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1.
The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco lines have been evaluated by feeding the lepidopteran Spodoptera littoralis throughout its larval life. Specific conditions were selected to study the long-term effects of feeding larvae on transgenic plants expressing the inhibitor at various levels. The data obtained led to the establishment of three relevant parameters to be considered during the experimentation: (i) the PI content of the plant lines to be used; (ii) the developmental stage of larvae sensitive to that PI content; (iii) the ratio of MTI-2/proteases sufficient to inhibit gut proteases. The experimental data obtained from feeding S. littoralis larvae using these conditions led to two main results. First, when L2 S. littoralis larvae were fed on high MTI-2 expressing tobacco plants, no effects on larval development were detected but there was a significantly reduced fertility. When the same larvae were fed on low expressing MTI-2 tobacco plants, only a less marked lowering of fertility was observed. Second, after the first generation, no differences in protease activity were observed in insects derived from larvae fed on high or low MTI-2 expressing tobacco lines, suggesting that genetic traits observed in previous studies were not inherited.  相似文献   

2.
Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators feeding on crop pests, through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the serine protease inhibitor, mustard trypsin inhibitor -2 (MTI-2), on the predatory ground beetle Pterostichus madidus were investigated, using diamondback moth, Plutella xylostella as the intermediary pest species. As expected, oilseed rape expressing MTI-2 had a deleterious effect on the development and survival of the pest. However, incomplete pest mortality resulted in survivors being available to predators at the next trophic level, and inhibition studies confirmed the presence of biologically active transgene product in pest larvae. Characterization of proteolytic digestive enzymes of P. madidus demonstrated that adults utilize serine proteases with trypsin-like and chymotrypsin-like specificities; the former activity was completely inhibited by MTI-2 in vitro. When P. madidus consumed prey reared on MTI-2 expressing plants over the reproductive period in their life cycle, no significant effects upon survival were observed as a result of exposure to the inhibitor. However, there was a short-term significant inhibition of weight gain in female beetles fed unlimited prey containing MTI-2, with a concomitant reduction of prey consumption. Biochemical analyses showed that the inhibitory effects of MTI-2 delivered via prey on gut proteolysis in the carabid decreased with time of exposure, possibly resulting from up-regulation of inhibitor-insensitive proteases. Of ecological significance, consumption of MTI-2 dosed prey had no detrimental effects on reproductive fitness of adult P. madidus.  相似文献   

3.
This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI.  相似文献   

4.
When investigating resistance to herbivorous insects in transgenic plants, it is primarily the damaging stage of the insect that is targeted. In our study, we showed that small effects of the transgenic plant on the pest performance might lead to a clear effect on the pest population level. Pollen beetle, Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae), larvae were reared on three transgenic lines of Brassica napus L. (Brassicaceae) expressing pea (Pisum sativum L.) lectin at 0.2, 0.6, and 0.7% of total soluble protein in anthers, and then monitored until the adult stage. Two lines without the gene construct and consequently containing no pea lectin were used as controls. The egg viability, larval weight, development time, and survival rate were recorded. In a separate experiment, the development time from egg to adult, adult weight, and winter survival of pollen beetles developed on intact plants were recorded. Survival of larvae to adult stage was significantly lower on the transgenic plant lines, independent of lectin level. Survival during hibernation was only 2.4% and was not affected by plant line. Combining previously published results from adult feeding and oviposition assays with the new data on larval development we summarize the effect of the transgenic oilseed rape on the pollen beetle, from oviposition in spring to overwintered adults of the new generation. Net reproductive rate was then shown to be reduced by half on the transgenic oilseed rape compared to the control plants.  相似文献   

