Transgenic tobacco plants carrying a baculovirus enhancin gene slow the development and increase the mortality of Trichoplusia ni larvae

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.     

Transgenic tobacco transformed with the Trichopusia ni Granulovirus Enhancin gene affects insect development

Transgenic tobacco transformed with the Trichoplusia ni granulovirus enhancin gene has been demonstrated to enhance baculovirus infection in larvae. In this paper we describe the effect of the long-term feeding of lyophilized transgenic tobacco material to Pseudaletia separata and Spodoptera exigua larvae. Our results demonstrated that the baculovirus enhancin gene products have potential for use in insect pest management.     

粘虫颗粒体病毒增效蛋白基因片段优化及功能

【目的】研究转宿主粘虫颗粒体病毒(Pseudaletia unipuncta granulovirus,Pu GV-Ps)增效蛋白基因截短片段优化及其增效作用,探索增效蛋白基因的合理利用途径。【方法】生物信息学分析增效蛋白结构域,构建增效蛋白基因截短片段原核表达载体,分析目的基因片段表达产物的表达水平、围食膜蛋白降解效能和增强活性,进一步明确Pu GV-Ps增效蛋白基因的功能区域。【结果】Pu GV-Ps增效蛋白含有M60-like结构域、锌离子催化域和糖蛋白结合域,并包含13个潜在的糖基化位点。以此为依据设计P69(短截M60-like结构域)和P77(短截糖蛋白结合域)2个截短片段,构建了表达载体p ET15b-P69和p ET15bP77,原核表达量明显高于全长基因P104。表达产物纯化蛋白围食膜降解活性表明,P69对斜纹夜蛾围食膜大分子蛋白降解程度高于P77,但两者均低于P104。病毒增强苏云金杆菌(Bt)实验表明,截短片段的表达产物提高了Bt对小菜蛾的毒力,但增强活性显著低于P104。【结论】研究结果表明,Pu GV-Ps增效蛋白基因N端M60-like结构域和C端糖蛋白结合域对其增效作用的发挥都具有一定功能,这些结构对维持增效蛋白的构象也发挥了一定的作用,截短片段P69有利于保持Pu GV-Ps增效蛋白的活性、提高表达水平。该研究结果对增效蛋白的工业化生产具有一定的指导意义。     

Ectopically expressed leaf and bulb lectins from garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) protect transgenic tobacco plants against cotton leafworm (<Emphasis Type="Italic">Spodoptera littoralis</Emphasis>)

The insecticidal activity of the leaf (ASAL) and bulb (ASAII) agglutinins from Allium sativum L. (garlic) against the cotton leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) was studied using transgenic tobacco plants expressing the lectins under the control of the constitutive CaMV35S promoter. PCR analysis confirmed that the garlic lectin genes were integrated into the plant genome. Western blots and semi-quantitative agglutination assays revealed lectin expression at various levels in the transgenic lines. Biochemical analyses indicated that the recombinant ASAL and ASAII are indistinguishable from the native garlic lectins. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the ectopically expressed ASAL and ASAII significantly (P < 0.05) reduced the weight gain of 4th instar larvae of S. littoralis. Further on, the lectins retarded the development of the larvae and their metamorphosis, and were detrimental to the pupal stage resulting in weight reduction and lethal abnormalities. Total mortality was scored with ASAL compared to 60% mortality with ASAII. These findings suggest that garlic lectins are suitable candidate insect resistance proteins for the control of S. littoralis through a transgenic approach.     

Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests

A member of the potato proteinase inhibitor II (PPI II) gene family that encodes for a chymotrypsin iso-inhibitor has been introduced into tobacco (Nicotiana tabacum) usingAgrobacterium tumefaciens-mediated T-DNA transfer. Analysis of the primary transgenic plants (designated R₀) confirmed that the introduced gene is being expressed and the inhibitor accumulates as an intact and fully functional protein. For insect feeding trials, progeny from the self-fertilization of R₀ plants (designated R₁) were used. Leaf tissue, either from transgenic or from control (non-transgenic) plants, was fed to larvae ofChrysodeixis eriosoma (Lepidoptera: Noctuidae, green looper),Spodoptera litura (F.) (Lepidoptera: Noctuidae) andThysanoplusia orichalcea (F.) (Lepidoptera: Noctuidae) and insect weight gain (increase in fresh weight) measured. Consistently,C. eriosoma larvae fed leaf tissue from transgenic plants expressing thePPI II gene grew slower than insects fed leaf tissue from non-transgenic plants or transgenic plants with no detectablePPI II protein accumulation. However, larvae of bothS. litura andT. orichalcea consistently demonstrated similar or faster growth when fed leaf tissue from transgenic plants compared with those fed non-transgenic plants. In agreement with the feeding trials, the chymotrypsin iso-inhibitor extracted from transgenic tobacco effectively retarded chymotrypsin-like activity measured inC. eriosoma digestive tract extracts, but not in extracts fromS. litura. We conclude, therefore, that for certain insects the use of chymotrypsin inhibitors should now be evaluated as an effective strategy to provide field resistance against insect pests in transgenic plants, but further, that a single proteinase inhibitor gene may not be universally effective against a range of insect pests. The significance of these observations is discussed with respect to the inclusion of chymotrypsin inhibitors in the composite of insect pest resistance factors that have been proposed for introduction into crop plants.     

Minor modifications to the cry1Ac9 nucleotide sequence are sufficient to generate transgenic plants resistant to Phthorimaea operculella

Minor modifications were made sequentially to the nucleotide sequence of truncated cry1Ac9 to produce cry1Ac9^A (one nucleotide change) and then cry1Ac9^B (seven nucleotide changes). The derivative genes under the control of the CaMV 35S promoter were transformed into Nicotiana tabacum in order to determine whether these modified genes conferred resistance on the resulting transgenic tobacco plants to larvae of the potato tuber moth (Phthorimaea operculella). Over two trials with PTM larvae on the transgenic plants expressing the cry 1Ac9^b gene, lower larval growth, development and survival was evident for most of the lines compared to the control plants. In the second trial, for four of these lines (7, 25, 26 and 28) larval growth rates were very low (0.28, 0.3, 0.42 and 0.28, respectively) compared to the control growth rate (4.18) and leaf damage was minimal. Northern analysis and RT‐PCR analysis showed that higher levels of cry 1Ac9 mRNA were present in the transgenic tobacco lines containing cry 1Ac9^b than in the tobacco lines containing cry 1Ac9^a. These results suggest that certain minor modifications to the nucleotide sequence of cry 1 Ac9 are sufficient to improve the stability of its mRNA when expressed in tobacco and that this increase in steady state mRNA is sufficient to confer significant resistance to PTM larvae.     

Pea Lectin Expressed Transgenically in Oilseed Rape Reduces Growth Rate of Pollen Beetle Larvae

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.     

Overexpression of glutamate decarboxylase in transgenic tobacco plants confers resistance to the northern root-knot nematode

Previous research suggests that the endogenous synthesis of gamma-aminobutyrate (GABA), a naturally occurring inhibitory neurotransmitter, serves as a plant defense mechanism against invertebrate pests. Here, we tested the hypothesis that elevated GABA levels in engineered tobacco confer resistance to the northern root nematode (Meloidogyne hapla). This nematode species was chosen because of its sedentary nature and economic importance in Canada. We derived nine phenotypically normal, homozygous lines of transgenic tobacco (Nicotiana tabacum L.), which contain one or two copies of a full-length, chimeric tobacco glutamate decarboxylase (GAD) cDNA or a mutant version that lacks the autoinhibitory calmodulin-binding domain, under the control of a chimeric octopine synthase/mannopine synthase promoter. Regardless of experimental protocol, uninfected transgenic lines consistently contained higher GABA concentrations than wild-type controls. Growth chamber trials revealed that 9–12 weeks after inoculation of tobacco transplants with the northern root-knot nematode, mature plants of five lines possessed significantly fewer egg masses on the root surface when the data were expressed on both root and root fresh weight bases. Therefore, it can be concluded that constitutive transgenic expression of GAD conferred resistance against the root-knot nematode in phenotypically normal tobacco plants, probably via a GABA-based mechanism.     

