Degradation of Cry1Ac protein within transgenic Bacillus thuringiensis rice tissues under field and laboratory conditions

To clarify the environmental fate of the Cry1Ac protein from Bacillus thuringiensis subsp. kurstaki (Bt) contained in transgenic rice plant stubble after harvest, degradation was monitored under field conditions using an enzyme-linked immunosorbent assay. In stalks, Cry1Ac protein concentration decreased rapidly to 50% of the initial amount during the first month after harvest; subsequently, the degradation decreased gradually reaching 21.3% when the experiment was terminated after 7 mo. A similar degradation pattern of the Cry1Ac protein was observed in rice roots. However, when the temperature increased in April of the following spring, protein degradation resumed, and no protein could be detected by the end of the experiment. In addition, a laboratory experiment was conducted to study the persistence of Cry1Ac protein released from rice tissue in water and paddy soil. The protein released from leaves degraded rapidly in paddy soil under flooded conditions during the first 20 d and plateaued until the termination of this trial at 135 d, when 15.3% of the initial amount was still detectable. In water, the Cry1Ac protein degraded more slowly than in soil but never entered a relatively stable phase as in soil. The degradation rate of Cry1Ac protein was significantly faster in nonsterile water than in sterile water. These results indicate that the soil environment can increase the degradation of Bt protein contained in plant residues. Therefore, plowing a field immediately after harvest could be an effective method for decreasing the persistence of Bt protein in transgenic rice fields.     

盐胁迫对Bt棉棉蕾杀虫蛋白表达的影响

为探讨土壤盐分对转Bt基因抗虫棉棉蕾抗虫性的影响程度,采用盆栽试验,以2个Bt棉品种‘新棉33B’(盐敏感)和‘中07’(耐盐)为试验材料,设置5个土壤盐分水平(0、0.15%、0.30%、0.45%和0.60%), 研究盐分对蕾期Bt棉棉蕾中杀虫蛋白含量、Bt基因相对表达量及氮代谢相关酶和物质代谢的影响.结果表明: 棉蕾中Bt杀虫蛋白含量随土壤盐分的升高而降低,与对照相比(0%),2个不同类型棉花品种在土壤盐分0.30%及以上时,棉蕾中杀虫蛋白含量显著下降,且随土壤盐分水平的升高,Bt 杀虫蛋白含量下降幅度增大.土壤盐分胁迫提高了棉蕾中Bt基因的相对表达量.在相同土壤盐分水平下,盐敏感品种棉蕾中Bt杀虫蛋白含量受土壤盐分影响较大.杀虫蛋白表达量下降幅度大的品种,其可溶性蛋白含量、谷氨酸丙酮酸转氨酶(GPT)和谷氨酸草酰乙酸转氨酶(GOT)活性下降幅度较大,游离氨基酸含量、蛋白酶和肽酶活性上升幅度也较高.说明土壤盐分胁迫下,Bt棉棉蕾蛋白质合成能力下降,分解能力增强,可能是导致杀虫蛋白表达量下降的主要原因.     

Field Evolved Resistance in Helicoverpa armigera (Lepidoptera: Noctuidae) to Bacillus thuringiensis Toxin Cry1Ac in Pakistan

Helicoverpa armigera (Hübner) is one of the most destructive pests of several field and vegetable crops, with indiscriminate use of insecticides contributing to multiple instances of resistance. In the present study we assessed whether H. armigera had developed resistance to Bt cotton and compared the results with several conventional insecticides. Furthermore, the genetics of resistance was also investigated to determine the inheritance to Cry1Ac resistance. To investigate the development of resistance to Bt cotton, and selected foliar insecticides, H. armigera populations were sampled in 2010 and 2011 in several cotton production regions in Pakistan. The resistance ratios (RR) for Cry1Ac, chlorpyrifos, profenofos, cypermethrin, spinosad, indoxacarb, abamectin and deltamethrin were 580-fold, 320-, 1110-, 1950-, 200-, 380, 690, and 40-fold, respectively, compared with the laboratory susceptible (Lab-PK) population. Selection of the field collected population with Cry1Ac in 2010 for five generations increased RR to 5440-fold. The selection also increased RR for deltamethrin, chlorpyrifos, profenofos, cypermethrin, spinosad, indoxacarb, abamectin to 125-folds, 650-, 2840-, 9830-, 370-, 3090-, 1330-fold. The estimated LC_50s for reciprocal crosses were 105 µg/ml (Cry1Ac-SEL female × Lab-PK male) and 81 g µg/ml (Lab-PK female × Cry1Ac-SEL male) suggesting that the resistance to Cry1Ac was autosomal; the degree of dominance (D_LC) was 0.60 and 0.57 respectively. Mixing of enzyme inhibitors significantly decreased resistance to Cry1Ac suggesting that the resistance to Cry1Ac and other insecticides tested in the present study was primarily metabolic. Resistance to Cry1Ac was probably due to a single but unstable factor suggesting that crop rotation with non-Bt cotton or other crops could reduce the selection pressure for H. armigera and improve the sustainability of Bt cotton.     

