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Find video protocols related to scientific articles indexed in Pubmed.
Mining Tissue-specific Contigs from Peanut (Arachis hypogaea L.) for Promoter Cloning by Deep Transcriptome Sequencing.
Plant Cell Physiol.
PUBLISHED: 09-16-2014
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Peanut (Arachis hypogaea L.), one of the most important oil legumes in the world, is heavily damaged by white grubs. Tissue-specific promoters are needed to incorporate insect resistance genes into peanut by genetic transformation to control the subterranean pests. Transcriptome sequencing is the most effective way to analyze differential gene expression in this non-model species and contribute to promoter cloning. The transcriptomes of the roots, seeds and leaves of peanut were sequenced using Illumina technology. A simple digital expression profile was established based on number of transcripts per million clean tags (TPM) from different tissues. Subsequently, 584 root-specific candidate transcript assembly contigs (TACs) and 316 seed-specific candidate TACs were identified. Among these candidate TACs, 55.3% were root-specific and 64.6% were seed-specific by semi-quantitative RT-PCR analysis. Moreover, the consistency of semi-quantitative RT-PCR with the simple digital expression profile was correlated with the length and TPM value of TACs. The results of gene ontology showed that some root-specific TACs are involved in stress resistance and respond to auxin stimulus, whereas, seed-specific candidate TACs are involved in embryo development, lipid storage and long-chain fatty acid biosynthesis. One root-specific promoter was cloned and characterized. We developed a high-yield screening system in peanut by establishing a simple digital expression profile based on Illumina sequencing. The feasible and rapid method presented by this study can be used for other non-model crops to explore tissue-specific or spatially specific promoters.
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Mining rare and ubiquitous toxin genes from a large collection of Bacillus thuringiensis strains.
J. Invertebr. Pathol.
PUBLISHED: 08-06-2014
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There has been considerable effort made in recent years for research groups and other organizations to build up large collections of strains of Bacillus thuringiensis in the search for genes encoding novel insecticidal toxins, or encoding novel metabolic pathways. Whilst next generation sequencing allows the detailed genetic characterization of a bacterial strain with relative ease it is still not practicable for large strain collections. In this work we assess the practicability of mining a mixture of genomic DNA from a two thousand strain collection for particular genes. Using PCR the collection was screened for both a rare (cry15) toxin gene as well as a more commonly found gene (vip3A). The method was successful in identifying both a cry15 gene and multiple examples of the vip3A gene family including a novel member of this family (vip3Aj). A number of variants of vip3Ag were cloned and expressed, and differences in toxicity observed despite extremely high sequence similarity.
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Genomic sequencing identifies novel Bacillus thuringiensis Vip1/Vip2 binary and Cry8 toxins that have high toxicity to Scarabaeoidea larvae.
Appl. Microbiol. Biotechnol.
PUBLISHED: 07-14-2014
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The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), which has previously been shown to encode the cry8Ga toxin gene, is active against both Holotrichia oblita and Holotrichia parallela. Recombinant Cry8Ga however is only weakly toxic to these insect pests suggesting the involvement of additional toxins in the native strain. We report that through the use of Illumina sequencing three additional, and novel, genes, namely vip1Ad1, vip2Ag1, and cry8-like, were identified in this strain. Although no protein corresponding to these genes could be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the HBF-18 proteome, reverse transcription (RT)-PCR indicated that all three genes were transcribed in the native strain. The two vip genes were cloned and expressed and, as with other Vip1/2 toxins, appeared to function as a binary toxin and showed strong activity against H. oblita, H. parallela and Anomala corpulenta. This is the first report to demonstrate that the Vip1/Vip2 binary toxin is active against these Scarabaeoidea larvae. The cry8-like gene appeared to be a C-terminally truncated form of a typical cry8 gene and was not expressed in our usual recombinant Bt expression system. When however the missing C-terminal region was replaced with the corresponding sequence from cry8Ea, the resulting hybrid expressed well and the toxin was active against the three test insects.