Soybean Kunitz, C-II and PI-IV inhibitor genes confer different levels of insect resistance to tobacco and potato transgenic plants

In modern, highly intensive agriculture, the control of insect pests is basically achieved with the application of chemical pesticides. Heavy reliance on this sole strategy is associated with several drawbacks, and the development of alternative or complementary methods to chemical control is desirable. In this work, three soybean genes (KTi ₃ , C-II and PI-IV)coding for serine proteinase inhibitors were isolated by PCR and transferred to Agrobacterium tumefaciens EHA 105, which in turn was used for transforming tobacco leaf and potato tuber discs. Biochemical assays confirmed that transgenic plants synthesized serine proteinase inhibitors; rates of expression varied among plants. The level of insect resistance (tested with Spodoptera littoralis Boisduval) was particularly high in tobacco, where many plants caused the death of all larvae. In potatoes, larval mortality was much less frequently achieved, but the results were still encouraging in that larval weight gain was reduced by 50% in the presence of adequate amounts of inhibitor. When 8-day-old larvae were fed different KTi ₃ -expressing tobacco plants, a highly significant (P<0.01) correlation was observed between inhibitor content and larval live weight. Larval weight gain was found to be dependent on midgut proteolytic activity. On the basis of the evidence collected, it is suggested that further work is required to identify more specific inhibitors for the main proteinases of the target insect. Received: 30 March 1998 / Accepted: 9 December 1999     

Biology and life table studies of the oriental tobacco budworm,Helicoverpa assulta (Lepidoptera: Noctuidae), influenced by different larval diets

Development, survivorship, pupal weight, oviposition, and life table parameters of the oriental tobacco budworm, Helicoverpa assulta Guenée, were evaluated in the laboratory on an artificial diet, pepper (Capsicum frutescens L.), and tobacco (Nicotiana tobacum L.). We found that the average developmental time of immature stages was longest on tobacco (36.2 d), intermediate on pepper (34.4 d), and shortest on artificial diet (33.5 d). Immature survival from egg to pupa varied from 31% on tobacco, 43% on pepper, and 74% on artificial diet. Pupal weight ranged from 197.4 mg/pupa on tobacco, 233.1 mg/pupa on pepper and 253.4 mg/pupa on artificial diet. The average numbers of eggs laid by adults reared as larvae on the artificial diet, pepper, or tobacco were 614, 421 and 334 eggs/female, respectively. Numbers of remaining eggs in ovaries of the adult females reared as larvae on the artificial diet, pepper, or tobacco were 16, 26, and 42 eggs/female, respectively. The longevity of adult females developed from larvae reared on the three diets was not significantly different, whereas the longevity of male adults from the larvae reared on artificial diet was longer (16.8 d) than that for males reared on tobacco (13.8 d) and pepper (13.3 d). The intrinsic, finite, gross, and net rates of increase were highest for females reared as larvae on artificial diet, lowest for females emerging from larvae reared on tobacco, and intermediate for females emerging from larvae reared on pepper. Generation times and doubling time of H. assulta were shortest for larvae fed artificial diet, intermediate from larvae reared on pepper, and longest from larvae reared on tobacco. We concluded that the artificial diet was the most suitable larval diet of H. assulta followed by pepper, and tobacco.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

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.     