5.
Chloroplast Transformation in Oilseed Rape   总被引:24,自引:0,他引:24  
The chloroplast transformation vector pNRAB carries two expression cassettes for the spectinomycin resistance gene aadA and the insect resistance gene cry1Aa10. The two cassettes are sited between the rps7 and ndhB targeting fragments. Biolistic delivery of the vector DNA, followed by spectinomycin selection, yielded chloroplast transformants at a frequency of four in 1000 bombarded cotyledon petioles. PCR analysis and Southern blot of PCR products confirmed the site-specific integration of aadA and cry1Aa10 into the chloroplast genomes of transgenic oilseed rape. When transgenic oilseed rape leaves were fed to second instar Plutella xylostera larvae, 47% mortality was observed against this insect and the surviving larvae had significantly lower weight than the control. This is the first report of chloroplast transformation in oilseed rape and the introduction of novel genes between the rps7 and ndhB genes in the chloroplast genome. This offers an opportunity for improvement of oilseed rape by chloroplast genetic engineering.  相似文献   

6.
This study was done to assess insect growth and mortality on tobacco plants transformed with baculovirus enhancin genes, as a first step toward the possible use of enhancin transgenes as part of an insect control system. Enhancin genes from Trichoplusia ni or Helicoverpa armigera baculoviruses were introduced into tobacco via Agrobacterium tumefaciens with kanamycin selection. PCR analyses of genomic DNA confirmed the presence of the enhancin genes in the kanamycin-resistant plants; however, the expression of the genes was very low and could be detected only with RT-PCR. Bioassays with Trichoplusia ni larvae showed that larval growth and development was significantly slower on some transgenic lines and that larval mortality was higher. The majority of the enhancin-transgenic plants had little or no inhibitory effect. The low expression of enhancin in plants carrying current expression cassettes and the relevance of these results to pest management are discussed.  相似文献   

7.
Transgenic cotton lines were developed for high-level expression of a synthetic cry1EC gene from a wound inducible promoter. The tobacco pathogenesis related promoter PR-1a was modified by placing CaMV35S promoter on its upstream in reverse orientation. The resultant chimeric promoter CaMV35S(r)PR-1a expressed constitutively and was further up-regulated at the site of feeding by insects. It was induced more rapidly by treatment with salicylic acid (SA). The CaMV35S(r)PR-1a cry1EC expressing transgenic lines of cotton showed 100% mortality of Spodoptera litura larvae. The tightly regulated low-level expression of PR-1a was modified to a highly expressing constitutive expression by CaMV35S placed in reverse orientation. Salicylic acid treatment and wounding enhanced the expression further by the chimeric promoter. The leaves expressed more δ-endotoxin around the sites of insect bites. The levels of expression and induction varied among different transgenic lines, suggesting position effect. Some of the transgenic lines that expressed Cry1EC from the chimeric promoter at a low level also showed 100% mortality when induced with salicylic acid. A highly expressing insect bite and wound inducible promoter is desirable for developing insect resistant transgenic plants.  相似文献   

8.
A giant taro proteinase inhibitor (GTPI) cDNA was expressed in transgenic tobacco using three different gene constructs. The highest expression level obtained was ca. 0.3% of total soluble protein when the cDNA was driven by the Arabidopsis rbcS ats1 promoter. Repeated feeding trials with Helicoverpa armigera larvae fed on clonally derived T0 and T1 plants expressing GTPI demonstrated that, relative to those fed on control plants, some growth inhibition (22–40%) occurs, but there was no increase in larval mortality. Proteinase activities of larvae fed on GTPI-expressing tobacco or GTPI-containing diet were examined to monitor the spectrum of digestive proteinases in the midgut. Total proteinase activity was reduced by 13%, but GTPI-insensitive proteinase activity was increased by up to 17%. Trypsin was inhibited by 58%, but chymotrypsin and elastase were increased by 26% and 16% respectively. These results point to an adaptive mechanism in this insect that elevates the levels of other classes of proteinases to compensate for the trypsin activity inhibited by dietary proteinase inhibitors.  相似文献   