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Characterization of one novel cry8 gene from Bacillus thuringiensis strain Q52-7.
World J. Microbiol. Biotechnol.
PUBLISHED: 06-18-2014
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Bacillus thuringiensis (Bt) is the most widely used insecticidal microbe due to its specific toxicity and safe use with respect to animals and the environment. In this study, we isolated Bt strain Q52-7 from a soil sample collected in the Qian Shan District, Liao Ning Province, China. We observed that the Q52-7 strain produced spherical crystals. The Bt Q52-7 strain had high toxicity against Asian Cockchafer (Holotrichia parallela), exhibiting an LC50 of 3.80 × 10(9) cfu/g, but is not toxic for Anomala corpulenta Motschulsky and Holotrichia oblita. Using general cry8 primers, we amplified a 1.3 kb fragment with the polymerase chain reaction. Specific primers were designed for the amplified fragment to clone the full-length coding region. A novel gene, cry8Na1, had 69 % sequence similarity with cry8Ca1. cry8Na1 gene was successfully expressed in the HD-73(-) acrystalliferous mutant of Bt subsp. Kurstaki HD-73. Bioassays demonstrated that the Cry8Na1 protein is highly toxic for the H. parallela, with a 50 % lethal concentration of 8.18 × 10(10) colony forming units per gram.
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Cultivable gut bacteria of scarabs (Coleoptera: Scarabaeidae) inhibit Bacillus thuringiensis multiplication.
Environ. Entomol.
PUBLISHED: 05-07-2014
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The entomopathogen Bacillus thuringiensis is used to control various pest species of scarab beetle but is not particularly effective. Gut bacteria have diverse ecological and evolutionary effects on their hosts, but whether gut bacteria can protect scarabs from B. thuringiensis infection remains poorly understood. To investigate this, we isolated 32 cultivable gut bacteria from Holotrichia oblita Faldermann, Holotrichia parallela Motschulsky, and Anomala corpulenta Motschulsky, and analyzed their effect on B. thuringiensis multiplication and Cry toxin stability. 16S rDNA analysis indicated that these gut bacteria belong to the Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes phyla. A confrontation culture analyses of the 32 isolates against three scarab-specific B. thuringiensis strains showed that the majority of the scarab gut bacteria had antibacterial activity against the B. thuringiensis strains. The Cry toxin stability analysis results showed that while several strains produced proteases capable of processing the scarab-specific toxin Cry8Ea, none were able to completely degrade it. These results suggest that gut bacteria can potentially affect the susceptibility of scarabs to B. thuringiensis and that this should be considered when considering future control measures.
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Screening of cry-type promoters with strong activity and application in Cry protein encapsulation in a sigK mutant.
Appl. Microbiol. Biotechnol.
PUBLISHED: 04-03-2014
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To optimize the expression of cry genes in a Bacillus thuringiensis sigK mutant failing in crystal releasing, the transcriptional activity of the cry promoters cry1A, cry3A, cry4A, and cry8E was compared using lacZ gene fusions. A beta-galactosidase assay indicated that the cry8E promoter showed the highest transcriptional activity. A novel Escherichia coli-B. thuringiensis shuttle vector pHT315-8E21b was constructed for cry gene expression using the cry8E promoter and the multiple cloning sites from vector pET21b, based on vector pHT315. SDS-PAGE analysis showed that the expression of the cry1Ac gene directed by the cry8E promoter was increased by approximately 2.4-fold over the expression directed by the cry3A promoter. The cry1Ba gene was expressed in the sigK mutant with the constructed vector pHT315-8E21b. Normal bipyramidal crystals encapsulated in mother cell were observed by transmission electron microscopy (TEM). The encapsulated Cry1Ba protein expressed in the sigK mutant showed activity against Ostrinia furnacalis and Plutella xylostella similar to that of the released Cry1Ba protein expressed in the acrystalliferous strain HD73 and can be protected from inactivation by UV light. All these results suggest that the cry8E promoter can be an efficient transcriptional element for cry gene expression in sigK mutants and can be utilized for the construction of a genetically engineered strain.