Development of a novel‐type transgenic cotton plant for control of cotton bollworm

The transgenic Bt cotton plant has been widely planted throughout the world for the control of cotton budworm Helicoverpa armigera (Hubner). However, a shift towards insect tolerance of Bt cotton is now apparent. In this study, the gene encoding neuropeptide F (NPF) was cloned from cotton budworm H. armigera, an important agricultural pest. The npf gene produces two splicing mRNA variants—npf1 and npf2 (with a 120‐bp segment inserted into the npf1 sequence). These are predicted to form the mature NPF1 and NPF2 peptides, and they were found to regulate feeding behaviour. Knock down of larval npf with dsNPF in vitro resulted in decreases of food consumption and body weight, and dsNPF also caused a decrease of glycogen and an increase of trehalose. Moreover, we produced transgenic tobacco plants transiently expressing dsNPF and transgenic cotton plants with stably expressed dsNPF. Results showed that H. armigera larvae fed on these transgenic plants or leaves had lower food consumption, body size and body weight compared to controls. These results indicate that NPF is important in the control of feeding of H. armigera and valuable for production of potential transgenic cotton.     

Cadmium resistance in tobacco plants expressing the MuSI gene

MuSI, a gene that corresponds to a domain that contains the rubber elongation factor (REF), is highly homologous to many stress-related proteins in plants. Since MuSI is up-regulated in the roots of plants treated with cadmium or copper, the involvement of MuSI in cadmium tolerance was investigated in this study. Escherichia coli cells overexpressing MuSI were more resistant to Cd than wild-type cells transfected with vector alone. MuSI transgenic plants were also more resistant to Cd. MuSI transgenic tobacco plants absorbed less Cd than wild-type plants. Cd translocation from roots to shoots was reduced in the transgenic plants, thereby avoiding Cd toxicity. The number of short trichomes in the leaves of wild-type tobacco plants was increased by Cd treatment, while this was unchanged in MuSI transgenic tobacco. These results suggest that MuSI transgenic tobacco plants have enhanced tolerance to Cd via reduced Cd uptake and/or increased Cd immobilization in the roots, resulting in less Cd translocation to the shoots.     

The expression in tobacco plants of Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF) alters growth and development of the tobacco budworm,Heliothis virescens

The production and characterisation of transgenic tobacco plantsexpressing a precursor of a regulatory peptide from Aedesaegypti (Trypsin Modulating and Oostatic Factor, Aea-TMOF) whichinterferes with the development of tobacco budworm larvae is described. Tobaccoplants were transformed with a synthetic gene containing 6 TMOF units spaced bydibasic residues, Arg-Arg, as potential post-translational cleavage sites.Peptide extracts from transgenic plants had TMOF activity and inhibitedin vitro the biosynthesis of serine proteases. Thisactivity was consistently present in T1 plants and absent in control plants.Tobacco budworm larvae, fed with transgenic leaves showed a reduced growth ratecompared to those fed with control plants. The low rather than acute toxicityofthis low impact gene is discussed in the context of alternative integrated pestmanagement strategies.     

Influence of transgenic cottons with <Emphasis Type="Italic">Bacillus thuringiensis cry1Ac</Emphasis> gene on the natural enemies of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Transgenic cotton has been released for cultivation in several parts of the world to increase crop productivity. However, concerns have been raised regarding the possible undesirable effects of genetically modified crops on non-target organisms in the eco-system. Therefore, we studied the effects of transgenic cottons with cry1Ac gene from Bacillus thuringiensis Berliner (Bt) on the natural enemies of cotton bollworm/legume pod borer, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) under field and laboratory conditions. There was no apparent effect of transgenic cotton on the relative abundance of predatory spiders (Clubiona sp. and Neoscona sp.), coccinellid (Cheilomenes sexmaculatus Fab.), and the chrysopid (Chrysoperla carnea Stephens). However, the abundance of spiders, coccinellids, and chrysopids was quite low in insecticide protected plots towards end of the cropping season. There was a significant reduction in cocoon formation and adult emergence of the ichneumonid parasitoid, Campoletis chlorideae Uchnida reared on H. armigera larvae fed on the leaves of transgenic cottons before and after parasitization. However, no Bt toxins were detected in H. armigera larvae and the parasitoid cocoons with enzyme linked immunosorbent assay. Reduction in cocoon formation was because of early mortality of the H. armigera larvae due to Bt toxins in the leaves of transgenic cotton. There was a slight reduction in adult weight and fecundity, and prolongation of the larval period when the parasitoid was raised on H. armigera larvae fed on the leaves of transgenic cotton before and after parasitization. Survival and development of C. chlorideae was also poor when H. armigera larvae were fed on the leaves of cotton hybrid Mech 184. The adverse effects of transgenic cotton on survival and development of C. chlorideae were largely due to early mortality, and possibly poor nutritional quality of H. armigera larvae due to toxic effects of the transgene.     

