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A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between
two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed
in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total
cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 × 2.0 μm. Bioassay
showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein
against third-instar larvae of Plutella xylostella, with an LC50 (after 48 h) value of 5.12 μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensis
cry gene and other foreign toxin genes and recombinant strains with high toxicity.
LiQiu Xia and XiaoShan Long contributed equally to this work. 相似文献
3.
José D. Antonino de SouzaJr. Sona Jain Claudia Maria Fontes de Oliveira Constância F. Ayres Wagner Alexandre Lucena 《BioControl》2009,54(3):467-473
Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed
at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were
isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient
against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein
pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis
israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk).
Handling Editor: Helen Roy. 相似文献
4.
Antonio Ventura-Suárez Ramón Cruz-Camarillo Joanne Rampersad David R. Ammons Edgar O. López-Villegas Jorge E. Ibarra Luz I. Rojas-Avelizapa 《Current microbiology》2011,62(1):307-312
Bacillus thuringiensis is a bacterium best known for its production of crystal-like bodies comprised of one or more Cry-proteins, which can be toxic
to insects, nematodes or cancer cells. Although strains of B. thuringiensis have occasionally been observed with filamentous appendages attached to their spores, appendages in association with their
parasporal bodies are extremely rare. Herein we report the characterization of Bt1-88, a bacterial strain isolated from the
Caribbean that produces a spore–crystal complex containing six long appendages, each comprised of numerous thinner filaments
approximately 10 nm in diameter and 2.5 μm in length. Each of the multi-filament appendages was attached to a single, small
parasporal body located at one end of the bacterial spore. Biochemical tests, 16S rDNA gene sequencing, and the identification
of two Cry proteins by partial protein sequencing (putatively Cry1A and Cry2A), unambiguously identified Bt1-88 as a strain
of B. thuringiensis. Bt1-88 represents the second reported strain of B. thuringiensis possessing a parasporal body/appendage phenotype characterized by one or more long appendages, comprised of numerous filaments
in association with a parasporal body. This finding suggests that Bt1-88 is a member of a new phenotypic class of B. thuringiensis, in which the parasporal body may perform a novel structural role through its association with multi-filament appendages. 相似文献
5.
S. B. Hu P. Liu X. Z. Ding L. Yan Y. J. Sun Y. M. Zhang W. P. Li L. Q. Xia 《Applied microbiology and biotechnology》2009,82(6):1157-1167
Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively
express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal
peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine–Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry−B. The recombinant plasmid was stably maintained over 240 generations in Cry−B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies.
The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l−1 Na2CO3–0.2% β-mercaptoethanol buffer at a wide range of alkaline pH values, and what’s more, the chitinase inclusions potentiated
the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera. 相似文献
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Changlong Shu Guixin Yan Rongyan Wang Jie Zhang Shuliang Feng Dafang Huang Fuping Song 《Applied microbiology and biotechnology》2009,84(4):701-707
A new polymerase chain reaction–restriction fragment length polymorphism method for the identification of cry8-type genes from Bacillus thuringiensis has been established by designing a pair of new universal primers. By this method, a novel gene, cry8Ga1, encoding a polypeptide of 1,157 amino acids with a deduced molecular mass of 131.2 kDa was identified and cloned from B. thuringiensis HBF-18. Recombinant B. thuringiensis strain HD8G, harboring cry8Ga1, has insecticidal activity against larvae of Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. This is the first report of a Cry toxin that has insecticidal activity to Melolonthidae pest H. oblita. 相似文献
7.
E. S. Boulygina A. N. Ignatov S. V. Tsygankova E. V. Korotkov B. B. Kuznetsov 《Microbiology》2009,78(6):703-710
The interspecies relationships between Bacillus thuringiensis strains producing different types of δ-endotoxins were studied using a range of molecular-biological methods. Analysis of
the 16S rRNA nucleotide sequence, the 16S to 23S rRNA intergenic spacer sequence, and the 5′-terminal region of 23S rRNA allowed
the studied strains to be subdivided into three groups based on the pattern of nucleotide substitutions. In terms of the pattern
of substitutions, the strains of the first group are similar to the B. thuringiensis type strain ATCC 10792T, the strains of the second group are practically identical to B. anthracis and the B. cereus type strain ATCC 14579T, whereas the third group combines strains of B. thuringiensis subsp. morrisoni with the cry2 gene and strains of B. thuringiensis subsp. tenebrionis with the cry3 gene. PCR fingerprinting with the use of six different primer systems ((GTG)5, REP, ERIC, and DIR) confirmed the presence of three statistically relevant groups, whose structure correlated with that
suggested by the analysis of ribosomal operon regions. 相似文献
8.
