出苏云金杆菌4.0718菌株伴孢晶体中所含的Cry1Ac和Cry2Aa蛋白,其精确分子量分别为134160Da和71097Da。     

Evaluating the Insecticidal Genes and Their Expressed Products in Bacillus thuringiensis Strains by Combining PCR with Mass Spectrometry

By a combination of PCR and mass spectrometry, a total of five cry genes (cry1Aa, cry1Ac, cry2Aa, cry2Ab, and cry1Ia) were detected in genomic DNA from the wild-type Bacillus thuringiensis strain 4.0718, and three protoxins (Cry1Aa, Cry1Ac, and Cry2Aa) were identified in the strain's parasporal crystals. These results indicated that this complementary method may be useful in evaluating B. thuringiensis strains at both the gene and protein levels.     

Characterization of cultured insect cells selected by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> crystal toxin

Summary Selection for resistance against Bacillus thuringiensis (Bt) Cry1Ac10 in the Trichoplusia ni (Hübner) cell line BTI-TN-5B1-4 (TnH5) was tested, and the development of resistance in the selected cells was like a S-form curve. Monitoring at the Cry1Ac10 50th challenge, the resistance ratio was 1, 294-fold as many as that of initial cells. But the resistance to Cry1Ac10 declined gradually when the selection was relaxed. The resistance declined rapidly at the low level of resistance and slowly at the high level of resistance. This resistant cell had high resistance to all the tested solubilized trypsin-treated mixture of crystal multitoxins of B. thuringiensis subsp. aizawai GC-91, an engineering bacterium of Bt, B. thuringiensis subsp. aizawai HD-133 and B. thuringiensis subsp. kurstaki HD-1, and low cross-resistance (19.7-fold) to activated Cry1C. Both N-acetyl-d-galactosamine (GalNAc) and tunicamycin did not inhibit the toxicity of Cry1Ac10 to the susceptible TnH5 cells. Comparison of the total proteins of the selected resistant cells with that of the nonselected susceptible cells by two-dimensional electrophoresis analysis showed that were obvious differences among the 11 protein expression. These results strongly suggest that there exists an unknown mechanism of resistance in the cell line that was different from the reported mechanisms in insects.     

<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Protein Cry6B (BGSC ID 4D8) is Toxic to Larvae of <Emphasis Type="Italic">Hypera postica</Emphasis>

Insecticidal proteins produced by strains of Bacillus thuringenesis are specific toward target pests. One of the Bt proteins, Cry 1Ac has been used successfully for controlling crop predation by polyphagous pests Helicoverpa armigera. Structurally, Bt proteins consist of three domains; domain I and III are fairly homologous in various Bt proteins while domain II is hypervariable. The hypervariable domain II is believed to be responsible for specificity toward target pest. Successful deployment of Bt proteins requires knowledge of its specificity toward the insect. Various Bt proteins have been characterized for activity against coleopteran pests. Some Bt proteins of class Cry6 have been found to be active against potato weevil. We have evaluated the activity of Cry6B protein (BGSC-4D8) against lucerne weevil, Hypera postica, which is a major pest of forage crop Medicago sativa. Results revealed that the purified Cry6B protein is significantly active against the coleopteran pest with LC₅₀ value 280 ng/μl. The leaves coated with the purified Cry6 toxin were three times less damaged as compared with the negative control.     

