Impact of transgenic oilseed rape expressing oryzacystatin-1 (OC-1) and of insecticidal proteins on longevity and digestive enzymes of the solitary bee Osmia bicornis

The risk that insect-resistant transgenic plants may pose for solitary bees was assessed by determining longevity of adult Osmia bicornis (O. rufa) chronically exposed to transgenic oilseed rape expressing oryzacystatin-1 (OC-1) or to the purified insecticidal proteins recombinant rOC-1, Kunitz soybean trypsin inhibitor (SBTI), Galanthus nivalis agglutinin (GNA), or Bacillus thuringiensis toxin Cry1Ab dissolved in sugar solution (at 0.01 and 0.1%, w:v, Cry1Ab only at 0.01%). Compared to control bees, longevity was significantly reduced by SBTI and GNA at both concentrations and by rOC-1 at 0.1%, but not by Cry1Ab or rOC-1 at 0.01%. Longevity on the OC-1 oilseed rape was not significantly different from the control plants. The effects of SBTI and rOC-1 on longevity were investigated through characterization of the digestive proteinases of O. bicornis and analysis of the response in proteinase profiles to ingestion of these proteinase inhibitors. A relatively complex profile of at least four types of soluble proteolytic enzymes was identified. Serine proteinases were found to be predominant, with metallo and especially cysteine proteinases making a smaller albeit significant contribution. The compensatory response to in vivo enzyme inhibition was similar for SBTI and rOC-1 although less pronounced for rOC-1. It consisted of a non-specific overproduction of native proteinases, both sensitive and insensitive, and the induction of a novel aspartic proteinase.     

Distribution and residual activity of two insecticidal proteins, avidin and aprotinin, expressed in transgenic tobacco plants, in the bodies and frass of Spodoptera litura larvae following feeding

To understand how a major cosmopolitan pest responds to two very different insecticidal proteins and to determine whether herbivorous insects and their frass could be environmental sources of recombinant proteins from transgenic plants, Spodoptera litura (Fab.) (Lepidoptera, Noctuidae) larvae were fed on tobacco leaves expressing either the biotin-binding protein, avidin, or the protease inhibitor, aprotinin. Control larvae received non-transgenic tobacco. Samples of larvae were taken after 5, 6 or 7 days’ feeding and frass was collected after two 24-h periods at 6 and 7 days. Insects in all treatments grew significantly during the experiment, but the avidin-fed larvae were significantly smaller than the others on Day 7. Avidin was found in all samples of avidin-fed larvae (7.0±0.86 ng mg⁻¹, n=45), at a lower level than in their frass (31.9±5.08 ng mg⁻¹, n=30), and these frass levels were lower than those of the the leaves fed to the larvae (69.0±6.71 ng mg⁻¹, n=45). All of the avidin detected in these samples was capable of binding biotin. On average, between 10 and 28% of avidin was recovered with the methods used, whereas almost full recovery of aprotinin was effected. Aprotinin levels in larvae (8.2±0.53 ng mg⁻¹, n=45) were also lower than aprotinin levels in frass (77.4±6.9 ng mg⁻¹, n=30), which were somewhat lower than those in the leaves fed to the larvae (88.6±2.51 ng mg⁻¹, n=45). Approximately half the trypsin-binding ability of aprotinin was lost in larvae, and in frass, aprotinin had lost about 90% of its ability to bind trypsin.     

转基因抗虫烟草研究进展

烟草为模式植物,也是外源杀虫基因最早转化成功的植物。文章从转Bt内毒素基因,植物凝集素GNA,Plec,AHA基因,蛋白酶抑制剂PIⅠ,PIⅡ,MTI,SKTI基因,昆虫特异性神经毒素基因,几丁质酶基因,畸形细胞分泌蛋白基因以及双抗虫基因等方面综述了转基因抗虫烟草的抗虫性、转基因抗虫烟草的经济性状等,展望了转基因抗虫烟草的研究和应用前景,以期对烟草害虫的治理尤其是对其他转基因抗虫作物的培育和研究有借鉴作用。     