Xiaopeng Zhang Zian Liang Zafar Ali Siddiqui Yuhua Gong Ziniu Yu Shouwen Chen 《Journal of industrial microbiology & biotechnology》2009,36(6):815-820
Spodoptera exigua is one of the most renowned agricultural pest insects and relatively insensitive to Bacillus thuringiensis subsp. kurstaki strains which are widely used commercial products to control lepidopterans such as Heliothis armigera. In the current study, we have developed a new and efficient approach to screen and breed a B. thuringiensis subsp. kurstaki strain exhibiting high toxicity against S. exigua while retaining its high toxicity against H. armigera. UV and diethyl sulfate methods were used for mutagenesis, followed by an agar plug plate diffusion assay for preliminary
screening of Zwittermicin A over-producing mutants, from which we obtained a mutant strain, designated here as B. thuringiensis subsp. kurstaki D1-23, with high toxicity against S. exigua. The toxicity of D1-23 against S. exigua and H. armigera was improved by 115.4 and 25.9%, respectively, compared to its parental commercial strain BMB005. 相似文献
9.
Xiaoyan Liu Donghai Peng Yi Luo Lifang Ruan Ziniu Yu Ming Sun 《Applied microbiology and biotechnology》2009,82(4):765-772
Shuttle vectors for Bacillus thuringiensis or Bacillus cereus usually cannot hold fragments larger than 20 kb. With the development of genome research, shuttle vectors with higher loading
capacity are necessary. We constructed an Escherichia coli to B. thuringiensis shuttle vector, pEMB0557, with a large loading capacity. This vector incorporated the ori60 replicon from B. thuringiensis subsp. kurstaki YBT-1520, erythromycin resistance (B. thuringiensis), and chloromycetin resistance (E. coli) genes. A bacterial artificial chromosome library of B. thuringiensis strain CT-43 was constructed and pEMB0557 was able to accommodate at least a 70-kb DNA fragment. Simultaneously, the cry1B gene on a 40-kb fragment could express a 140-kDa protein in plasmid-cured B. thuringiensis BMB171. Due to its high capacity and utility in expressing exogenous genes, pEMB0557 will be useful in cloning (especially
silencing genes) and expressing large DNA fragments (e.g., gene clusters) in B. thuringiensis. Plasmid pEMB0557 provides a new tool for B. thuringiensis genome or B. cereus group research. 相似文献
10.
The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence
analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein,
with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain
of B. thuringiensis (HD-73−), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming
crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC50) of the two mutants were 0.2334 × 108 and 0.2591 × 108 colony-forming units g−1, considerably lower than the LC50 of the wild-type strain HBF-1 (0.9583 × 108 CFU g−1) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 108 CFU g−1). 相似文献
11.
The Crystal Proteins from Bacillus thuringiensis subsp. thompsoni Display a Synergistic Activity Against the Codling Moth, Cydia pomonella 总被引:1,自引:0,他引:1
Crystal proteins from Bacillus thuringiensis subsp. thompsoni strain HnC are active against the codling moth, Cydia pomonella, a major pest of orchards. Inclusion bodies purified from strain HnC displayed an LC50 of 3.34 × 10−3μg/μl. HnC-purified crystals were tenfold more active than Cry2Aa and Cry1Aa toxins, and 100-fold more toxic than Cry1Ab.
The 34-kDa and 40-kDa proteins contained in HnC inclusion bodies were shown to act synergistically. The toxicity of crystal
proteins produced by the recombinant B. thuringiensis strain BT-OP expressing the full-length native operon was about tenfold higher than that of the 34-kDa protein. When the
gene encoding the non-insecticidal 40-kDa protein, which is not active, was introduced into the recombinant strain producing
only the 34-kDa protein, the toxicity was raised tenfold and was similar to that of the strain BT-OP.
Received: 25 August 1999 / Accepted: 5 October 1999 相似文献
12.
Analyses of the haemolymph of the larvae ofSpodoptera littoralis Boisduval with an amino acid analyser indicated the presence of a group of amino acids, some of which showed an obvious quantitative
decrease as a result of treatment withBacillus thuringiensis Berliner such as threonine, serine, asparagine, alanine, valine, methionine, isoleucine, leucine, phenyl alanine and ornithine.