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An improved PCR-restriction fragment length polymorphism (RFLP) method for the identification of cry1-type genes.
Appl. Environ. Microbiol.
PUBLISHED: 08-30-2013
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The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.
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A genetically modified broad-spectrum strain of Bacillus thuringiensis toxic against Holotrichia parallela, Anomala corpulenta and Holotrichia oblita.
World J. Microbiol. Biotechnol.
PUBLISHED: 06-26-2013
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Cry8Ea1 from Bacillus thuringiensis strain Bt185 has insecticidal activity against Holotrichia parallela. Cry8Ca2 from strain HBF-1 is effective against Anomala corpulenta. Cry8Ga1 from strain HBF-18 is toxic to H. oblita. Given the need to broaden the spectrum of B. thuringiensis, a broad-spectrum coleopteran active strain of B. thuringiensis, BIOT185, engineered to express multiple cry genes, including cry8Ea1, cry8Fa1 and cry8Ca2, was created by homologous recombination introducing the cry8Ca2 into the B. thuringiensis strain Bt185 by Liu et al. (Appl Microbiol Biotechnol 87:243-249, 2010). To further broaden the spectrum, an engineered B. thuringiensis strain BIOT1858G was constructed by introducing the recombinant plasmid pSTK-8G containing cry8Ga1 into the engineered B. thuringiensis strain BIOT185. PCR and Southern blotting demonstrated that the cry8Ga1 gene was transferred to the novel strain BIOT1858G. SDS-PAGE and RT-PCR confirmed the normal expression and transcription of the cry8Ga1 gene in addition to the cry8Ea1, cry8Fa1 and cry8Ca2 genes. Bioassays of BIOT1858G indicated that the recombinant strain broadened the spectrum against not only H. parallela susceptible to the Cry8E protein and A. corpulenta susceptible to the Cry8C protein but also H. oblita susceptible to the Cry8G protein. The pesticide could also decrease the cost of production and field labor.
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Transcriptional regulation and characteristics of a novel N-acetylmuramoyl-L-alanine amidase gene involved in Bacillus thuringiensis mother cell lysis.
J. Bacteriol.
PUBLISHED: 04-19-2013
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In Bacillus thuringiensis, a novel N-acetylmuramoyl-L-alanine amidase gene (named cwlB) was detected, and the CwlB protein was purified and characterized. Reverse transcription-PCR (RT-PCR) results indicated that cwlB and an upstream gene (named cwlA) formed one transcriptional unit. 5 rapid amplification of cDNA ends (5-RACE)-PCR and transcriptional fusions with the lacZ gene indicated that transcription of the operon was directed by a promoter, P(cwlA), which is located upstream from the cwlA gene and that the transcription start site is a single 5-end nucleotide residue T located 25 nucleotides (bp) upstream from the cwlA translational start codon. Moreover, the activity of P(cwlA) was controlled by ?(K). Morphological analysis suggested that the mutation of cwlB could delay spore release compared to the timing of spore release in the wild-type strain. Western blot assay demonstrated that purified CwlB bound to the B. thuringiensis cell wall. Observations with laser confocal microscopy and a green fluorescent protein-based reporter system demonstrated that the CwlB protein localizes to the cell envelope. All results suggest that the CwlB protein is involved in mother cell lysis in B. thuringiensis.
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Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73.
Genome Announc
PUBLISHED: 03-22-2013
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Bacillus thuringiensis is a Gram-positive bacterium that produces intracellular protein crystals toxic to a wide variety of insect larvae. We report the complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73 from the Centre OILB (Institut Pasteur, France), which belongs to serotype 3ab and is toxic to lepidopteran larvae.
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Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis.