9.
Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) were investigated on larvae of the solitary bee Osmia bicornis (= O. rufa). Furthermore, recombinant OC-1 (rOC-1), the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA) were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1%) in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products.  相似文献   

10.
Larvae of Baris coerulescens Scop. (Coleoptera: Curculionidæ) exhibit a complex array of gut proteinase activities comprising cysteine and serine proteinases. The major cysteine proteinase activity, showing an optimum at pH 6.0, corresponds to at least 4 different proteinases. On the contrary, the minor serine proteinase activity, with an optimum at pH 9.0, seems to be due essentially to a single proteinase. The cysteine proteinase inhibitor oryzacystatin I (OC-I) inhibits completely the cysteine proteinase activity in vitro. However, larval growth and survival were not significantly different on control and transgenic oilseed rape plants expressing high levels of active OC-I. In larvae grown on transgenic plants, cysteine proteinase activity was dramatically decreased, whereas serine proteinase activity was increased by more than 2-fold, when compared to larvae raised on control plants. For both activities, no new proteinase was detected in insects fed plants expressing OC-I. These results suggest that partial compensation of the inhibition of cysteine proteinase activity by the increase in serine proteinase activity allowed the larvae to overcome the effects of OC-I consumption. This case illustrates problems that could arise when trying to achieve high levels of protection for plants against Coleopteran pests possessing a complex digestive proteinase pool.  相似文献   

11.
Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) on the predatory ladybird Harmonia axyridis were investigated using diamondback moth Plutella xylostella as the pest species. As expected, oilseed rape expressing OC-1 had no effects on either development or survival of the pest, which utilizes serine digestive proteases. Immunoassays confirmed accumulation of the transgene product in pest larval tissues at levels of up to 3 ng per gut. Characterization of proteolytic digestive enzymes of H. axyridis demonstrated that larvae and adults utilize cysteine and aspartic proteases; the former activity was completely inhibited by oryzacystatin in vitro. However, when H. axyridis larvae consumed prey reared on OC-1 expressing plants over their entire life cycle, no significant effects upon survival or overall development were observed. The inhibitor initially stimulated development, with a shortening of the developmental period of the second instar by 27% (P < 0.0001) accompanied by a 36% increase in weight of second instar larvae (P = 0.007). OC-1 had no detrimental effects on reproductive fitness of adult H. axyridis. Interestingly there was a significant increase in consumption of OC-1 dosed prey. The results show that prey reared on transgenic plants expressing a protein which inhibited ladybird digestive enzymes in vitro had no effects in vivo; the ladybird was able to up-regulate digestive proteases in response to the inhibitor.  相似文献   

12.
Different parts of genetically transformed tomato (Lycopersicon esculentum L.) plants that express the tobacco anionic peroxidase were compared for insect resistance with corresponding wild type plants. Leaf feeding by first instar Helicoverpa zea and Manduca sexta was often significantly reduced on intact transgenic plants and/or leaf disks compared to wild type plants, but the effect could depend on leaf age. Leaves of transgenic plants were generally as susceptible to feeding damage by third instar Helicoverpa zea (Boddie) and Manduca sexta (L.) as wild type plants. Green fruit was equally susceptible to third instar larvae of H. zea in both type plants, but fruit of transgenic plants were more resistant to first instar larvae as indicated by significantly greater mortality. Basal stem sections were more resistant to neonate larvae of H. zea and adults of Carpophilus lugubris Murray compared to wild type plants as indicated by significantly greater mortality and/or reduced feeding damage. Thus, tobacco anionic peroxidase activity can increase plant resistance to insects in tomato, a plant species closely related to the original source plant species, when expressed at sufficiently high levels. However, the degree of resistance is dependent on the size of insect and plant tissue involved.  相似文献   