Expression of Bacillus thuringiensis Cry1Ac protein in cotton plants, acquisition by pests and predators: a tritrophic analysis

1. Studies have shown that Cry proteins of the bacterium Bacillus thuringiensis expressed in transgenic plants can be acquired by nontarget herbivores and predators. A series of studies under field and controlled conditions was conducted to investigate the extent to which Cry1Ac protein from Bt transgenic cotton reaches the third trophic level and to measure the amount of protein that herbivores can acquire and expose to predators. 2. Levels of Cry1Ac in Bt cotton leaves decreased over the season. Among herbivores (four species), Cry1Ac was detected in lepidopteran larvae and the amount varied between species. Among predators (seven species), Cry1Ac was detected in Podisus maculiventris and Chrysoperla rufilabris. 3. In the greenhouse, only 14% of the Cry1Ac detected in the prey (Spodoptera exigua larvae) was subsequently found in the predator P. maculiventris. Detection of Cry1Ac protein in Orius insidiosus, Geocoris punctipes and Nabis roseipennis was probably limited by the amount of prey consumed that had fed on Bt cotton. 4. Purified Cry1Ac was acquired by the small predatory bug G. punctipes but at much higher concentration than found in plants or in lepidopteran larvae. 5. Bt protein was shown to move through prey to the third trophic level. Predatory heteropterans acquired Cry1Ac from prey fed Bt cotton, but acquisition was dependent on the concentration of Cry1Ac conveyed by the prey and the amount of prey consumed. The type and availability of prey capable of acquiring the protein, coupled with the generalist feeding behaviour of the most common predators in the cotton ecosystem, probably constrain the flow of Cry1Ac through trophic levels.     

Bacillus thuringiensis protein transfer between rootstock and scion of grafted poplar

Bacillus thuringiensis (Bt) Cry1Ac protein is a toxin against different leaf‐eating lepidopteran insects that attack poplar trees. In the present study, the mode of migration of the Bt‐Cry1Ac protein within poplar grafts was investigated. Grafting was done using Pb29 (transgenic poplar 741 with cry1Ac genes), CC71 (transgenic poplar 741 with cry3A genes), non‐transgenic poplar 741 and non‐transgenic Populus tomentosa, either as scion or as rootstock. In order to detect migration of Bt‐Cry1Ac protein from one portion of the graft union to different tissues in the grafted plant, ELISA analysis was employed to assess the content of Bt‐Cry1Ac protein in the phloem, xylem, pith and leaves of the grafted poplar. To further verify migration of Bt‐Cry1Ac protein, Clostera anachoreta larvae, which are susceptible to Bt‐Cry1Ac protein, were fed leaves from the control graft (i.e., graft portion that originally did not contain Bt‐Cry1Ac protein). The results showed that Bt‐Cry1Ac protein was transported between rootstock and scion mainly through the phloem. Migration of Bt‐Cry1Ac protein in the grafted union was also evidenced in that the leaves of the control graft did have a lethal effect on C. anachoreta larvae in laboratory feeding experiments.     

Age-related increase in levels of insecticidal protein in the progenies of transgenic oilseed rape and its efficacy against a susceptible strain of diamondback moth

Many crops transformed with insecticidal genes isolated from Bacillus thuringiensis (Bt) show resistance to targeted insect pests. The concentration of Bt endotoxin proteins in plants is very important in transgenic crop efficacy and risk assessment. In the present study, changes in levels of Cry1Ac protein in the leaves of transgenic Bt oilseed rape (Brassica napus) carrying a Bt cry1Ac gene under the control of the cauliflower mosaic virus 35S promoter were quantified during vegetative growth by enzyme‐linked immunosorbent assay. Plants were grown in a glasshouse, sampled at 2, 4, 5 and 6 weeks, and the concentration of Cry1Ac was quantified in basal, top and previous top leaves. The mean concentration differed between sowing dates when Cry1Ac concentration was expressed as ng g^?1 fresh leaf weight but not when expressed as ng mg^?1 total soluble protein. It was demonstrated that Cry1Ac concentration increased significantly as the leaf aged, while the total soluble plant protein decreased significantly. Levels of Cry1Ac were therefore higher in leaves at the base of the plants than in leaves close to the growing point. However, even young leaves with very low Cry1Ac concentrations caused high mortality in the larvae of a Cry1Ac‐susceptible laboratory strain of the diamondback moth. The feeding area of leaves consumed by larvae in vivo and in situ was similar. Leaf damage caused by sampling (i.e. artificially) or by feeding of larvae did not affect the levels of Cry1Ac in the leaves under the experimental conditions in this study.     