EFFECTS OF BT RICE ON THE FOOD CONSUMPTION,GROWTH AND SURVIVAL OF CHILO SUPPRESSAUS LARVAE UNDER DIFFERENT TEMPERATURES*

Abstract The Bt transgenic rice line, i.e. KMD1 containing a synthetic cry 1 Ab gene from Bacillus thuringiensis Berliner was tested in the laboratory for evaluating its effects on larval food consumption, growth and survival of striped stem borer (SSB), Chilo suppressalis (Walker) under different temperatures. The food consumption and body weight growth as well as the survival of both 3rd and 5th instar larvae fed on Bt rice were significantly reduced, as compared with those on the control. The temperature showed no marked effect on both food consumption and body weight growth of larvae fed on Bt rice stems since the onset of 3rd instar, conversely had significant effects as since the initial stage of 5th instar. In contrast, the temperature did not distinctly influence the survival of both 3rd and 5th instar larvae. There was a linearly positive relationship between the corrected mortality of larvae and the accumulative amount of Bt rice tissues ingested by larvae.     

转双基因烟草对棉铃虫的杀虫活性评价

以含Ｂｔ杀虫蛋白基因（单基因）烟草和常规烟草为对照,系统测定了含Ｂｔ与豇豆胰蛋白酶抑制剂蛋白基因（双基因）的抗虫烟草对棉铃虫不同龄期幼虫的杀虫活性。结果表明：１￣３龄幼虫取食转双基因烟草３ｄ后死亡率为８０．５％￣９９．３％,取食６ｄ后死亡率达１００％,均显著高于转单基因烟草。２龄幼虫取食转基因烟草３ｄ后死亡率为８０．５％￣９９．３％,取食６ｄ后死亡率达１００％,均显著高于转单基因烟草。２龄幼虫取食     

Assessing the utilization of a carbohydrate food source and the impact of insecticidal proteins on larvae of the green lacewing, Chrysoperla carnea

A concern with the widespread use of insecticidal transgenic crops is their potential to adversely affect non-target organisms, including biological control agents such as larvae of the green lacewing, Chrysoperla carnea (Neuroptera: Chrysopidae). Since the insecticidal proteins expressed by the current transgenic plants are active only after ingestion, dietary bioassays are required to test direct effects on non-target organisms. After showing that C. carnea larvae utilize carbohydrate foods, we exposed them to insecticidal proteins dissolved in a sucrose solution. Feeding on snowdrop lectin (Galanthus nivalis agglutinin, GNA) as a model compound, the larvae were negatively affected in a number of life-table parameters. Interestingly, GNA caused a prolongation in first instar development, but had no effect on subsequent utilization of prey resulting in an increased weight of second instars. Comparable studies with avidin, a biotin-binding protein, revealed strong effects on C. carnea survival at the concentration tested. Despite the fact that the proteolytic digestion of C. carnea larvae is reported to be dominated by serine proteases, ingestion of soybean trypsin inhibitor (SBTI) did not cause any detrimental effects. Similarly, two Cry proteins derived from Bacillus thuringiensis (Cry1Ac and Cry1Ab) did not cause negative effects on C. carnea, what is consistent with earlier studies. The here presented bioassay provides a valuable tool to assess direct impacts of insecticidal proteins to C. carnea larvae and other predators that are known to feed on carbohydrate solutions.