Bt水稻田重要非靶标节肢动物暴露于Cry2Aa蛋白的程度分析

根据风险=危险×暴露的原理,在实验室条件下评价转基因作物对非靶标节肢动物影响时,所选择的代表性非靶标生物通常是在农田系统中较高地暴露于转基因外源杀虫蛋白的节肢动物种.为了弄清Bt稻田主要节肢动物暴露于Cry蛋白的程度,选择合适的非靶标节肢动物,用于转基因抗虫水稻的风险评价,本文采用酶联免疫技术检测了水稻不同生长期从转cry2Aa基因水稻田采集的不同节肢动物体内Cry2Aa蛋白的含量.结果表明: 不同节肢动物种体内的Cry蛋白含量差异显著.一些节肢动物体内不含Cry蛋白,而一些节肢动物体内含有较高的Cry蛋白;相对于花期后采集的节肢动物,在Bt水稻花期采集的节肢动物,特别是捕食性节肢动物体内的Cry蛋白含量较高;寄生性节肢动物体内未检测到Cry蛋白.这为在实验室条件下评价转基因水稻对农田非靶标节肢动物的影响奠定了基础.     

Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) using T-DNA probes was applied to localize transgenes onto specific chromosomes and confirm the steady integration of transferred genes in three genetically modified (GM) rice lines, LS28 (event LS30-32-20-1), Cry1Ac1 (event C7-1-9-1) and LS28×Cry1Ac1 (event L/C1-1-3-1), which are a rice leaf blast-resistant single trait GM line, a leaf folder-resistant single trait GM line, and a rice leaf blast-resistant and leaf folder-resistant stacked GM hybrid line, respectively. The FISH signals were clearly detected on the arms of one homologous chromosome pair for LS28, and on the arms of another chromosome pair for Cry1Ac1 when using the transformation vector pSBM AtCK containing the rice leaf blast-resistant gene (LS28) and pMJ-RTB containing the leaf folder-resistant gene (mCry1Ac1) as a probe, respectively. As expected, we detected two pairs of FISH signals, each on the arms of different chromosome pairs in the stacked GM rice line LS28×Cry1Ac1 when using both pSBM AtCK and pMJ-RTB as probes. These results indicate that the transgenes are located at specific homologous loci and show position stability among generations in both single trait and stacked GM rice lines. The usefulness and the necessity of FISH to detect inserted genes in transformed plants will be discussed for the purpose of future studies to develop breeding programs and conduct risk assessment of GM plants.     

Molecular characterization of lepidopteran pest-resistant transgenic rice events expressing synthetic Cry1Ac

The insecticidal toxin gene of Bacillus thuringiensis (Bt) is one of the most commonly used in the development of genetically modified (GM) crops. In this research, we analyzed Bt rice showing lepidopteran pest-resistance. The Bt gene is a synthetic Cry1Ac composed of optimal codons for plants, and the Bt protein is targeted to the chloroplast by a transit peptide. Three Cry1Ac rice events (C103-3, C127-1, and C7-1) were analyzed for molecular characterization. C103-3 contains two copies of T-DNA where the left border (LB) region is truncated. Both C7-1 and C127-1 have a single copy of T-DNA, but a part of the vector backbone DNA is inserted into the genome of C127-1; thus, only C7-1 had intact T-DNA. Progenies of C7-1 crossed with the original cultivar, Nakdong, and double-haploid lines from anther culture of lines crossed with the elite cultivar, Dongjin, were analyzed for T-DNA flanking genomic DNA and genotyping. Results showed that an intact T-DNA region without the vector backbone was inserted into the genome and was stably inherited through generations. The C7-1 homozygous event could be used as breeding material to develop GM rice with pest resistance.     