Susceptibility of Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera: Crambidae) to four Bacillus thuringiensis toxins

Sugarcane borer, Diatraea saccharalis (F.), is a primary corn stalk borer pest targeted by transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the mid-southern region of the United States. Recently, genes encoding for Cry1A.105 and Cry2Ab2 Bt proteins were transferred into corn plants (event MON 89034) for controlling lepidopteran pests. This new generation of Bt corn with stacked-genes of Cry1A.105 and Cry2Ab2 will become commercially available in 2009. Susceptibility of Cry1Ab-susceptible and -resistant strains of D. saccharalis were evaluated on four selected Bt proteins including Cry1Aa, Cry1Ac, Cry1A.105, and Cry2Ab2. The Cry1Ab-resistant strain is capable of completing its larval development on commercial Cry1Ab-expressing corn plants. Neonates of D. saccharalis were assayed on a meridic diet containing one of the four Cry proteins. Larval mortality, body weight, and number of surviving larvae that did not gain significant weight (<0.1 mg per larva) were recorded after 7 days. Cry1Aa was the most toxic protein against both insect strains, followed in decreasing potency by Cry1A.105, Cry1Ac, and Cry2Ab2. Using practical mortality (larvae either died or no significant weight gain after 7 days), the median lethal concentration (LC₅₀) of the Cry1Ab-resistant strain was estimated to be >80-, 45-, 4.1-, and −0.5-fold greater than that of the susceptible strain to Cry1Aa, Cry1Ac, Cry1A.105 and Cry2Ab2 proteins, respectively. This information should be useful to support the commercialization of the new Bt corn event MON 89034 for managing D. saccharalis in the mid-southern region of the United States.     

Toxicity and Receptor Binding Properties of Bacillus thuringiensisδ-Endotoxins to the Midgut Brush Border Membrane Vesicles of the Rice Leaf Folders, Cnaphalocrocis medinalis and Marasmia patnalis

Pesticidal activity and receptor-binding properties of Bacillus thuringiensis toxins to rice leaf folders, Cnaphalocrocis medinalis and Marasmia patnalis, were investigated. Saturation and competition binding experiments were done with iodine (¹²⁵1)-labeled Bt proteins and brush border membrane vesicles prepared from the midgut of C. medinalis and M. patnalis. The results show saturable, specific, and high-affinity binding of all toxins except Cry2A toxin. Cry1Aa and Cry2A toxins were bound with low affinity but with high binding site concentration. Heterologous competition experiments showed that Cry1Aa, Cry1Ab, and Cry1Ac recognized or shared the same binding site that is different from the binding site for Cry2A toxin. Iodine (¹²⁵I)-labeled Cry1Ac and Cry1Ab toxins were used in ligand blot experiments to detect specific binding proteins in brush border membrane vesicles of C. medinalis and M. patnalis. Cry1Ab toxin protein binds to 205-kDa and 200-kDa proteins respectively in case of C. medinalis and M. patnalis. The apparent molecular mass of the protein bound to labeled Cry1Ac toxins was identified as a 120-kDa protein in both C. medinalis and M. patnalis. Received: 10 April 2000 / Accepted: 23 May 2000     

Development of ELISA for the Determination of Transgenic Bt‐Cottons Using Antibodies against Cry1Ac Protein from Bacillus thuringiensis HD‐73