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.     

Seasonal expression profiles of insecticidal protein and control efficacy against Helicoverpa armigera for Bt cotton in the Yangtze River valley of China

Seasonal levels of Bacillus thuringiensis (Bt) insecticidal protein and its control efficacy against Helicoverpa armigera (Hübner) in Bt transgenic cotton GK19 (carrying a Cry1Ac/Cry1Ab fused gene) and BG1560 (carrying a Cry1Ac gene) were investigated in Tianmen County, Hubei Province, located in the Yangtze River valley of China, in 2001 and 2002. The results showed that the toxin content in Bt cotton changed significantly over time, and that the structure, growth stage, and variety were significant sources of variability. Generally, insecticidal protein levels were high during the early stages of cotton growth; they declined in mid-season, and rebounded in late season. On most dates sampled, the toxin contents in leaf, square, petal, and stamens (including nonovule pistil tissue) were much higher than those in ovule and boll. Compared with BG1560, the expression of Cry1Ac/Cry1Ab protein in GK19 was more variable during the whole growth period of cotton. The field evaluation on larval population dynamics of H. armigera in Bt and conventional cotton showed that the larval densities in BG1560 and GK19 fields decreased, respectively, 92.04 and 81.85% in 2001, and 96.84 and 91.80% in 2002.     

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.     

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.     

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.     

Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests

Cry1Ac protoxin (the active insecticidal toxin in both Bollgard and Bollgard II cotton [Gossypium hirsutum L.]), and Cry2Ab2 toxin (the second insecticidal toxin in Bollgard II cotton) were bioassayed against five of the primary lepidopteran pests of cotton by using diet incorporation. Cry1Ac was the most toxic to Heliothis virescens (F.) and Pectinophora gossypiella (Saunders), demonstrated good activity against Helicoverpa zea (Boddie), and had negligible toxicity against Spodoptera exigua (Hübner) and Spodoptera frugiperda (J. E. Smith). Cry2Ab2 was the most toxic to P. gossypiella and least toxic to S. frugiperda. Cry2Ab2 was more toxic to S. exigua and S. frugiperda than Cry1Ac. Of the three insect species most sensitive to both Bacillus thuringiensis (Bt) proteins (including H. zea), P. gossypiella was only three-fold less sensitive to Cry2Ab2 than Cry1Ac, whereas H. virescens was 40-fold less sensitive to Cry2Ab2 compared with CrylAc. Cotton plants expressing Cry1Ac only and both Cry1Ac and Cry2Ab2 proteins were characterized for toxicity against H. zea and S.frugiperda larvae in the laboratory and H. zea larvae in an environmental chamber. In no-choice assays on excised squares from plants of different ages, second instar H. zea larvae were controlled by Cry1Ac/Cry2Ab2 cotton with mortality levels of 90% and greater at 5 d compared with 30-80% mortality for Cry1Ac-only cotton, depending on plant age. Similarly, feeding on leaf discs from Cry1Ac/Cry2Ab2 cotton resulted in mortality of second instars of S.frugiperda ranging from 69 to 93%, whereas exposure to Cry1Ac-only cotton yielded 20-69% mortality, depending on plant age. When cotton blooms were infested in situ in an environmental chamber with neonate H. zea larvae previously fed on synthetic diet for 0, 24, or 48 h, 7-d flower abortion levels for Cry1Ac-only cotton were 15, 41, and 63%, respectively, whereas for Cry1Ac/Cry2Ab2 cotton, flower abortion levels were 0, 0, and 5%, respectively. Cry1Ac and Cry2Ab2 concentrations were measured within various cotton tissues of Cry1Ac-only and Cry1Ac/Cry2Ab2 plants, respectively, by using enzyme-linked immunosorbent assay. Terminal leaves significantly expressed the highest, and large leaves, calyx, and bracts expressed significantly the lowest concentrations of Cry1Ac, respectively. Ovules expressed significantly the highest, and terminal leaves, large leaves, bracts, and calyx expressed significantly (P < 0.05) the lowest concentrations of Cry2Ab2. These results help explain the observed differences between Bollgard and Bollgard II mortality against the primary lepidopteran cotton pests, and they may lead to improved scouting and resistance management practices, and to more effective control of these pests with Bt transgenic crops in the future.     