The distinct properties of natural and GM cry insecticidal proteins

The Cry toxins are a family of crystal-forming proteins produced by the bacterium Bacillus thuringiensis. Their mode of action is thought to be to create pores that disrupt the gut epithelial membranes of juvenile insects. These pores allow pathogen entry into the hemocoel, thereby killing the insect. Genes encoding a spectrum of Cry toxins, including Cry mutants, Cry chimaeras and other Cry derivatives, are used commercially to enhance insect resistance in genetically modified (GM) crops. In most countries of the world, such GM crops are regulated and must be assessed for human and environmental safety. However, such risk assessments often do not test the GM crop or its tissues directly. Instead, assessments rely primarily on historical information from naturally occurring Cry proteins and on data collected on Cry proteins (called ‘surrogates’) purified from laboratory strains of bacteria engineered to express Cry protein. However, neither surrogates nor naturally occurring Cry proteins are identical to the proteins to which humans or other nontarget organisms are exposed by the production and consumption of GM plants. To-date there has been no systematic survey of these differences. This review fills this knowledge gap with respect to the most commonly grown GM Cry-containing crops approved for international use. Having described the specific differences between natural, surrogate and GM Cry proteins this review assesses these differences for their potential to undermine the reliability of risk assessments. Lastly, we make specific recommendations for improving risk assessments.     

Whole Genome Sequencing of Elite Rice Cultivars as a Comprehensive Information Resource for Marker Assisted Selection

Current advances in sequencing technologies and bioinformatics revealed the genomic background of rice, a staple food for the poor people, and provided the basis to develop large genomic variation databases for thousands of cultivars. Proper analysis of this massive resource is expected to give novel insights into the structure, function, and evolution of the rice genome, and to aid the development of rice varieties through marker assisted selection or genomic selection. In this work we present sequencing and bioinformatics analyses of 104 rice varieties belonging to the major subspecies of Oryza sativa. We identified repetitive elements and recurrent copy number variation covering about 200 Mbp of the rice genome. Genotyping of over 18 million polymorphic locations within O. sativa allowed us to reconstruct the individual haplotype patterns shaping the genomic background of elite varieties used by farmers throughout the Americas. Based on a reconstruction of the alleles for the gene GBSSI, we could identify novel genetic markers for selection of varieties with high amylose content. We expect that both the analysis methods and the genomic information described here would be of great use for the rice research community and for other groups carrying on similar sequencing efforts in other crops.     

Assessment of transgene flow in tomato and potential effects of genetically modified tomato expressing Cry3Bb1 toxins on bumblebee feeding behaviour

One of the concerns surrounding the commercial release of genetically modified (GM) crops is the escape of transgenes into agricultural or semi‐natural habitats through vertical gene flow, as this may cause environmental or economic problems. There is also the concern that GM crops may affect pollinators and the pollination services they provide. Despite the growing commercial interest of GM tomato (Solanum lycopersicum), gene flow has been assessed only sparsely in tomato. To evaluate the likelihood of gene flow from GM tomato plants to sexually compatible plants, and to assess whether bumblebee activity is affected by GM tomato, three experiments were conducted under greenhouse conditions, using a Bt‐tomato expressing the insecticidal Cry3Bb1 protein as model system: (a) artificial crosses between a GM tomato line, two wild tomato relatives (Solanum hirsutum and Solanum nigrum) and a non‐GM tomato variety; (b) bumblebee‐mediated crosses between GM and non‐GM tomato plants and (c) visual observations of bumblebees' feeding behaviour. No hybrids were obtained between the GM tomato line and S. hirsutum and S. nigrum. In an experimental design where non‐GM receptor plants outnumbered GM plants by approximately 3:1, the bumblebee‐mediated cross‐fertilisation rate between GM and non‐GM tomato plants was measured at 4.3 ± 5.47%. No significant differences in feeding behaviour of bumblebees foraging on GM and non‐GM tomato plants were observed. Therefore, we conclude that: (a) the probability of transgene introgression between the GM tomato line used in this study and its wild relatives S. hirsutum and S. nigrum is negligible; (b) bumblebee activity can mediate cross‐fertilisation between GM and non‐GM tomato and (3) the Cry3Bb1‐expressing tomato line tested does not adversely affect the feeding behaviour of bumblebees.     

The microRNA-7322-5p/p38/Hsp19 axis modulates Chilo suppressalis cell-defences against Cry1Ca: an effective target for a stacked transgenic rice approach