The area cultivated with Bt‐cottons expressing Cry1Ac gene increases year by year in China and other countries. To evaluate any potential adverse impacts on the environment from the release of Bt (Bacillus thuringiensis) technology, the development of a method for easily detecting the activity of the Cry1Ac toxins is of particular interest. The aim of this study was to develop sandwich‐ELISA for the detection of Cry1Ac protein in Bt‐cotton tissues. A specific antibody was obtained from rabbits inoculated with Cry1Ac protein derived from Bt strain HD‐73 and a secondary antibody conjugated to HRP could combine the Bt Cry1Ac protein specifically. The limit of detection was 5 ng/mL and there were no cross‐reactions between the positive control of Cry1Ab/1Ac, Cry1C, Cry2A, Cry3Bb1 and Cry9C. Extracts of proteins from cotton leaves were used to evaluate the suitability of the assay. Tris‐borate buffer and sodium carbonate buffer were employed for the extraction of protein, the limit absorbance of detection was 0.134 and 0.449, respectively, and the latter produced a higher background. The results showed that cultivars GK‐12, GK‐22, insect‐resistant cotton, bivalent transgenic cotton and shiyuan 321 assayed positively and NON was the negative sample. The PCR method was used for the validation of the developed assay. Although both methods allowed the same results to be obtained, ELISA needed simple equipment and took less time. The developed immunoassay method is considered reliable for the detection of Bt Cry1Ac protein.     

Species-specific differences in oak foliage affect preference and performance of gypsy moth caterpillars

Laboratory feeding experiments using two transgenic Bacillus thuringiensis (Bt) rape cultivars (Bt‐Westar and Bt‐Oscar) both expressing the Cry1Ac protein, and the corresponding untransformed lines, were carried out to study the effects of transgenic Bt rape on the non‐target herbivore Athalia rosae (L.) (Hymenoptera: Tenthredinidae). Furthermore, Cry1Ac protein concentration in Bt rape leaves, A. rosae larvae fed Bt rape, their faeces, eonymph instars, pupae, and adults were quantified using an enzyme‐linked immunosorbent assay (ELISA). There were no significant differences in mortality, larval development, and weight between transgenic Bt rape and non‐transgenic rape fed A. rosae. Additionally, we did not detect any significant differences in the fecundity and fertility of adult females either fed as larvae with transgenic Bt or with non‐transgenic rape. However, results of the ELISA indicated that Cry1Ac protein was detectable in larvae and faeces (Bt‐Westar 1.1 ± 0.2 and Bt‐Oscar 0.3 ± 0.2 µg Cry1Ac protein/g fresh weight) although this was less than in the leaf material, where concentrations were 2.2 ± 0.8 µg Cry1Ac protein/g fresh weight for Bt‐Westar and 7.5 ± 2.9 µg Cry1Ac protein/g fresh weight in Bt‐Oscar. In contrast, Cry1Ac protein could not be detected in eonymphs, pupae, or adults of A. rosae. Our results suggest that Cry1Ac protein in Bt rape does not have a significant effect on the herbivore A. rosae but the protein is still detectable after ingestion and excretion by these herbivores, thus providing the possibility of exposure to organisms other than herbivores.     

Expressing a moth abcc2 gene in transgenic Drosophila causes susceptibility to Bt Cry1Ac without requiring a cadherin-like protein receptor

Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins’ mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 (Px-abcc2), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD₅₀ concentrations of Cry1Ac toxin or protoxin, plus purified CR12-MPED cloned from M. sexta or P. xylostella. The M. sexta CR12-MPED protein acted synergistically with Cry1Ac protoxin and activated toxin significantly more effectively than the P. xylostella peptide. This work demonstrates ABCC2 is the major functional Cry1Ac receptor for P. xylostella and the importance of CaLP proteins in Bt mode of action may vary between different lepidopteran species.     