J. Invertebr. Pathol.
PUBLISHED: 03-18-2013
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A pooled clone method was developed to screen for cry2A genes. This metagenomic method avoids the need to analyse isolated Bacillus thuringiensis strains by performing gene specific PCR on plasmid-enriched DNA prepared from a pooled soil sample. Using this approach the novel holotype gene cry2Ah1 was cloned and characterized. The toxin gene was over-expressed in Escherichia coli Rosetta (DE3) and the expressed toxin accumulated in both the soluble and insoluble fractions. The soluble Cry2Ah1 was found to have a weight loss activity against Ostrinia furnacalis, and a growth inhibitory activity to both Cry1Ac-susceptible and resistant Helicoverpa armigera populations.
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A chimeric cry8Ea1 gene flanked by MARs efficiently controls Holotrichia parallela.
Plant Cell Rep.
PUBLISHED: 03-06-2013
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Peanuts transformed with the synthetic cry8Ea1 gene flanked by MARs are a potentially effective control strategy against white grubs. Cry8Ea1 protein levels of the construct containing MARs were increased by 2.5 times. White grubs are now recognized as the most important pests of peanut worldwide. A synthetic cry8Ea1 gene, which was toxic to Holotrichia parallela larvae, was expressed in chimeric peanut roots using an Agrobacterium rhizogenes-mediated transformation system. The relative mRNA and protein levels of the cry8Ea1 gene were confirmed by quantitative real-time PCR and ELISA, respectively. The effects of matrix attachment regions (MARs) on the expression and activity of the cry8Ea1 gene were analyzed. The average expression level of cry8Ea1 in peanut roots was higher for the plants harboring constructs flanked by MARs from tobacco. Moreover, differing from previous studies, the synthetic cry8Ea1 gene flanked by MARs showed more variation in protein levels than mRNA levels. These composite plants containing cry8Ea1 gene flanked by MARs exhibited a high toxicity against Holotrichia parallela larvae as shown by bioassay analysis, thus offering a potential effective combination to control subterranean insects in peanuts.
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Characterization of cry9Da4, cry9Eb2, and cry9Ee1 genes from Bacillus thuringiensis strain T03B001.
Appl. Microbiol. Biotechnol.
PUBLISHED: 02-15-2013
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Three cry9 genes, cry9Da4, cry9Eb2, and cry9Ee1, were cloned from Bacillus thuringiensis strain T03B001 using a high-resolution melting analysis method. All three cry9 genes were overexpressed in Escherichia coli Rosetta (DE3), and the expressed products Cry9Eb2 and Cry9Ee1 were shown to be toxic to Plutella xylostella and Ostrinia furnacalis, but not to Helicoverpa armigera or Colaphellus bowringi. The bioassay of Cry9Eb2 and Cry9Ee1 against Cry1Ac-resistant P. xylostella strains indicated that both novel Cry9 toxins exhibited no cross-resistance with Cry1Ac. Cry9Eb2 and Cry9Ee1 can be applied not only for P. xylostella and O. furnacalis control, but also for the Cry1Ac-resistance management of pests.
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A pangenomic study of Bacillus thuringiensis.
J Genet Genomics
PUBLISHED: 07-24-2011
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Bacillus thuringiensis (B. thuringiensis) is a soil-dwelling Gram-positive bacterium and its plasmid-encoded toxins (Cry) are commonly used as biological alternatives to pesticides. In a pangenomic study, we sequenced seven B. thuringiensis isolates in both high coverage and base-quality using the next-generation sequencing platform. The B. thuringiensis pangenome was extrapolated to have 4196 core genes and an asymptotic value of 558 unique genes when a new genome is added. Compared to the pangenomes of its closely related species of the same genus, B. thuringiensis pangenome shows an open characteristic, similar to B. cereus but not to B. anthracis; the latter has a closed pangenome. We also found extensive divergence among the seven B. thuringiensis genome assemblies, which harbor ample repeats and single nucleotide polymorphisms (SNPs). The identities among orthologous genes are greater than 84.5% and the hotspots for the genome variations were discovered in genomic regions of 2.3-2.8Mb and 5.0-5.6Mb. We concluded that high-coverage sequence assemblies from multiple strains, before all the gaps are closed, are very useful for pangenomic studies.
