Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance

We evaluated the insecticidal toxicity of Cry1Aa, Cry1Ab and Cry1Ac toxins against neonate larvae of sugarcane shoot borer Chilo infuscatellus Snellen (Lepidoptera: Crambidae) in vitro on diet surface. With the lowest LC₅₀ value, Cry1Ab emerged as the most effective among the three toxins. Sugarcane cultivars Co 86032 and CoJ 64 were transformed with cry1Ab gene driven by maize ubiquitin promoter through particle bombardment and Agrobacterium-mediated transformation systems. Gene pyramiding was also attempted by retransforming sugarcane plants carrying bovine pancreatic trypsin inhibitor (aprotinin) gene, with cry1Ab. Southern analysis confirmed multiple integration of the transgene in case of particle bombardment and single site integration in Agrobacterium-mediated transformants. The expression of cry1Ab was demonstrated through Western analysis and the toxin was quantified using ELISA. The amount of Cry1Ab protein in different events varied from 0.007 to 1.73% of the total soluble leaf protein; the events transformed by Agrobacterium method showed significantly higher values. In in vivo bioassay with neonate larvae of shoot borer, transgenics produced considerably lower percentage of deadhearts despite suffering feeding damage by the borer compared with the untransformed control plants. Expressed Cry1Ab content was negatively related to deadheart damage. Aprotinin-expressing sugarcane pyramided with cry1Ab also showed reduction in damage. The potential of producing sugarcane transgenics with cry1Ab and aprotinin genes resistant to early shoot borer was discussed in the light of the results obtained.     

Fate of recombinant DNA and Cry1Ab protein after ingestion and dispersal of genetically modified maize in comparison to rapeseed by fallow deer (Dama dama)

The fate of recombinant DNA in fallow deer (Dama dama) was investigated by feeding a diet of isogenic or genetically modified (GM) maize expressing Cry1Ab protein against the European corn borer (Ostrinia nubilalis). To study the degradability of ingested DNA, polymerase chain reaction (PCR) assays were introduced to detect fragments of the endogenous, highly abundant chloroplast-specific rubisco gene, the maize-specific zein gene and the recombinant cry1Ab gene. PCR analysis revealed that small chloroplast- and maize-specific DNA fragments were detectable in contents of rumen, abomasums, jejunum, caecum and colon and occasionally in visceral tissues. In contrast, no fragments of the recombinant cry1Ab gene were detectable in gastrointestinal (GI) contents. The Cry1Ab protein was analysed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting technique. Neither ELISA nor immunoblotting yielded positive signals of immunoactive Cry1Ab protein in GI contents and tissues of fallow deer fed with GM maize. In conclusion, after uptake of GM maize, neither cry1Ab-specific gene fragments nor Cry1Ab protein were detected in the GI tract of fallow deer, indicating complete digestion of the GM maize. Additional investigations on the germination capacity of conventional rapeseed and maize seed after ingestion by fallow deer and faecal excretion (endozoochory) were performed to draw conclusions regarding a potential spreading of germinable GM crop seed by deer. Germination tests revealed that germinable rapeseed kernels were detectable in faeces; in contrast, no intact maize seeds were found in faeces.     

Cloning and characterization of truncated <Emphasis Type="Italic">cry1Ab</Emphasis> gene from a new indigenous isolate of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The insecticidal crystal protein(s) encoded by cry gene(s) of Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. A new 3′-truncated cry1Ab gene was cloned from an indigenous isolate of Bt, A19-31. Nucleotide sequencing and homology search revealed that the deduced amino acid sequence of Cry1Ab toxin of Bt strain A19-31 had a variation of two amino acid residues with the holotype sequence, Cry1Ab1. Expression of the 3′-truncated cry1Ab gene was studied in an acrystalliferous strain of Bt (4Q7). SDS-PAGE and immunostrip analysis of spore-crystal mixture revealed a low level expression of the 3′-truncated cry1Ab gene. Insecticidal activity assay showed that the recombinant 3′-truncated cry1Ab gene product was toxic to larvae of both Helicoverpa armigera and Spodoptera litura.     