13.
The resistance of a transgenic line of oilseed rape expressing constitutively the cysteine proteinase inhibitor oryzacystatin I (OCI) was assessed against Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae). The levels of OCI expression in the transformed line averaged 0.2% and 0.05% of total soluble protein in leaves and petioles respectively. In vitro analyses showed that P. chrysocephala larvae use both cysteine and serine proteinases for protein digestion, and that all the cysteine proteolytic activity is OCI-sensitive. However, bioassays showed that adults fed identically on leaf discs from control or transformed plants. When larvae were reared on transgenic plants expressing OCI, they showed an increase in weight gain compared to those reared on control plants. Furthermore, those larvae from transgenic plants exhibited a 2-fold increase in both cysteine and serine proteolytic activity as a reponse to the presence of OCI. The plasticity of insect digestive physiology and feeding behaviour are discussed, as well as the relevance of engineering a genotype expressing both types of proteinase inhibitors.  相似文献   

14.
The cabbage stem flea beetle (CSFB), Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae), is one of the most important pests in European winter oilseed rape production. Adult beetles feed on young leaves whereas larvae mine within the petioles and stems. Larval infestation can cause significant crop damage. In this study, the host quality for CSFB of four oilseed rape (Brassica napus L.) cultivars and seven other brassicaceous species with different glucosinolate (GSL) profiles was assessed under controlled conditions. Larval instar weights and mortality were measured after 14 and 21 days of feeding in the petioles of test plants. To study the impact of GSL on the performance of larvae, the GSL contents in petioles from non-infested and infested plants were analysed before, and 21 days after, the start of larval infestation. Larval performance was not significantly different between the four cultivars of oilseed rape, but differed considerably among the other brassicaceous species tested. In comparison to the weight of larvae in the standard B. napus cv. Robust, the larval weight was higher in turnip rape (Brassica rapa L. var. silvestris) and significantly reduced in white mustard (Sinapis alba L.), oil radish (Raphanus sativa L. var. oleiformis), and cabbage (Brassica oleracea L. convar. capitata var. alba). The duration of larval development increased in white mustard and oilseed radish. The GSL profiles of the petioles showed little difference between non-infested and infested plants of oilseed rape whereas the content of aliphatic GSL increased in the infested turnip rape plants. In contrast, the aliphatic and benzenic GSL decreased in infested Indian rape (B. rapa subsp. dichotoma Roxb.). Larval weight was not correlated with the total GSL content of plants, neither before infestation nor 21 days after. Larval weight was positively correlated with progoitrin and 4-hydroxyglucobrassicin. White mustard, which provides inferior host quality for larval development, has the potential to introduce insect resistance into high-yielding oilseed rape cultivars in breeding programmes.  相似文献   

15.
In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T0-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T2-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T2-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.  相似文献   

16.
Transgenic poplar (Populus nigra, cv. Jean Pourtet) plants were recovered as a result of Agrobacterium tumefaciens-mediated transformation performed with EHA105 pBI-KUN strain. Plasmid pBI-KUN contains a 650 bp insert derived from the soybean (Glycine max L.) KTi3, gene, coding for a Kunitz trypsin proteinase inhibitor. A total of 58 independent transgenic lines were obtained from 200 co-cultivated leaf explants. Southern blot hybridization analysis demonstrated the presence of KTi3 gene in the poplar genome. Northern blot analysis of different kanamycin-resistant plantlets confirmed the accumulation of KTi3 mRNA and revealed different levels of expression. The trypsin inhibitory activity was determined in poplar transgenic tissues by means of specific assay. Moreover, the trypsin-like digestive proteinases of the polyphagous moth Lymantria dispar (Lepidoptera, Lymantriidae) and Clostera anastomosis (Lepidoptera, Notodontidae) were detected and inhibited in vitro by Kunitz proteinase inhibitor from selected transgenic plants. Two insect bioassays were performed on P. nigra transgenic plant lines, using larvae of the above mentioned insects. In both cases larval mortality and growth as well as pupal weight were not significantly affected when the insects were fed on transgenic leaves and control leaves, respectively.  相似文献   