Glutamic acid, cystine, β-alanine, γ-butyric acid and histidine contents of the haemolymph of diseased larvae were higher
than those in the haemolymph of normal individuals. This increase in the content of some amino acids in infested larvae may
be attributed to the possible dissolution of protein crystals ofBacillus thuringiensis. The activity of proteolytic enzymes in the gut may be partly involved in the reaction.
B. thuringiensis was found to affect the concentration of some ions of the haemolymph ofS. littoralis. So, a marked decrease in sodium and potassium concentration was observed 4–5 days after treatment withB. thuringiensis. The change in Na+/K+ ratio in healthy and treated larvae possibly indicate their interference, in the larval toxicity withB. thuringiensis. An obvious decrease in the concentration of magnesium and zinc was also observed in the larval haemolymph after feeding
on a diet containing theB. thuringiensis preparations. The pH value of the haemolymph however showed no change after treatment withB. thuringiensis.
Résumé L'analyse de l'hémolymphe des chenilles deSpodoptera littoralis Boisduval à l'aide d'un analyseur d'acides aminés montre une diminution significative de la quantité de certains acides aminés à la suite d'un traitement parBacillus thuringiensis Berliner, tels que: thréonine, sérine, asparagine, alanine, valine, methionine, isoleucine, leucine, phenylalanine et ornithine. La teneur en acide glutamique, cystine, β alanine, γ acide butyrique et histidine de l'hémolymphe des larves traitées est plus élevée que celle des chenilles témoins. Cette augmentation est peut-être liée à la dissolution des protéines du cristal deB. thuringiensis. B. thuringiensis affecte également la concentration de certains ions dans l'hémolymphe deS. littoralis. On observe une nette réduction du taux de sodium et de potassium 4 à 5 jours après le traitement. Il est possible que cette modification du rapport Na+/K+ chez les larves malades traduise l'interférence de ces deux ions dans les phénomènes de toxicité deB. thuringiensis. Une diminution nette de la concentration en magnésium et en zinc est également notée dans l'hémolymphe des chenilles après alimentation sur un milieu traité parB. thuringiensis. Le pH de l'hémolymphe n'est pas affecté.相似文献
13.
Casique-Arroyo G Bideshi D Salcedo-Hernández R Barboza-Corona JE 《Antonie van Leeuwenhoek》2007,92(1):1-9
Bacillus thuringiensis subsp. kurstaki HD-73 was transformed with the homologous endochitinase gene chiA74 of B. thuringiensis subsp. kenyae LBIT-82 under the regulation of its own promoter and Shine–Dalgarno sequence. The plasmid, pEHchiA74, which harbors chiA74, was detected by southern blot analysis and showed high segregational stability when the recombinant strain was grown in
a medium without antibiotic. The recombinant bacterium transformed with pEHchiA74 showed an improvement in chitinolytic activity
three times that of the wild-type strain. Expression of ChiA74 did not have any deleterious effect on the crystal morphology
and size, but sporulation and Cry1Ac production in rich medium (nutrient broth with glucose) was reduced by approximately
30%. No significant increase in the toxicity of the transformant bacterium toward Plutella xylostella was detected using the same amount of total protein. However, it is possible that ChiA74 synthesis compensated for the decrease
in net Cry1Ac synthesis and toxicity observed with the recombinant strain. 相似文献
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We characterized a novel Bacillus thuringiensis isolate native to Argentina (FCC 41) that exhibits a mosquitocidal activity higher than the reference B. thuringiensis subsp. israelensis. This isolate shows a rounded crystal harboring two major proteins of about 70–80 kDa. Moreover, we cloned and sequenced
the encoding gene of one of the crystal proteins (Cry) consisting of an open reading frame of 2061 pb that encodes a protein
of 687 amino acid residues. The deduced amino acid sequence has a predicted relative molecular mass of 78 kDa and is 52% and
45% identical to those of the reported Cry24Aa and Cry24Ba sequences, respectively. The novel Cry protein was designated as
Cry24Ca, which also exhibited larvicidal activity against Aedes aegypti when its encoding gene was expressed in an Escherichia coli host strain. 相似文献
16.
Camila C. Davolos Ana M. Guidelli-Thuler Irlan L. de Abreu Janete A. D. Sena Manoel V. F. Lemos 《Biotechnology letters》2009,31(12):1891-1897
Some pest management programs employ PCR to identify cry1 genes from Bacillus thuringiensis to predict bacterial toxicity towards different insect pests. However, due to changes on the mode of action of the Cry proteins,
new primers had to be designed to detect the new genes. Therefore, an ‘in-silico’ study of genetic sequences from five cry1 subclasses was carried out and characterized by molecular tools. The design of new primers allows for more precise selection
of B. thuringiensis isolates, helping to better direct the programs employing biological control. 相似文献
17.