Expression of a Bacillus thuringiensis cry1Ab gene in transgenic white spruce and its efficacy against the spruce budworm (Choristoneura fumiferana)

A synthetic version of the cry1Ab gene from Bacillus thuringiensis (Bt) was introduced into white spruce (Picea glauca) by microprojectile bombardment. A plasmid carrying the cry1Ab gene, driven by a ubiquitin (maize) promoter, was co-transferred with a plasmid containing the gus–nptII fusion gene as a screenable selection marker. Molecular analysis of the transgenic lines showed a high level (more than 90%) of co-integration of the cry1Ab gene with the screenable marker. A wide range of expression levels of the cry1Ab gene and corresponding endotoxin was obtained. Accumulation of the Cry1Ab protein was evaluated in embryogenic tissue, the needles of somatic seedlings and in the needles of 5-year-old field-grown trees of individual lines. Laboratory and field insect feeding trials suggest that several spruce transgenic lines were lethal to spruce budworm larvae.     

Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants

In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2–3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.     

Molecular Cloning of a New Crystal Protein Gene cry1Af1 of Bacillus thuringiensis NT0423 from Korean Sericultural Farms

A new cry1Ab-type gene encoding the 130 kDa protein of Bacillus thuringiensis NT0423 bipyramidal crystals was cloned, sequenced, and expressed in a crystal-negative B. thuringiensis host. Hybridization experiments revealed that the crystal protein gene is located on a 44 MDa plasmid of B. thuringiensis NT0423. A strong positive signal detected on the 6.6 kb HindIII fragment from B. thuringiensis NT0423 plasmid DNA was cloned and sequenced. The cry1Ab-type gene, designated cry1Af1, consisted of open reading frame of 3453 bp, encoding a protein of 1151 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 130,215 Da. With both Bt I and Br II promoter sequences were found, the B. thuringiensis NT0423 crystal protein gene promoter closely aligned with those of cry1A-type crystal protein gene. When compared with known sequences of other Cry and Cyt proteins, the Cry1Af1 protein showed maximum 93% sequence identity to Cry1Ab protein of B. thuringiensis subsp. kurstaki. The expressed Cry1Af1 protein in a crystal-negative B. thuringiensis host appears to have strong insecticidal activity against lepidopteran larvae (Plutella xylostella). Crystals containing Cry1Af1 were about six times more toxic than the wild-type crystals of B. thuringiensis NT0423. Received: 20 February 2001 / Accepted: 17 April 2001     

Cloning, Characterization, and Expression of a New cry1Ab Gene from DOR Bt-1, an Indigenous Isolate of Bacillus thuringiensis

A new cry1Ab gene was cloned from the promising local isolate, DOR Bt-1, a Bacillus thuringiensis strain isolated from castor semilooper (Achaea janata L.) cadavers from castor bean (Ricinus communis L.) field. The nucleotide sequence of the cloned cry1Ab gene indicated that the open reading frame consisted of 3,465 bases encoding a protein of 1,155 amino acid residues with an estimated molecular weight of 130 kDa. Homology comparisons revealed that the deduced amino acid sequence of cry1Ab had a variation of seven amino acid residues compared to those of the known Cry1Ab proteins in the NCBI database and this gene has been designated as cry1Ab26 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry1Ab26 was cloned into pET 29a(+) vector and expressed in E. coli strain BL21 (DE3) under the control of T7 promoter with IPTG induction. ELISA, SDS-PAGE, and Western blot analysis confirmed the expression of 130-kDa protein. Insect bioassays with neonate larvae of Helicoverpa armigera showed that the partially purified Cry1Ab26 caused 97 % mortality within 5 days of feeding.     

Transcriptional silencing and developmental reactivation of cry1Ab gene in transgenic rice

One transgenic rice line lacking Cry1Ab expression product was screened in the progenies of Agrobacterium-transformed transgenic rice variety Zhong 8215 with a cry1Ab gene under field releasing conditions by using GUS histochemical assay and Western blot. Molecular hybridization results revealed that the cry1Ab gene was silenced in the transgenic rice variety Zhong 8215 and two copies of ubiquitin promoter were integrated into the rice genome. The silencing of cry1Ab gene in transgenic rice was found to be due to the methylation of the ubiquitin promoter as revealed by methylation analysis. Meanwhile, different concentrations of demethylation reagent 5-azacytidine combining with different treatment time were employed to treat the silenced transgenic rice seeds. The results indicated that 5-azacytidine could reactivate 8%–30% of the silenced transgenic rice plants and the expression level of the reactivated cry1Ab transgene could reach as high as 0.147% of the total soluble protein. Treatment with low concentration of 5-azacytidine (45 mg/L for 1 d and 2 d) could lead to the highest reactivation ratio and the highest expression level of the cry1Ab gene.     