Bacillus thuringiensis (Bt)-secreted crystal (Cry) toxins form oligomeric pores in host cell membranes and are a common element in generating insect-resistant transgenic crops. Although Cry toxin function has been well documented, cellular defences against pore-formation have not been as well developed. Elucidation of the processes underlying this defence, however, could contribute to the development of enhanced Bt crops. Here, we demonstrate that Cry1Ca-mediated downregulation of microRNA-7322-5p (miR-7322-5p), which binds to the 3′ untranslated region of p38, negatively regulates the susceptibility of Chilo suppressalis to Cry1Ca. Moreover, Cry1Ca exposure enhanced phosphorylation of Hsp19, and hsp19 downregulation increased susceptibility to Cry1Ca. Further, Hsp19 phosphorylation occurs downstream of p38, and pull-down assays confirmed the interactions between Hsp19 and Cry1Ca, suggesting that activation of Hsp19 by the miR-7322-5p/p38/Hsp19 pathway promotes Cry1Ca sequestration. To assess the efficacy of targeting this pathway in planta, double-stranded RNA (dsRNA) targeting C. suppressalis p38 (dsp38) was introduced into a previously generated cry1Ca-expressing rice line (1CH1-2) to yield a single-copy cry1Ca/dsp38 rice line (p38-rice). Feeding on this rice line triggered a significant reduction in C. suppressalis p38 expression and the line was more resistant to C. suppressalis than 1CH1-2 in both short term (7-day) and continuous feeding bioassays as well as field trials. These findings provide new insights into invertebrate epithelium cellular defences and demonstrate a potential new pyramiding strategy for Bt crops.     

Concentrated Protein Body Product Derived from Rice Endosperm as an Oral Tolerogen for Allergen-Specific Immunotherapy—A New Mucosal Vaccine Formulation against Japanese Cedar Pollen Allergy

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.     

A built-in mechanism to mitigate the spread of insect-resistance and herbicide-tolerance transgenes into weedy rice populations

Background

The major challenge of cultivating genetically modified (GM) rice (Oryza sativa) at the commercial scale is to prevent the spread of transgenes from GM cultivated rice to its coexisting weedy rice (O. sativa f. spontanea). The strategic development of GM rice with a built-in control mechanism can mitigate transgene spread in weedy rice populations.

Methodology/Principal Findings

An RNAi cassette suppressing the expression of the bentazon detoxifying enzyme CYP81A6 was constructed into the T-DNA which contained two tightly linked transgenes expressing the Bt insecticidal protein Cry1Ab and the glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), respectively. GM rice plants developed from this T-DNA were resistant to lepidopteran pests and tolerant to glyphosate, but sensitive to bentazon. The application of bentazon of 2000 mg/L at the rate of 40 mL/m², which is approximately the recommended dose for the field application to control common rice weeds, killed all F₂ plants containing the transgenes generated from the Crop-weed hybrids between a GM rice line (CGH-13) and two weedy rice strains (PI-63 and PI-1401).

Conclusions/Significance

Weedy rice plants containing transgenes from GM rice through gene flow can be selectively killed by the spray of bentazon when a non-GM rice variety is cultivated alternately in a few-year interval. The built-in control mechanism in combination of cropping management is likely to mitigate the spread of transgenes into weedy rice populations.     

转Bt水稻土壤跳虫群落组成及其数量变化

以转Bt水稻恢复系"克螟稻"(Cry1Ab纯合基因型)和"华恢1号"(Cry1Ab+Cry1Ac融合基因型)以及融合基因型转Bt水稻杂交系"Bt汕忧63",及其对照亲本水稻"秀水11"、"明恢63"和"汕优63"稻田土壤跳虫类群为对象,系统研究转Bt水稻种植下土壤跳虫群落组成及其数量动态变化,以评价不同基因型和不同育种品系转Bt水稻种植下稻田土壤生态安全性。结果表明,转Bt水稻种植导致土壤跳虫个别稀有类群的消失,并显著影响半土生和真土生类群以及土壤跳虫总量,但对群落多样性、均匀度和种类丰富度等影响不显著。与对照亲本相比,Cry1Ab转Bt稻田半土生类群和土壤跳虫总量及其种类丰富度指标显著增加了54.7%、44.4%和26.7%;Cry1Ab+Cry1Ac转Bt杂交稻田球角跳属百分比和真土生跳虫数量显著增加了212.3%和180.4%。就恢复系处理而言,与Cry1Ab转Bt水稻相比,Cry1Ab+Cry1Ac转Bt水稻种植导致棘跳属、球角跳属和原等跳属百分比以及半土生跳虫数量分别显著降低了62.1%、56.7%、61.8%和43.4%,同时,显著提高了裔符跳属百分比达88.2%。就Cry1Ab+Cry1Ac融合基因型转Bt水稻而言,与恢复系相比,转Bt杂交稻种植导致球角跳属和原等跳属百分比,半土生类群和土壤跳虫总量及其种类丰富度和群落多样性显著增加了312.9%和171.6%,302.4%和233.2%,以及54.0%和26.7%,同时,显著降低了裔符跳属百分比达65.5%。     