转基因植物环境监测进展

近20年来,转基因植物的商业化应用规模越来越大,而转基因生物安全问题依然是转基因植物产业进一步发展的最主要制约因素。转基因植物在商业化应用之前虽然预先进行了风险评估,但是,包括环境监测在内的风险管理措施是确保转基因植物安全应用的必要手段。在转基因作物大规模应用近20年之后,其在靶标生物抗性、对生物多样性的影响、基因漂移、在生态系统中的长期存留等方面产生的环境风险已经渐渐显现出来,表明风险评估无法为转基因植物应用提供足够的安全保障,还必须通过开展系统而长期的环境监测,明确转基因植物在生产应用后的实际环境影响。联合国环境规划署和欧盟等已经制定了转基因植物环境监测的法规和技术指南,一些国家实施了系统的转基因植物环境监测。对转基因植物所产生的环境风险以及环境监测应包括的内容进行了综述。     

Effects of transgenic Bt corn litter on the earthworm Lumbricus terrestris

A 200-day study was carried out to investigate the impact of transgenic Bacillus thuringiensis (Bt) corn on immature and adult Lumbricus terrestris in the field and in the laboratory. Another objective of this study was to develop test methods that could be used for standard testing of the impact of transgenic plants on different earthworm species in the field and in the laboratory. For this purpose two different experiments were involved, a laboratory experiment with adult L. terrestris and a field experiment with immature L. terrestris. No lethal effects of transgenic Bt corn on immature and adult earthworms were observed. Immature L. terrestris in the field had a very similar growth pattern when fed either (Bt+) or (Bt-) corn litter. No significant differences in relative weights of (Bt+) and (Bt-) corn-fed adult L. terrestris were observed during the first 160 days of the laboratory trial, but after 200 days adult L. terrestris had a significant weight loss of 18% of their initial weight when fed (Bt+) corn litter compared to a weight gain of 4% of the initial weight of (Bt-) corn-fed earthworms. Further studies are necessary to see whether or not this difference in relative weight was due to the Bt toxin or other factors discussed in the study. Degradation of Cry1Ab toxin in corn residues was significantly slower in the field than at 10 degrees C in the laboratory. Enzyme-linked immunosorbent assay results indicated that earthworms in both experiments were exposed to the Bt toxin throughout the whole experimental time.     

Tier-1 assays for assessing the toxicity of insecticidal proteins produced by genetically engineered plants to non-target arthropods

In assessing an insect-resistant genetically engineered （IRGE） crop before its commercialization, researchers normally use so-called ＂Tier-1 assays＂ as the initial step to determine the effects of the crop on non-target organisms. In these tests, the insecticidal proteins （IPs） produced by the IRGEs are added to the diets of test organisms in the laboratory. Test organisms in such assays can be directly exposed to much higher concentrations of the test IPs than they would encounter in the field. The results of Tier-1 assays are thus more conservative than those generated in studies in which the organisms are exposed to the IPs by feeding on IRGE plant tissue or in the case of predators or parasites, by feeding on invertebrate prey or hosts that have fed on IRGE plant tissue. In this report, we consider three important factors that must be considered in Tier-1 assays： （i） methods for delivery of the IP to the test organisms; （ii） the need for and selection of compounds used as positive controls; and （iii） methods for monitoring the concentration, stability and bioactivity of the IP during the assay. We also analyze the existing data from Tier-1 assays regarding the toxicity of Bt Cry proteins to non-target arthropod species. The data indicate that the widely used Bt proteins have no direct toxicity to non-target organisms     

Constitutive expression of a cowpea trypsin inhibitor gene,CpTi, in transgenic rice plants confers resistance to two major rice insect pests

The gene encoding a cowpea trypsin inhibitor (CpTI), which confers insect resistance in trangenic tobacco, was introduced into rice. Expression of the CpTi gene driven by the constitutively active promoter of the rice actin 1 gene (Act1) leads to high-level accumulation of the CpTI protein in transgenic rice plants. Protein extracts from transgenic rice plants exhibit a strong inhibitory activity against bovine trypsin, suggesting that the proteinase inhibitor produced in transgenic rice is functionally active. Small-scale field tests showed that the transgenic rice plants expressing the CpTi gene had significantly increased resistance to two species of rice stem borers, which are major rice insect pests. Our results suggest that the cowpea trypsin inhibitor may be useful for the control of rice insect pests.     