Gene expression and insect resistance in transgenic broccoli containing a Bacillus thuringiensis cry1Ab gene with the chemically inducible PR-1a promoter

We produced 49 broccoli plants (Brassica oleracea L. ssp. italica) containing a Bacillus thuringiensis cry1Ab gene under control of the chemically inducible PR-1a promoter from tobacco. Most of them showed substantial or complete control of neonate diamondback moth larvae, regardless of whether the transgene was induced or not. Ten plants were selected for detailed study via northern and western analysis and insect bioassays. They expressed the cry1Ab gene and gave complete insect control when treated with the chemical inducers INA (2,6-dichloroiso-nicotinic acid) or BTH (1,2,3-benzothiadiazole-7-carbothioic acid S-methyl ester); however, leaves treated with water alone were also partially or completely protected from insect damage. Transgenic progeny plants showed greater inducibility than primary transformants at the molecular level. Two progeny lines produced cry1Ab mRNA and Cry1Ab protein and gave insect control only after induction, both when detached leaves and intact plants were tested. The relevance of these results to resistance management strategies is discussed.     

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.     

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest,Chilo suppressalis (Lepidoptera: Crambidae)

Two transgenic rice lines (T2A‐1 and T1C‐19b) expressing cry2A and cry1C genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis (Walker) (Lepidoptera: Crambidae). The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a cry1Ab/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A‐1 > Huahui 1 > T1C‐19b among rice lines, and leaf > stem > root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar: higher level at early stages than at later stages with an exception that high Cry1C level in T1C‐19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the jointing stage with an exception for T1C‐19b providing an excellent performance at the maturing stage. By comparison, T1C‐19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A‐1, being close to Huahui 1. The results suggest cry1C is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C‐19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.     

Cloning of cry2Aa and cry2Ab genes from new isolates of Bacillus thuringiensis and their expression in recombinant Bacillus thuringiensis and Escherichia coli strains

Bacillus thuringiensis (Bt) is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of Bt are promising candidates for management of resistance development in insects due to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. Two insecticidal crystal protein genes of Bt, viz. cry2Aa and cry2Ab were cloned from new isolates of Bt, 22-4 and 22-11, respectively. Expression of both the genes was studied in an acrystalliferous strain of Bt (4Q7) by fusing the cry2Aa and cry2Ab genes downstream of cry2Aa promoter and orf1 + orf2 sequences. Western blot analysis revealed a low level expression of the cloned cry2Aa and cry2Ab genes in the recombinant Bt strains. High-level expression of cry2Aa and cry2Ab genes was achieved in the recombinant E. coli by cloning the cry2A genes under the control of the T7 promoter.     

Cry2A toxins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> expressed in insect cells are toxic to two lepidopteran insects

The cry2Aa and cry2Ab genes from a Brazilian Bacillus thuringiensis strain were introduced into the genome of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in order to evaluate the heterologous proteins expression in insect cells and their toxicity to different insects. The recombinant viruses (vAcCry2Aa and vSynCry2Ab) were amplified in Trichoplusia ni (BTI-Tn5B1-4) cells and used to infect Spodoptera frugiperda larvae. Total extracts from S. frugiperda infected with the recombinant viruses were analysed by SDS-PAGE, which detected the presence of polypeptides around 65 kDa. Cuboid-shaped protein crystals were observed in insect extracts by light and scanning electron microscopy. Bioassays, using the heterologous proteins showed toxicity against second instar A. gemmatalis larvae (Cry2Aa) with a LC₅₀ of 1.03 μg/ml and second instar S. frugiperda larvae (Cry2Ab) with a LC₅₀ of 3.45 μg/ml. No toxic activity was detected for Aedes aegypti and Culex quinquenfaciatus.     

Evaluating the Insecticidal Genes and Their Expressed Products in Bacillus thuringiensis Strains by Combining PCR with Mass Spectrometry

By a combination of PCR and mass spectrometry, a total of five cry genes (cry1Aa, cry1Ac, cry2Aa, cry2Ab, and cry1Ia) were detected in genomic DNA from the wild-type Bacillus thuringiensis strain 4.0718, and three protoxins (Cry1Aa, Cry1Ac, and Cry2Aa) were identified in the strain's parasporal crystals. These results indicated that this complementary method may be useful in evaluating B. thuringiensis strains at both the gene and protein levels.     

Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.

Bacillus thuringiensis (Bt) strain FJAT-12 was a novel Bt strain isolated by Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Science. In this study, a new cry2Ab gene was cloned from Bt strain FJAT-12 and named as cry2Ab30 by Bt delta-endotoxin Nomenclature Committee. The sequencing results showed there were two mutations in conservative sites which led to two amino acids modification. Homology modeling indicated that the two changes were located in β-sheet of Domain II. A prokaryotic expression vector pET30a-cry2Ab30 was constructed and the expressed protein was analyzed by western blot using Cry2Ab antibody. The expression conditions including IPTG concentration, revolution and temperature were optimized to get the highest expression level by SDS-PAGE and BandScan. The bioassay results also showed that the Cry2Ab30 toxin had high insecticidal activity against Plutella xylostella and the LC₅₀ value was 0.0103 μg.mL^?1. The two mutations in β-sheet of Domain II might contribute to insecticidal activity of Cry2Ab30 toxin against Plutella xylostella.     

Cry2Ab蛋白对龟纹瓢虫的安全性研究

【目的】转基因作物对非靶标昆虫的影响是转基因作物环境安全评价的重要内容,研究Cry2Ab蛋白对龟纹瓢虫的影响,对转基因作物的环境安全评价具有重要意义。【方法】采用实验动物学、分子生物学等方法,研究Cry2Ab蛋白对龟纹瓢虫发育历期、成虫体重、雌雄比例及体内氨基酸种类和含量的影响。【结果】与蔗糖对照组相比,Cry2Ab蛋白对龟纹瓢虫不同龄期的发育历期、成虫体重和雌雄比例均无明显差异,对体内氨基酸种类和含量也没有显著差异。【结论】Cry2Ab蛋白对龟纹瓢虫的生长发育及代谢无显著影响。     

Fate of genetically modified maize and conventional rapeseed,and endozoochory in wild boar (Sus scrofa)

Feeding experiments were carried out to investigate the digestive fate of transgenic DNA and novel protein in wild boar applying polymerase chain reaction (PCR) and immunodiagnostic techniques. Furthermore, the dispersal of viable maize and rapeseed (endozoochory) was studied. A diet containing conventional rapeseed, and either genetically modified (GM) maize expressing Cry1Ab protein (Bt176) or non-GM isogenic maize was offered. By conventional and quantitative PCR both chloroplast-specific plant DNA (rubisco) and cry1Ab gene fragments were detected only in gastrointestinal content. Using an enzyme-linked immunosorbent assay (ELISA) positive signals of immunoactive Cry1Ab protein were detected in digesta samples. Analysis of endozoochory showed that excreted maize seeds retain their germination capacity only in extremely rare cases and no intact rapeseed was found in faeces. A possible dispersal of viable seeds by wild boars is highly unlikely.     

Survival of Helicoverpa armigera larvae on and Bt toxin expression in various parts of transgenic Bt cotton (Bollgard II) plants

There is no conclusive evidence that Helicoverpa spp. (Lepidoptera: Noctuidae) in Australia have evolved significant levels of resistance to Bollgard II^® cotton (which expresses two Bt toxin genes, cry1Ac and cry2Ab). However, there is evidence of surviving larvae on Bollgard II cotton in the field. The distribution and survival of early‐instar Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae were examined on whole Bollgard II and non‐Bt cotton plants in greenhouse bioassays. The expression of Cry toxins in various parts of Bollgard II plants was compared to the survival of larvae in those locations. Only 1% of larvae survived after 6 days on greenhouse‐grown Bollgard II plants compared to 31% on non‐Bt cotton plants. Overall, and across all time intervals, more larvae survived on reproductive parts (squares, flowers, and bolls) than on vegetative parts (leaves, stems, and petioles) on Bollgard II plants. The concentration of Cry1Ac toxin did not differ between plant structures, whereas Cry2Ab toxin differed significantly, but there was no relationship between the level of expression and the location of larvae. This study provides no evidence that lower expression of Cry toxins in the reproductive parts of plants explains the survival of H. armigera larvae on Bollgard II cotton.