Multi-generation effects of Bt rice on Anagrus nilaparvatae, a parasitoid of the nontarget pest Nilapavarta lugens

Little is known about the potential cumulative long-term effects of transgenic crops on nontarget organisms. In the present laboratory study, the potential cumulative effects of transgenic Bacillus thuringiensis (Bt) rice on parasitoids in successive generations were observed for an egg parasitoid, Anagrus nilaparvatae parasitizing eggs of Nilaparvata lugens (St?l) (Hemiptera: Delphacidae) feeding on Bt rice. Enzyme-linked immunosorbent assay test confirmed that Cry1Ab insecticidal protein could be detected in newly eclosed parasitoid adults. However, no significant effect on the fecundity of Anagrus nilaparvatae Pang et Wang (Hymenoptera: Mymaridae) was observed between Bt and non-Bt rice. Developmental times of both genders of A. nilaparvatae parasitizing host eggs laid in Bt (KMD1 and KMD2) rice lines were significantly prolonged from first generation to second generation, but not always prolonged from third generation to 11th generation as compared with the control rice line. Furthermore, the sex ratio of A. nilaparvatae progeny from the first generation to 11th generation in three rice lines was not significantly different. In general, our results suggested that the effect of Bt rice on this parasitoid could be negligible.     

Normal expression of insect-resistant transgene in progeny of common wild rice crossed with genetically modified rice: its implication in ecological biosafety assessment

Transgene outflow from genetically modified (GM) rice to its wild relatives may cause undesirable ecological consequences. Understanding the level of transgene expression in wild rice following gene flow is important for assessing such consequences, providing that transgene escape from GM rice cannot be prevented. To determine the expression of a transgene in common wild rice (Oryza rufipogon), we analyzed the content of Cry1Ac protein in three GM rice lines containing a Bt transgene, their F₁ hybrids with common wild rice and F₂ progeny at different growth stages, using the sandwich enzyme-linked immunosorbent assay. The average content of Cry1Ac protein in leaf samples of the wild rice lines ranged between 0.016 and 0.069% during the entire growth period, whereas that in stems varied between 0.12 and 0.39%. A great variation in Cry1Ac protein content was detected among individuals of F₁ hybrids and F₂ progeny, with some wild individuals showing higher level of Bt toxin than the cultivated GM rice. The results suggest that the Bt transgene can express normally in the interspecific hybrids between insect-resistant GM rice and common wild rice, and may have similar effects on the target insects as in GM rice.     

Host-plant mediated effects of transgenic maize on the insect parasitoid Campoletis sonorensis (Hymenoptera: Ichneumonidae)

Determining the impact of genetically modified (GM) crops on beneficial organisms is an important aspect of the environmental risk assessment of GM crops. In the present study, the impact of Bt maize expressing Cry1Ab on the development and behaviour of the parasitoid Campoletis sonorensis was compared to individuals reared on hosts fed conventionally bred plants partially resistant to the European corn borer (Ostrinia nubilalis Hübner) and on susceptible maize hybrids. Adult parasitoids reared on Bt maize-fed Spodoptera frugiperda larvae were significantly smaller (15–30%) than those reared in hosts fed either of the conventional maize hybrids. The magnitude of this effect was dependent on the size of the host at oviposition and its subsequent growth rate. The development time of C. sonorensis was not affected by the maize treatment. In choice tests, female parasitoids displayed no preference for hosts fed a specific maize hybrid. No Cry1Ab was detected within adult parasitoids.     

Lack of Detectable Allergenicity in Genetically Modified Maize Containing “Cry” Proteins as Compared to Native Maize Based on In Silico & In Vitro Analysis