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Identification of the minimal active fragment of the Cry1Ah toxin.
Biotechnol. Lett.
PUBLISHED: 09-28-2010
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cry1Ah1, a novel holo-type gene cloned from Bacillus thuringiensis strain BT-8, encoded a protein exhibiting strong insecticidal activity against lepidopteran insects. To identify the minimal active fragment of the Cry1Ah toxin, 9 pairs of primers were designed to generate different PCR products. Seven PCR products were amplified by different primers using the cry1Ah1 gene as a template and cloned into a pET-21b vector. These positive clones were separately transformed into Escherichia coli. Insecticidal activity against 2nd-instar larvae of Plutella xylostella was performed using the leaf-dip bioassay: the minimal active fragment of the Cry1Ah toxin was located between amino acid residues 50I and 639E.
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[Comparison of midgut bacterial community between Bt-resistant and sensitive Helicoverpa armigera].
Wei Sheng Wu Xue Bao
PUBLISHED: 08-07-2010
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To investigate the midgut bacterial community of sensitive and resistant Helicoverpa armigera for the emergence of resistance to Bacillus thuringiensis.
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Construction of a Bacillus thuringiensis engineered strain with high toxicity and broad pesticidal spectrum against coleopteran insects.
Appl. Microbiol. Biotechnol.
PUBLISHED: 01-28-2010
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A newly engineered strain, denominated BIOT185, was constructed through integrating the cry8Ca2 gene into the endogenous plasmid of BT185 (contains cry8Ea1) by homologous recombination. The thermosensitive plasmid vector was eliminated by the rising temperature of recombinant cultures. No antibiotic gene or other unnecessary genes were introduced to the new strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry8Ca2 gene was expressed normally and produced a 130-kDa protein in the BIOT185 strain. Bioassay results showed that the new strain had high toxicity to the pests Anomala corpulenta and Holotrichia parallela, which often damage the same fields.
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[Characterization of Bacillus thuringiensis spoIII D gene mutation].
Wei Sheng Wu Xue Bao
PUBLISHED: 12-25-2009
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Construction and characterization of a spoIII D gene deletion mutant of Bacillus thuringiensis.
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Characterization of a novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and Holotrichia parallela.
Appl. Microbiol. Biotechnol.
PUBLISHED: 01-24-2009
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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.
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Current patents related to Bacillus thuringiensis insecticidal crystal proteins.
Recent Pat DNA Gene Seq
PUBLISHED: 01-20-2009
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This work categorizes a number of patents related to Bacillus thuringiensis insecticidal crystal proteins. The patents are classified into groups according to the type of toxins appearing in the claims. The purpose of the summary is to promote the application of B. thuringiensis insecticidal crystal proteins and the development of patentable technologies.
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An engineered Bacillus thuringiensis strain with insecticidal activity against Scarabaeidae (Anomala corpulenta) and Chrysomelidae (Leptinotarsa decemlineata and Colaphellus bowringi).
Biotechnol. Lett.