Susceptibility of field populations of sugarcane borer from non‐Bt and Bt maize plants to five individual Cry toxins

Abstract Sugarcane borer, Diatraea saccharalis (F.), is a major target of transgenic maize expressing Bacillus thuringiensis (Bt) proteins in South America and the US mid‐south region. Resistance development in target pest populations is a major threat to the sustainable use of Bt crops. In our field trials in 2009, a significant number of live borers and plant injury from D. saccharalis were observed in an experimental SmartStax? maize line. The objective of this study was to assess the relative susceptibility of two field populations of D. saccharalis collected from non‐Bt and Bt maize plants containing SmartStax? traits to five individual Cry proteins. The five Bt proteins included two proteins (Cry1A.105 and Cry2Ab2) that were expressed in SmartStax? maize plants and three other common Bt proteins (Cry1Aa, Cry1Ab and Cry1Ac) that were not produced in SmartStax?. Larval mortality and growth inhibition on Bt diet of the fourth generation after field collections were evaluated 7 days after release of neonates on the diet surface. The laboratory bioassays showed that 50% lethal concentration (LC₅₀) values for Cry1A.105 and Cry2Ab2 for the population originated from Bt plants were 3.55‐ and 1.34‐fold greater, respectively, than those of the population collected from non‐Bt plants. In contrast, relative to the population from non‐Bt plants, the LC₅₀ of the population sampled from Bt plants were 3.85‐, 2.5‐ and 1.64‐fold more sensitive to Cry1Aa, Cry1Ab and Cry1Ac, respectively. The results did not provide clear evidence to conclude that the observed field survival of D. saccharalis on Bt plants was associated with increased levels of resistance.     

二化螟钙黏蛋白CAD1的原核表达及与Cry1Ac、Cry2Aa蛋白的结合

【目的】二化螟是水稻的重要害虫之一,钙黏蛋白(cadherin,CAD)是一类重要的Bt杀虫蛋白受体,在获得二化螟钙黏蛋白基因(Cs CAD1)的基础上,明确Cs CAD1蛋白与Cry1Ac和Cry2Aa蛋白的结合能力。【方法】利用PCR技术克隆Cs CAD1基因片段,将构建的p ET-28a-(+)-Cs CAD1重组质粒转入原核表达菌株BL21(DE3)中,IPTG诱导表达。目的蛋白经Ni柱亲和纯化后SDS-PAGE电泳检测,利用western blot和ligand blot技术分析其与Cry1Ac和Cry2Aa蛋白的结合能力。【结果】重组载体可在表达菌株BL21中表达一个约44 ku的蛋白,原核表达载体构建成功。SDS-PAGE显示该蛋白条带单一,且纯度较好。Ni柱亲和层析纯化该目的蛋白后进行Ligand blot分析,结果显示Cs CAD1重组蛋白可以与Cry1Ac和Cry2Aa蛋白结合。【结论】Cs CAD1蛋白可以与Cry1Ac和Cry2Aa蛋白结合,是潜在的Cry蛋白受体,所得结果有助于阐明Cry1Ac和Cry2Aa蛋白对二化螟的作用机制。     

Bacillus thuringiensis plants expressing Cry1Ac,Cry2Ab and Cry1F are not toxic to the assassin bug,Zelus renardii

Cotton‐ and maize‐producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), have been commercialized since 1996. Bt plants are subjected to environmental risk assessments for non‐target organisms, including natural enemies that suppress pest populations. Here, we used Cry1F‐resistant Spodoptera frugiperda (J.E. Smith) and Cry1Ac and Cry2Ab‐resistant Trichoplusia ni (Hübner) as prey for the assassin bug, Zelus renardii (Kolenati), a common predator in maize and cotton fields. In tritrophic studies, we assessed several fitness parameters of Z. renardii when it fed on resistant S. frugiperda that had fed on Bt maize expressing Cry1F or on resistant T. ni that had fed on Bt cotton expressing Cry1Ac and Cry2Ab. Survival, nymphal duration, adult weight, adult longevity and female fecundity of Z. renardii were not different when they were fed resistant‐prey larvae (S. frugiperda or T. ni) reared on either a Bt crop or respective non‐Bt crops. ELISA tests demonstrated that the Cry proteins were present in the plant at the highest levels, at lower levels in the prey and at the lowest levels in the predator. While Z. renardii was exposed to Cry1F and Cry1Ac and Cry2Ab when it fed on hosts that consumed Bt‐transgenic plants, the proteins did not affect important fitness parameters in this common and important predator.     