BackgroundGenetically modified, (GM) crops with potential allergens must be evaluated for safety and endogenous IgE binding pattern compared to native variety, prior to market release.ObjectiveTo compare endogenous IgE binding proteins of three GM maize seeds containing Cry 1Ab,1Ac,1C transgenic proteins with non GM maize.MethodsAn integrated approach of in silico & in vitro methods was employed. Cry proteins were tested for presence of allergen sequence by FASTA in allergen databases. Biochemical assays for maize extracts were performed. Specific IgE (sIgE) and Immunoblot using food sensitized patients sera (n = 39) to non GM and GM maize antigens was performed.ResultsIn silico approaches, confirmed for non sequence similarity of stated transgenic proteins in allergen databases. An insignificant (p> 0.05) variation in protein content between GM and non GM maize was observed. Simulated Gastric Fluid (SGF) revealed reduced number of stable protein fractions in GM then non GM maize which might be due to shift of constituent protein expression. Specific IgE values from patients showed insignificant difference in non GM and GM maize extracts. Five maize sensitized cases, recognized same 7 protein fractions of 88-28 kD as IgE bindng in both GM and non-GM maize, signifying absence of variation. Four of the reported IgE binding proteins were also found to be stable by SGF.ConclusionCry proteins did not indicate any significant similarity of >35% in allergen databases. Immunoassays also did not identify appreciable differences in endogenous IgE binding in GM and non GM maize.     

Binding Site Concentration Explains the Differential Susceptibility of Chilo suppressalis and Sesamia inferens to Cry1A-Producing Rice

Chilo suppressalis and Sesamia inferens are two important lepidopteran rice pests that occur concurrently during outbreaks in paddy fields in the main rice-growing areas of China. Previous and current field tests demonstrate that the transgenic rice line Huahui 1 (HH1) producing a Cry1Ab-Cry1Ac hybrid toxin from the bacterium Bacillus thuringiensis reduces egg and larval densities of C. suppressalis but not of S. inferens. This differential susceptibility to HH1 rice correlates with the reduced susceptibility to Cry1Ab and Cry1Ac toxins in S. inferens larvae compared to C. suppressalis larvae. The goal of this study was to identify the mechanism responsible for this differential susceptibility. In saturation binding assays, both Cry1Ab and Cry1Ac toxins bound with high affinity and in a saturable manner to midgut brush border membrane vesicles (BBMV) from C. suppressalis and S. inferens larvae. While binding affinities were similar, a dramatically lower concentration of Cry1A toxin binding sites was detected for S. inferens BBMV than for C. suppressalis BBMV. In contrast, no significant differences between species were detected for Cry1Ca toxin binding to BBMV. Ligand blotting detected BBMV proteins binding Cry1Ac or Cry1Ca toxins, some of them unique to C. suppressalis or S. inferens. These data support that reduced Cry1A binding site concentration is associated with a lower susceptibility to Cry1A toxins and HH1 rice in S. inferens larvae than in C. suppressalis larvae. Moreover, our data support Cry1Ca as a candidate for pyramiding efforts with Cry1A-producing rice to extend the activity range and durability of this technology against rice stem borers.     

Outcrossing potential between 11 important genetically modified crops and the Chilean vascular flora

The potential impact of genetically modified (GM) crops on biodiversity is one of the main concerns in an environmental risk assessment (ERA). The likelihood of outcrossing and pollen‐mediated gene flow from GM crops and non‐GM crops are explained by the same principles and depend primarily on the biology of the species. We conducted a national‐scale study of the likelihood of outcrossing between 11 GM crops and vascular plants in Chile by use of a systematized database that included cultivated, introduced and native plant species in Chile. The database included geographical distributions and key biological and agronomical characteristics for 3505 introduced, 4993 native and 257 cultivated (of which 11 were native and 246 were introduced) plant species. Out of the considered GM crops (cotton, soya bean, maize, grape, wheat, rice, sugar beet, alfalfa, canola, tomato and potato), only potato and tomato presented native relatives (66 species total). Introduced relative species showed that three GM groups were formed having: a) up to one introduced relative (cotton and soya bean), b) up to two (rice, grape, maize and wheat) and c) from two to seven (sugar beet, alfalfa, canola, tomato and potato). In particular, GM crops presenting introduced noncultivated relative species were canola (1 relative species), alfalfa (up to 4), rice (1), tomato (up to 2) and potato (up to 2). The outcrossing potential between species [OP; scaled from ‘very low’ (1) to ‘very high’ (5)] was developed, showing medium OPs (3) for GM–native relative interactions when they occurred, low (2) for GMs and introduced noncultivated and high (4) for the grape‐Vitis vinifera GM–introduced cultivated interaction. This analytical tool might be useful for future ERA for unconfined GM crop release in Chile.     