Transfer of Cry1Ac and Cry2Ab proteins from genetically engineered Bt cotton to herbivores and predators

With the cultivation of Bt cotton, the produced insecticidal Cry proteins are ingested by herbivores and potentially transferred along the food chain to natural enemies, such as predators. In laboratory experiments with Bollgard II cotton, concentrations of Cry1Ac and Cry2Ab were measured in Lepidoptera larvae (Spodoptera littoralis, Heliothis virescens), plant bugs (Euschistus heros), aphids (Aphis gossypii), whiteflies (Bemisia tabaci), thrips (Thrips tabaci, Frankliniella occidentalis), and spider mites (Tetranychus urticae). Tritrophic experiments were conducted with caterpillars of S. littoralis as prey and larvae of ladybird beetles (Harmonia axyridis, Adalia bipunctata) and lacewings (Chrysoperla carnea) as predators. Immunological measurements (ELISA) indicated that herbivores feeding on Bt cotton contained 5%–50% of the Bt protein concentrations in leaves except whiteflies and aphids, which contained no or only traces of Bt protein, and spider mites, which contained 7 times more Cry1Ac than leaves. Similarly, predators contained 1%–30% of the Cry protein concentration in prey. For the nontarget risk assessment, this indicates that Bt protein concentrations decrease considerably from one trophic level to the next in the food web, except for spider mites that contain Bt protein concentrations higher than those measured in the leaves. Exposure of phloem sucking hemipterans is negligible.     

Bt毒蛋白Cry1Ac在人造土壤中对赤子爱胜蚓毒理及生化影响

Bt毒素能通过转基因作物的花粉、根和残株进入土壤.为评估转基因作物对土壤动物的影响,本文模拟转基因棉的Bt毒素进入土壤的发生程度,用含不同浓度Bt毒蛋白Cry1Ac的人造土壤处理蚯蚓,测定蚯蚓存活率、重量变化及体内总蛋白含量和过氧化氢酶(CAT)、乙酰胆碱酯酶(AchE)、谷胱甘肽-S-转移酶(GST)和纤维素酶活性.结果表明,Bt毒蛋白对蚯蚓的生物量和生理水平影响均不明显,不存在急毒性和亚致死毒性影响,对蚯蚓比较安全.     

转cry1Ab/cry1Ac基因玉米Cry1Ab/Cry1Ac融合蛋白表达及对亚洲玉米螟的室内杀虫效果

【目的】室内抗螟性评价是转Bt基因抗虫玉米研发和安全性评价的重要环节。【方法】采用酶联免疫吸附测定法(ELISA)测定了转cry1Ab/cry1Ac基因玉米ZZM030心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量;采用室内生测法测定了分别取食转基因玉米ZZM030和非转基因玉米X249心叶后亚洲玉米螟Ostrinia furnacalis敏感品系ACB-BtS、Cry1Ab抗性品系ACB-AbR和Cry1Ac抗性品系ACB-AcR初孵幼虫的存活率。【结果】转基因抗虫玉米ZZM030 4叶期和8叶期心叶中Cry1Ab/Cry1Ac融合杀虫蛋白的表达量分别是10.62和2.94 μg/g FW。敏感品系亚洲玉米螟初孵幼虫取食转基因玉米ZZM030心叶2 d的存活率仅为23.6%,4 d后存活率为0,而取食非转基因对照玉米X249心叶4 d的存活率高达93.1%。Cry1Ab抗性品系和Cry1Ac抗性品系初孵幼虫取食转基因玉米ZZM030心叶6 d后的存活率分别为11.1%和12.5%,而取食非转基因玉米X249心叶6 d后的存活率分别为81.9%和77.8%。【结论】转cry1Ab/cry1Ac基因玉米ZZM030心叶中高表达的Cry1Ab/Cry1Ac融合蛋白对亚洲玉米螟初孵幼虫具有极高的杀虫效果。     