17.
Environmental risk assessment of transgenic crops is implemented under the Cartagena Protocol domestic law in accordance with guidelines for implementing the assessment established by the Ministry of Agriculture, Forestry and Fisheries (MAFF) and the Ministry of Environment (MOE) in Japan. Environmental risk assessments of transgenic crops are implemented based on the concept of ‘substantial equivalence’ to conventional crops. A unique requirement in Japan to monitor the production of harmful substances, or allelochemicals, is unparalleled in other countries. The potential for allelochemicals to be secreted from the roots of transgenic crops to affect other plants or soil microflora or for substances in the plant body to affect other plants after dying out must be evaluated. We evaluated the allelopathic potential of seven transgenic oilseed rape (Brassica napus L.) lines that express glufosinate tolerance in terms of substantial equivalence to conventional oilseed rape lines, and established evaluation methods. Our results indicate no potential production of allelochemicals for any of the seven transgenic oilseed rape lines compared with conventional oilseed rape lines.  相似文献   

18.
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.  相似文献   

19.
2000年7月中旬和8月中旬, 分别测定了采自田间的转CpTI-Bt基因双价抗虫棉(SGK321, 以下简称CpTI-Bt棉)和转Bt基因抗虫棉(中30,以下简称Bt棉)对棉铃虫Helicoverpa armigera幼虫存活、生长的影响。结果表明:7月中旬两种转基因抗虫棉抗虫效果均较好,尤其是CpTI-Bt棉棉叶和花瓣对4龄幼虫3天内致死率为92%以上;8月中旬两种转基因棉的抗虫活性均明显降低,且Bt棉的杀虫活性显著低于CpTI-Bt棉,其幼虫死亡率与对照受体棉中16的死亡率之间无显著差异,仅显著抑制了幼虫的生长;石远321(SGK321受体品系)的花瓣具有一定的抗虫活性,可显著降低取食幼虫的体重,甚至造成部分幼虫死亡; CpTI-Bt棉中,花瓣和棉叶的抗虫性明显高于蕾和铃心。对5龄幼虫取食棉铃1日后的营养指标测定结果显示: 两种转基因抗虫棉处理的幼虫相对生长率和相对取食量均显著低于石远321,但两者之间无显著差异; CpTI-Bt棉处理的幼虫近似消化率显著低于石远321和Bt棉,但其食物利用率显著高于石远321和Bt棉。  相似文献   

20.
A range of crops have been transformed with delta-endotoxin genes from Bacillus thuringiensis (Bt) to produce transgenic plants with high levels of resistance to lepidopteran pests. Parasitoids are important natural enemies of lepidopteran larvae and the effects of Bt plants on these non-target insects have to be investigated to avoid unnecessary disruption of biological control. This study investigated the effects of Cry1Ac-expressing transgenic oilseed rape (Brassica napus) on the solitary braconid endoparasitoid Cotesia plutellae in small-scale laboratory experiments. C. plutellae is an important natural enemy of the diamondback moth (Plutella xylostella), the most important pest of brassica crops world-wide. Bt oilseed rape caused 100% mortality of a Bt-susceptible P. xylostella strain but no mortality of the Bt-resistant P. xylostella strain NO-QA. C. plutellae eggs laid in Bt-susceptible hosts feeding on Bt leaves hatched but premature host mortality did not allow C. plutellae larvae to complete their development. In contrast, C. plutellae developed to maturity in Bt-resistant hosts fed on Bt oilseed rape leaves and there was no effect of Bt plants on percentage parasitism, time to emergence from hosts, time to adult emergence and percentage adult emergence from cocoons. Weights of female progeny after development in Bt-resistant hosts did not differ between plant types but male progeny was significantly heavier on wildtype plants in one of two experiments. The proportion of female progeny was significantly higher on Bt plants in the first experiment with Bt-resistant hosts but this effect was not observed again when the experiment was repeated.  相似文献   

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