Size distribution of allergenic Cry j 2 released from airborne <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> pollen grains during the pollen scattering seasons

Japanese cedar (Cryptomeria japonica) pollinosis is one of seasonal allergic rhinitis that mainly occurs in Japan. The pollinosis is caused by two main kinds of allergenic proteins called Cry j 1 and Cry j 2 which exist in Cryptomeria japonica pollen. In our previous study, we reported that the size-segregated of airborne fine allergenic Cry j 1 and morphological change of Cry j 1 due to the contact with rainfall. However, the study on airborne allergenic Cry j 2 in different particle sizes has not been identified until now. Therefore, the main aim of this study is to investigate the size distribution and scattering behavior of allergenic Cry j 2. The Cry j 2 particles were collected and determined in different particle sizes at the urban sampling points during the most severe pollination season of 2012 in Saitama, Japan. After the size-segregated Cry j 2 allergenic particles were collected using an Andersen high-volume (AHV) atmospheric sample, the airborne Cry j 2 concentrations were determined with a surface plasmon resonance (SPR) method. At the same time, the airborne Cryptomeria japonica pollens were also counted by the Durham pollen sampler. The higher concentrations of the allergenic Cry j 2 were detected even in particle sizes equal to or less than 1.1 μm (PM_1.1) than other particle sizes. The airborne particles ranges from 0.06 to 11 μm were also collected by a low-pressure impactor (LPI) atmospheric sampler. After that, the concentrations of Cry j 2 allergenic particles in fine particle sizes were measured by the SPR method either. With the help of this study, we have confirmed the existence of fine daughter allergenic particles, which clearly differ from the parent pollen grains in size, especially after the rainy days. It is possible that the daughter allergenic species will be released from the fractions of cell wall and burst pollen grains. We concluded that rainwater was one of the important factors that affects the release of pollen allergenic proteins of both Cry j 1 and Cry j 2 from the parent pollen grains.     

Expression of Cry1Ab and Cry2Ab by a Polycistronic Transgene with a Self-Cleavage Peptide in Rice

Insect resistance to Bacillus thuringiensis (Bt) crystal protein is a major threat to the long-term use of transgenic Bt crops. Gene stacking is a readily deployable strategy to delay the development of insect resistance while it may also broaden insecticidal spectrum. Here, we report the creation of transgenic rice expressing discrete Cry1Ab and Cry2Ab simultaneously from a single expression cassette using 2A self-cleaving peptides, which are autonomous elements from virus guiding the polycistronic viral gene expression in eukaryotes. The synthetic coding sequences of Cry1Ab and Cry2Ab, linked by the coding sequence of a 2A peptide from either foot and mouth disease virus or porcine teschovirus-1, regardless of order, were all expressed as discrete Cry1Ab and Cry2Ab at high levels in the transgenic rice. Insect bioassays demonstrated that the transgenic plants were highly resistant to lepidopteran pests. This study suggested that 2A peptide can be utilized to express multiple Bt genes at high levels in transgenic crops.     

Variant cry1Ab entomocidal Bacillus thuringiensis toxin gene facilitates the recovery of an increased number of lepidopteran insect resistant independent rice transformants against yellow stem borer (Scirpophaga incertulus) inflicted damage

The primary technical constraint plant scientists face in generating insect resistant transgenic crops with insecticidal Bacillus thuringiensis (Bt) crystal protein (Cry) genes remains failing to generate sufficiently large numbers of effective resistant transgenic plant lines. One possible means to overcome this challenge is through deployment of a Cry toxin gene that contains high levels of insecticidal specific activity for target insect pests. In the present study, we tested this hypothesis using a natural variant of the Cry1Ab toxin under laboratory conditions that possessed increased insecticidal potency against the yellow stem borer (YSB, Scirpophaga incertulus), one of the most damaging rice insect pests. Following adoption of a stringent selection strategy for YSB resistant transgenic rice lines under field conditions, results showed recovery of a significantly higher number of YSB resistant independent transgenic plant lines with the variant cry1Ab gene relative to transgenic plant lines harbouring cry1Ab berliner gene. Structural homology modelling of the variant toxin peptide with the Cry1Aa toxin molecule, circular dichroism spectral analysis, and hydropathy plot analysis indicated that serine substitution by phenylalanine at amino acid position 223 of the Cry1Ab toxin molecule resulted in a changed role for α-helix 7 in domain I of Cry1Ab for enhanced toxicity.