Similar genetic basis of resistance to Bt toxin Cry1Ac in Boll-selected and diet-selected strains of pink bollworm

Genetically engineered cotton and corn plants producing insecticidal Bacillus thuringiensis (Bt) toxins kill some key insect pests. Yet, evolution of resistance by pests threatens long-term insect control by these transgenic Bt crops. We compared the genetic basis of resistance to Bt toxin Cry1Ac in two independently derived, laboratory-selected strains of a major cotton pest, the pink bollworm (Pectinophora gossypiella [Saunders]). The Arizona pooled resistant strain (AZP-R) was started with pink bollworm from 10 field populations and selected with Cry1Ac in diet. The Bt4R resistant strain was started with a long-term susceptible laboratory strain and selected first with Bt cotton bolls and later with Cry1Ac in diet. Previous work showed that AZP-R had three recessive mutations (r1, r2, and r3) in the pink bollworm cadherin gene (PgCad1) linked with resistance to Cry1Ac and Bt cotton producing Cry1Ac. Here we report that inheritance of resistance to a diagnostic concentration of Cry1Ac was recessive in Bt4R. In interstrain complementation tests for allelism, F(1) progeny from crosses between AZP-R and Bt4R were resistant to Cry1Ac, indicating a shared resistance locus in the two strains. Molecular analysis of the Bt4R cadherin gene identified a novel 15-bp deletion (r4) predicted to cause the loss of five amino acids upstream of the Cry1Ac-binding region of the cadherin protein. Four recessive mutations in PgCad1 are now implicated in resistance in five different strains, showing that mutations in cadherin are the primary mechanism of resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona.     

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.     

Plant-toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae)

In Australia, transgenic cotton plants expressing the cry1Ac gene from Bacillus thuringiensis Berliner variety kurstaki are less toxic to first-instar Helicoverpa armigera (Hübner) after the plant is producing fruit. We developed two bioassay methods (leaf mush, leaf disk) to test if the physiological state of the plants explained changes in toxicity and a third method (diet incorporation) was developed to quantify the toxicity of Bt leaves when mixed in chickpea diet. Cry1Ac protein was less toxic to H. armigera larvae when the protein was mixed with leaves from fruiting versus presquare conventional cotton. Differences in LC50 varied from 2.4- to 726-fold, depending on the source of toxin and conventional plant material. These results suggest that plant-toxin interactions in fruiting cotton are reducing the toxicity of the Cry1Ac protein. The possible role of tannins in these changes is discussed.     

转Cry1Ac活性杀虫蛋白及慈菇蛋白酶抑制剂B基因的棉花

根据植物基因的结构特征,合成了CrylAc活性杀虫蛋白的编码序列并与内质网定位肽编码序列组成嵌合杀虫蛋白基因Bt29K.构建了含Bt29K基因及慈菇蛋白酶抑制剂B(API-B)基因表达框的双抗虫基因植物表达载体.通过根癌土壤杆菌(Agrobacteriumtumefaciens(Smith et Townsend)Conn LBA4404)介导转化了棉花(Gossypium hirsu-tun L.)的两个生产品种(系).根据抗棉铃虫(Heliothis armigera)试验及农艺性状的观察调查结果,经6代筛选,获得了抗棉铃虫90.0%～99.7%且农艺性状优良的9个双价抗虫棉纯合品系.分子生物学分析结果表明,两个抗虫基因在棉花基因组中的插入拷贝数为1个或2个.活性Cry1Ac和API-B蛋白在转基因抗虫棉株系中的表达量分别约占总可溶性蛋白的0.17%和0.09%.对双抗纯合系植株及仅转Bt基因的棉花纯合系抗虫性检测结果表明前者的抗虫性明显高于后者,因此推断本研究采用的双抗虫基因表达载体构建策略是合理的.     