Susceptibility of legume pod borer (LPB), Maruca vitrata to delta-endotoxins of Bacillus thuringiensis (Bt) in Taiwan

Baseline susceptibility of legume pod borer (LPB) to the insecticidal crystal proteins (ICPs) from Bacillus thuringiensis, viz, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca and Cry2Aa was assessed in Taiwan. Insect bioassays were performed by incorporating the Bt delta-endotoxins into the LPB artificial diet. The efficacy of different Bt delta-endotoxins against second instar larvae of LPB showed that the toxin Cry1Ab was the most potent toxin (LC(50) 0.207ppm), followed by Cry1Ca, Cry1Aa, Cry2Aa and Cry1Ac in descending order, with LC(50)s 0.477ppm, 0.812ppm, 1.058ppm and 1.666ppm, respectively. Hence, Cry1Ab and/or Cry1Ca toxins would provide effective control of early larval stages of LPB.     

Dynamic expression of green fluorescent protein and Bacillus thuringiensis Cry1Ac endotaxin in interspecific hybrids and successive backcross generations (BC1 and BC2) between transgenic Brassica napus crop and wild Brassica juncea

The persistence and stability of a transgene encoding a Bacillus thuringiensis (Bt) Cry1Ac insecticidal protein was investigated in hybrids between crop Brassica napus and a recurrent wild Brassica juncea population. Interspecific hybrids (F₁) and backcross progenies (BC₁, BC₂) containing green fluorescent protein (GFP) and Bt genes were successfully produced in the greenhouse. Stable Bt toxin levels were found in hybrid and advanced backcross progenies formed in wild B. juncea. Bt Cry1Ac concentration was significantly lower in BC₂ plants than in transgenic B. napus, F₁, BC₁, while no significant differences were detected among the latter three plant genotypes. A GFP marker gene was used as a scorable marker and indicator of Bt transgene expression. GFP fluorescence intensity was significantly correlated with Bt Cry1Ac concentration at the flowering stage and the pod formation stage in both transgenic oilseed rape hybrids and backcrossed progenies (BC₁, BC₂). It was demonstrated that GFP was a suitable marker for Bt protein in the backcross of B. juncea, which could facilitate the detection of gene flow and is useful in biosafety management.     

Fitness of Bt‐resistant cabbage loopers on Bt cotton plants

Development of resistance to the insecticidal toxins from Bacillus thuringiensis (Bt) in insects is the major threat to the continued success of transgenic Bt crops in agriculture. The fitness of Bt‐resistant insects on Bt and non‐Bt plants is a key parameter that determines the development of Bt resistance in insect populations. In this study, a comprehensive analysis of the fitness of Bt‐resistant Trichoplusia ni strains on Bt cotton leaves was conducted. The Bt‐resistant T. ni strains carried two genetically independent mechanisms of resistance to Bt toxins Cry1Ac and Cry2Ab. The effects of the two resistance mechanisms, individually and in combination, on the fitness of the T. ni strains on conventional non‐Bt cotton and on transgenic Bt cotton leaves expressing a single‐toxin Cry1Ac (Bollgard I) or two Bt toxins Cry1Ac and Cry2Ab (Bollgard II) were examined. The presence of Bt toxins in plants reduced the fitness of resistant insects, indicated by decreased net reproductive rate (R₀) and intrinsic rate of increase (r). The reduction in fitness in resistant T. ni on Bollgard II leaves was greater than that on Bollgard I leaves. A 12.4‐day asynchrony of adult emergence between the susceptible T. ni grown on non‐Bt cotton leaves and the dual‐toxin‐resistant T. ni on Bollgard II leaves was observed. Therefore, multitoxin Bt plants not only reduce the probability for T. ni to develop resistance but also strongly reduce the fitness of resistant insects feeding on the plants.