Persistence and biological activity in soil of the insecticidal proteins from Bacillus thuringiensis, especially from transgenic plants

The Dune System of Doñana National Park (SW Spain) exhibit a mosaic of environmental characteristics, with different plant communities, all under the same Mediterranean climate, creating an interesting field laboratory for the study of plant responses to stressing conditions. Fourteen woody plant populations were selected, belonging to either xerophytic or hygrophytic plant communities on stabilised dunes, where topography causes differences in soil water availability. Plants were tagged and morphological and ecophysiological measurements were recorded in winter and summer of 1999. Seasonal differences in ecophysiological measurements together with morphological variables were used as plant traits to identify the main adaptive responses of the species. Cluster analysis of traits separated three groups of plant strategies: spiny legume species; sclerophyll, and semideciduous species. In addition, another two kind of strategies have been found in the semideciduous group of species, those withstanding water shortages, attaining very negative water potentials, low photochemical efficiency, and leaf proline accumulation in summer, as opposed to those tolerating water deficit, with moderate seasonal differences in water potential, proline content and photochemical efficiency. The results of this study indicate that legume species behave as a different functional group and drought-semideciduous species present different adaptive responses under the same environmental stress. Ecophysiological measurements must be used as plant traits to detect functional groups under Mediterranean climate.     

Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco

Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under the control of the 35S CaMV promoter. Although the transgene was expressed and the protein was active in the homozygous lines selected, growth of Spodoptera exigua (Lepidoptera: Noctuidae) larvae reared on transgenic plants was not affected. The protease activity in larval midgut extracts after 2 days feeding on transformed tobacco leaves from the highest expressing plant showed a reduction of 25% in the trypsin-like activity compared to that from insects fed on non-transformed controls. The susceptibility of digestive serine-proteases to inhibition by BTI-CMe was confirmed by activity staining gels. This decrease was compensated with a significant induction of leucine aminopeptidase-like and carboxipeptidase A-like activities, whilechymotrypsin-, elastase-, and carboxipeptidase B-like proteases were not affected.     

Population-scale laboratory studies of the effect of transgenic plants on nontarget insects

Studies of the effects of insect-resistant transgenic plants on beneficial insects have, to date, concentrated mainly on either small-scale "worst case scenario" laboratory experiments or on field trials. We present a laboratory method using large population cages that represent an intermediate experimental scale, allowing the study of ecological and behavioural interactions between transgenic plants, pests and their natural enemies under more controlled conditions than is possible in the field. Previous studies have also concentrated on natural enemies of lepidopteran and coleopteran target pests. However, natural enemies of other pests, which are not controlled by the transgenic plants, are also potentially exposed to the transgene product when feeding on hosts. The reduction in the use of insecticides on transgenic crops could lead to increasing problems with such nontarget pests, normally controlled by sprays, especially if there are any negative effects of the transgenic plant on their natural enemies. This study tested two lines of insect-resistant transgenic oilseed rape (Brassica napus) for side-effects on the hymenopteran parasitoid Diaeretiella rapae and its aphid host, Myzus persicae. One transgenic line expressed the delta-endotoxin Cry1Ac from Bacillus thuringiensis (Bt) and a second expressed the proteinase inhibitor oryzacystatin I (OC-I) from rice. These transgenic plant lines were developed to provide resistance to lepidopteran and coleopteran pests, respectively. No detrimental effects of the transgenic oilseed rape lines on the ability of the parasitoid to control aphid populations were observed. Adult parasitoid emergence and sex ratio were also not consistently altered on the transgenic oilseed rape lines compared with the wild-type lines.     

Dependence of Asian honeybee on deciduous woody plants for pollen resource during spring to mid‐summer in northern Japan