A recombinant gene expressing a Cry1Ac-GFP fusion protein with a molecular mass of approximately 160 kD was constructed to
investigate the expression of cry1Ac, the localization of its gene product Cry1Ac, and its role in crystal development in Bacillus thuringiensis. The cry1Ac-gfp fusion gene under the control of the cry1Ac promoter was cloned into the plasmid pHT304, and this construct was designated pHTcry1Ac-gfp. pHTcry1Ac-gfp was transformed
into the crystal-negative strain, HD-73 cry−, and the resulting strain was named HD-73−(pHTcry1Ac-gfp). The gfp gene was then inserted into the large HD-73 endogenous plasmid pHT73 and fused with the 3′ terminal of the cry1Ac gene by homologous recombination, yielding HD-73Φ(cry1Ac-gfp)3534. Laser confocal microscopy and Western blot analyses showed for the first time that the Cry1Ac-GFP fusion proteins in
both HD-73−(pHTcry1Ac-gfp) and HD-73Φ(cry1Ac-gfp)3534 were produced during asymmetric septum formation. Surprisingly, the Cry1Ac-GFP fusion protein showed polarity and was
located near the septa in both strains. There was no significant difference between Cry1Ac-GFP and Cry1Ac in their toxicity
to Plutella xylostella larvae. 相似文献
18.
A transgenic strain of the nitrogen-fixing filamentous cyanobacterium Anabaena PCC 7120 protected expressed δ-endotoxin proteins of Bacillus thuringiensis subsp. israelensis from damage inflicted by UV-B, a sunlight component that penetrates Earth's ozone layer. This organism, which serves as a
food source to mosquito larvae and could multiply in their breeding sites, may solve the environment-imposed limitations of
B. thuringiensis subsp. israelensis as a mosquito biological control agent.
Received: 20 November 2001 / Accepted: 31 December 2001 相似文献
19.
Isolation and Characterization of Bacillus thuringiensis Strains from Aquatic Environments in Spain 总被引:1,自引:0,他引:1
Samples collected from aquatic environments from Spain were analyzed for the occurrence and dipteran toxicity of Bacillus thuringiensis. From a total of 41 samples, 122 isolates were obtained, yielding a B. thuringiensis index of 0.22. Isolates were assigned to 13 different serovars, with serovar thuringiensis (serotype H1) the most frequently
found. Toxicity tests carried out revealed that eight isolates (6.6% out of the total) were active against Tipula oleracea larvae. Serological tests assigned these toxic isolates to serovar thuringiensis. The toxicity found in these isolates against
the tipulid was approximately seven times lower than that shown by the standard strain B. thuringiensis ser. israelensis IPS-82. Implication of Cry2A protein in toxic activity is hypothesized.
Received: 3 December 1999 / Accepted: 5 January 2000 相似文献
20.
J. L. Gringorten S. S. Sohi L. Masson 《In vitro cellular & developmental biology. Animal》1999,35(5):299-303
Summary Eight continuous insect cell lines were tested for susceptibility to the δ-endotoxins of several lepidopteran-active strains
and cloned-gene products of Bacillus thuringiensis. The assays were performed on cells suspended in agarose gel, which allowed the toxins activated at pH 10.5 to be applied
directly in a high-pH buffer without causing solvent toxicity to the cells. The responses of the cell lines to the various
toxins produced activity spectra that were used to identify functionally similar and dissimilar toxin proteins.
IPRI-CF-1 and FPMI-MS-5, derived from neonate larvae of Choristoneura fumiferana and Manduca sexta, respectively, exhibited the greatest sensitivity to the toxins tested, whereas B. thuringiensis subsp. entomocidus had the broadest in vitro host range. Analysis of activity spectra led to the identification of the particular Cry protein
that was responsible for the broad toxicity of this subspecies and demonstrated a distinct difference in toxin composition
between two strains of subsp. sotto. The identical spectra observed for subsp. kurstaki HD-1 and NRD-12 is consistent with insect bioassay data obtained previously by other workers and supports the conclusion
that there is virtually no difference in activity between these two strains.
The in vitro assay system, referred to as the “lawn assay” and used to test B. thuringiensis activated toxins against insect cell lines, is particularly useful in mode-of-action studies and as a rapid, preliminary
test for the presence of specific cytolytic proteins, rather than as a method for screening toxins of wild-type strains for
insecticidal activity. The response of cells in vitro to B. thuringiensis toxins is often very different from that of the insect from which the cells were derived. 相似文献