多年种植Bt稻后外源蛋白在土壤中的积累

外源蛋白在环境中的残留与积累是转Bt基因作物环境安全评价的重要内容之一。我国已育成多个具有商业化前景的Bt稻品系,但目前多年种植Bt稻后Bt外源蛋白是否会在土壤中积累还不清楚。本研究在同一试验田连续9年种植了转cry1Ab/1Ac基因明恢63(华恢1号)和转cry2A基因明恢63水稻,采用酶联免疫吸附法(ELISA)跟踪监测了分蘖期和收获后60 d根际土中外源蛋白含量变化,试验第1年(2012年)和最后1年(2020年)还测定了苗期、开花期和成熟期根际土中外源蛋白含量。结果表明: 2012年,转cry1Ab/1Ac基因明恢63在苗期、分蘖期、开花期、成熟期和收获后60 d根际土中外源蛋白含量分别为1.25、1.77、1.97、1.71和0.30 ng·g^-1,2020年分别为1.30、1.69、2.03、1.77和0.43 ng·g^-1;2012年,转cry2A基因明恢63在苗期、分蘖期、开花期、成熟期和收获后60 d根际土中外源蛋白含量分别为0.91、1.52、1.53、1.37和0.12 ng·g^-1,2020年分别为0.95、1.43、1.61、1.40和0.15 ng·g^-1。多因素方差分析显示,时间效应对Bt外源蛋白积累不显著,而品种和生育期效应显著。Bt稻生长过程中根际土中可以检测出微量的Bt外源蛋白,但收获后60 d已经基本降解完毕,根际土中Bt外源蛋白含量不会随着种植时间的增加而累积。     

High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigera to Bt Soybean in Brazil

The Old World bollworm, Helicoverpa armigera (Hübner), was recently introduced into Brazil, where it has caused extensive damage to cotton and soybean crops. MON 87701 × MON 89788 soybean, which expresses the Bt protein Cry1Ac, was recently deployed in Brazil, providing high levels of control against H. armigera. To assess the risk of resistance to the Cry1Ac protein expressed by MON 87701 × MON 89788 soybean in Brazil, we conducted studies to evaluate the baseline susceptibility of H. armigera to Cry1Ac, in planta efficacy including the assessment of the high-dose criterion, and the initial resistance allele frequency based on an F₂ screen. The mean Cry1Ac lethal concentration (LC₅₀) ranged from 0.11 to 1.82 μg·mL⁻¹ of diet among all H. armigera field populations collected from crop seasons 2013/14 to 2014/15, which indicated about 16.5-fold variation. MON 87701 × MON 89788 soybean exhibited a high level of efficacy against H. armigera and most likely met the high dose criterion against this target species in leaf tissue dilution bioassays up to 50 times. A total of 212 F₂ family lines of H. armigera were established from field collections sampled from seven locations across Brazil and were screened for the presence of MON 87701 × MON 89788 soybean resistance alleles. None of the 212 families survived on MON 87701 × MON 89788 soybean leaf tissue (estimated allele frequency = 0.0011). The responses of H. armigera to Cry1Ac protein, high susceptibility to MON 87701 × MON 89788 soybean, and low frequency of resistance alleles across the main soybean-producing regions support the assumptions of a high-dose/refuge strategy. However, maintenance of reasonable compliance with the refuge recommendation will be essential to delay the evolution of resistance in H. armigera to MON 87701 × MON 89788 soybean in Brazil.     

Expression of Bt cry1Ac in transgenic oilseed rape in China and transgenic performance of intraspecific hybrids against Helicoverpa armigera larvae

The expression of a synthetic Bacillus thuringiensis ( Bt ) cry1Ac gene in oilseed rape (OSR, Brassica napus ) was monitored under field conditions in China, and performance against Helicoverpa armigera larvae was compared in intraspecific hybrids with a Chinese OSR variety. Leaf samples from transgenic OSR were collected at various developmental stages in two separate field experiments. The Bt Cry1Ac concentrations in the third uppermost leaves increased before pod formation stage and either increased or decreased after pod formation stage whereas the total soluble protein increased before and decreased after pod-fill in the later growing season. Spontaneously formed intraspecific hybrids between transgenic OSR and a Chinese conventional OSR were obtained in the field and transgenic status was confirmed by a green fluorescent protein (GFP) phenotype and polymerase chain reaction. A bioassay on the neonate larvae of a susceptible strain of H.   armigera was performed to test the efficacy of Bt Cry1Ac toxin in hybrid OSR plants. Both the original transgenic OSR and hybrid plants had a negative effect on body-weight gain of insect larvae. It was assumed that Bt Cry1Ac toxin concentration was similar in hybrids compared to the original transgenic OSR at the investigated developmental stages. The frequency of hybrid production and volunteerism could potentially enhance the evolution of insect pest tolerance in the field.