Apis cerana japonica Radoszkowski, endemic to Japan, is known to be one of the most important pollinators for wild plants and crops, such as buckwheat, in cool to warm temperate Japan. To determine the degree of dependence of A. cerana japonica on forest resources, we analyzed pollen brought back to nests in a typical “Satoyama” landscape with relatively high deciduous forest coverage in northern Japan. We divided the landscape elements of the study area into three types: deciduous forest, conifer plantation and open land according to landcover digital data, and each pollen taxon was assigned to one of these three types of landscape elements. We collected total pollen loads of 15.75 g (total of colonies A and B) in May (spring), 1.57 g (total of colonies A and C) in June (early summer), 19.03 g (total of colonies A, B and C) in July (mid‐summer) and 45.61 g (total of colonies A, B and D) in September (autumn). Deciduous forests are the most important foraging habitats for A. cerana japonica in the “Satoyama” landscape especially from spring to mid‐summer when mass flowering of tall trees and shrubs species provides rich floral resources for developing bee colonies. On the other hand, the bees frequently foraged from herbaceous plant species in autumn when flowering of tree species reduces and herbaceous plant species have flowering peaks. In turn, the bees provide pollination services to a number of wild flowers blooming in various forest layers ranging from the canopy to the understory layer.     

转Bt基因作物释放杀虫晶体蛋白对土壤生态安全的影响

随着转Bt基因抗虫作物的大面积推广种植,其环境安全性问题日益引起关注。转Bt基因作物在生长期内持续不断地向环境释放杀虫晶体蛋白,这些杀虫晶体蛋白积累一旦超过了昆虫的消耗及环境因子对其的钝化,就可能对非靶标昆虫或土壤微生物造成伤害。转Bt基因作物向土壤中释放杀虫晶体蛋白的途径主要有3种:根系分泌、花粉飘落和秸秆还田。释放到土壤中的Bt杀虫晶体蛋白能够迅速被土壤活性颗粒吸附,1～3 h就能达到吸附平衡。吸附态Bt杀虫晶体蛋白不易被土壤微生物或酶降解,导致杀虫活性持续时间显著延长。土壤微生物种群变化是衡量Bt作物对土壤生态影响的重要指标。研究表明,Bt作物根系分泌物或Bt生物体降解释放的杀虫晶体蛋白对于土壤蚯蚓、线虫、原生动物、细菌和真菌没有毒性,但可使丛枝菌根真菌(AMF)菌丝长度减小,不能形成侵染单元。Bt杀虫晶体蛋白对土壤酶活性的影响程度依这类蛋白的导入方式或Bt作物生育期的不同而呈现差异。土壤中Bt Cry1Ab蛋白能被部分后茬作物吸收,但不同的商品试剂盒检测结果存在差异。文章综述了Bt杀虫晶体蛋白在土壤中释放、吸附、残留特性及其对土壤动物、土壤微生物、土壤酶活性和后茬作物的影响,旨在为转Bt基因作物释放杀虫晶体蛋白的土壤生态安全评价提供参考依据。     

Performance of transgenic spring wheat plants and effects on non-target organisms under glasshouse and semi-field conditions

Abstract:  A convertible glasshouse was established to study annual transgenic plants under near-field environmental conditions while simultaneously ensuring a high level of biological containment. This system can provide a useful step in the assessment of transgenic plants prior to open-field experiments. Two transgenic wheat lines (cv. Bobwhite) were investigated and compared with their corresponding non-transformed wildtypes with respect to plant performance, expression of the transgenic trait and interactions with antagonists. The first line expressed snowdrop lectin [ Galanthus nivalis agglutinin (GNA)] for enhanced resistance to aphids, and the second one overexpressed the endogenous Lr10 gene to enhance resistance to leaf rust. Interestingly, 1000-kernel weight of Lr10 -transgenic plants was significantly reduced, indicating that the overexpression of the Lr10 gene caused a significant fitness cost for the plant. GNA-transgenic plants expressed the lectin at levels too low to affect the target aphids. A detached leaf bioassay with Lr10 -transgenic plants revealed an increased resistance to leaf rust. No differences in the performance of aphids or cereal leaf beetles on transgenic and non-transformed plants were recorded in the convertible glasshouse and in complementary glasshouse studies. Similarly, infection levels with powdery mildew did not differ between transgenic and non-transformed plants but Bobwhite plants were significantly more infected when compared with conventional Swiss spring wheat cultivars. Overall, the assessment revealed that for the plants investigated here, their genetic background had a stronger impact on the performance of a plant and its interactions with insect herbivores and pathogens than the expression of the transgene.     

Influence of thermal motion on 1H chemical shifts in proteins: the case of bovine pancreatic trypsin inhibitor.

The possible influence of thermal motion on 1H chemical shifts is discussed for a small stable protein, the bovine pancreatic Kunitz trypsin inhibitor (BPTI). The thermal effects on the aromatic side chains and on the backbone are treated separately. The thermal motion of the aromatic side chains is accounted for in terms of their rotation around the C(alpha)-C(beta) bond and the motion of each individual proton is interpreted as a ratio between the amount of ordered and quite disordered states. The influence of hydrogen bonds is introduced as an extra contribution to the chemical shifts of the bonded proton. Their contribution to the chemical shifts resulting from the polarization of the peptide bond is investigated, as is their influence on local flexibility. Finally, the relative importance of each contribution to the chemical shift information is compared.     

A synthesis of laboratory and field studies on the effects of transgenic Bacillus thuringiensis (Bt) maize on non‐target Lepidoptera

One of the major applications of transgenic crops in agriculture are the so‐called Bacillus thuringiensis Berliner (Bt) plants, in particular Bt maizes, which produce insecticidal Cry proteins that target specific orders, such as the Lepidoptera or Coleoptera. We reviewed publications that reported on the direct toxic effects of Bt‐maize and/or Cry proteins of current Bt‐maize events on larvae of non‐target butterflies and moths (Lepidoptera). In total, 20 peer‐reviewed publications were identified, of which 16 papers contributed laboratory‐based data and seven field‐based data. An adverse effect on caterpillars was recorded in 52% of all laboratory‐based and in 21% of all field‐based observations. The variables most often studied and having the highest occurrence of effects were larval survival, body mass, and developmental time. Parameters of the adult stage were under‐represented in the studies. Overall, 11 lepidopteran species were tested. The majority of the studies originated from the USA, with the Monarch butterfly being the most studied, whereas other species and other parts of the world were widely neglected. Laboratory experiments were often run under unrealistic conditions from an ecological point of view. Although the papers we reviewed indicated a potential hazard for Lepidoptera that are exposed to and feed on lepidopteran‐specific Bt‐maize pollen, a general conclusion on the level of risk for butterflies and moths cannot as yet be drawn. A comprehensive risk characterization would require thorough hazard identification, exposure assessment, and impact assessment. However, our review showed that even the basic level of hazard characterization is as yet incomplete. Reasons for this are the still‐limited numbers of publications and concurrent lack of knowledge, the restriction of data to only a few species, the over‐representation of North American species, and the identified limitations of both laboratory and field experiments. The findings of this review suggest that more realistic, ecologically meaningful, and detailed experiments and analyses are crucial to improve the present assessment of Bt‐maize cultivation effects on Lepidoptera.     

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.     

Effect of crop rotation and soil cover on alteration of the soil microflora generated by the culture of transgenic plants producing opines

The culture of transgenic Lotus corniculatus plants producing opines, which are bacterial growth substrates, leads to the selection of rhizospheric bacteria able to utilize these substrates. We have investigated the fate of the opine-utilizing community over time under different experimental conditions following elimination of selective pressure exerted by the transgenic plants. These plants were removed from the soil, which was either left unplanted or replanted with wild-type L. corniculatus or wheat plants. The density of opine-utilizing bacteria in the fallow soils remained essentially unchanged throughout the experiment, regardless of the soil of origin (soil planted with wild-type or transgenic plants). When wild-type Lotus plants were used to replace their transgenic counterparts, only the bacterial populations able to utilize the opines were affected. Long-term changes affecting the opine-utilizing bacterial community on Lotus roots was dependent upon the opine studied. The concentration of nopaline utilizers decreased, upon replacement of the transgenic plants, to a level similar to that of normal plants, while the concentration of mannopine utilizers decreased to levels intermediate between transgenic and normal plants. These data indicate that: (i) the opine-utilizing bacterial populations can be controlled in the rhizosphere via plant-exudate engineering; (ii) the interaction between the engineered plants and their root-associated micro-organisms is transgene specific; and (iii) alterations induced by the cultivation of transgenic plants may sometimes be persistent. Furthermore, opine-utilizing bacterial populations can be controlled by crop rotation. Therefore, favouring the growth of a rhizobacterium of agronomic interest via an opine-based strategy appears feasible.     

Multiple traits of agronomic importance in transgenic indica rice plants: analysis of transgene integration patterns,expression levels and stability

We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) -endotoxin genes cry1Ac and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R₀ and R₁ plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.