PUBLISHED: 01-14-2009
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A genetically-engineered Bacillus thuringiensis (Bt) strain, 3A-HBF, with a broad insecticidal spectrum was constructed by introducing the recombinant plasmid pSTK-3A containing cry3Aa7 into the wild-type Bt strain HBF-1 containing the cry8Ca2 gene. The Cry3Aa7 protein produced by strain 3A-HBF was verified by SDS-PAGE and Western blotting. Flat rectangular crystals of Cry3Aa7 protein were observed besides spherical crystals (Cry8Ca2). The plasmid pSTK-3A was stable when strain 3A-HBF was grown in medium without antibiotics. The growth rate of 3A-HBF was not significantly different from that of the recipient strain, HBF-1. Strain 3A-HBF showed toxicity against two families of pests, Scarabaeidae and Chrysomelidae pests, which are susceptible to Cry8Ca (Anomala corpulenta) and Cry3Aa (Leptinotarsa decemlineata and Colaphellus bowringi). The 50% lethal concentrations of 3A-HBF against A. corpulenta, L. decemlineata and C. bowringi were 0.730 x 10(8) c.f.u./g dry soil, 1.74 microg/ml and 1.15 microg/ml, respectively.
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Characterization of two novel cry8 genes from Bacillus thuringiensis strain BT185.
Curr. Microbiol.
PUBLISHED: 01-07-2009
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Two novel cry8-type genes, cry8Ea1 and cry8Fa1, obtained from a Holotrichia parallela-specific Bacillus thuringiensis strain, BT185, were characterized. Findings showed that cry8Ea1 and cry8Fa1 encoded polypeptides of 1164 and 1174 amino acid residues, respectively. The deduced amino acid sequences of both Cry8Ea1 and Cry8Fa1 polypeptides are the most similar to that of Cry8Ba1. Eight conserved blocks (blocks 1-8) exist in Cry8Ea1 and Cry8Fa1 polypeptides compared with known Cry proteins. Cry8Ea1 and the Cry8Fa1 toxins could form spheric crystals when they were expressed in the acrystalliferous mutant strain HD73(-). The spores and crystals from the recombinant strain containing cry8Ea1 were toxic to Holotrichia parallela, with an LC(50) of 0.0875 x 10(8) colony-forming units (CFU)/g. However, Cry8Fa1 expressed in the recombinant strain was not toxic to H. parallela, Anomala corpulenta, or H. oblita.
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[Comparison of codon optimizations of cry1Ah1 gene in rice].
Sheng Wu Gong Cheng Xue Bao
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cry1Ah1, one of holo-type cry genes, cloned in this laboratory from Bacillus thuringiensis strain has been patented in China, and it encoded a protein with strong insecticidal activity against certain lepidopteran insect pests, such as Chilo suppressalis. cry1Ah1 gene is exhibiting good application prospects. In order to improve the expression level of cry1Ah1 gene in rice, and investigate the effect of codon usage preference of gene expression, we designed five different optimized schemes for cry1Ah1 insecticidal critical fragment in accordance with bias of rice codon, to improve G+C content, removed the shear signal and unstable factors. Optimized cry1Ah1 genes were transformed into Escherichia coli Rosetta (DE3) respectively, and 65 kDa polypeptides was expressed normally in inclusion body separately. All of these expressed polypeptides showed insecticidal activity against 2nd-instar larvae of Plutella xylostella and neonate of Chilo suppressalis. After transformation with modified cry1Ah1 genes into Var nippobare, the transgenic rice seedlings were detected by PCR, the positive rate containing target gene was more than 87%. Afterwards, the results of real-time RT-PCR and ELISA assay indicated that the highest expression level of five modified cry1Ah1 genes was that using the highest frequent codons. Average expression amount of Cry1Ah1 polypeptides was 0.104% of total soluble proteins from the positive transgenic rice.
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PeanutDB: an integrated bioinformatics web portal for Arachis hypogaea transcriptomics.
BMC Plant Biol.
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The peanut (Arachis hypogaea) is an important crop cultivated worldwide for oil production and food sources. Its complex genetic architecture (e.g., the large and tetraploid genome possibly due to unique cross of wild diploid relatives and subsequent chromosome duplication: 2n = 4x = 40, AABB, 2800 Mb) presents a major challenge for its genome sequencing and makes it a less-studied crop. Without a doubt, transcriptome sequencing is the most effective way to harness the genome structure and gene expression dynamics of this non-model species that has a limited genomic resource.
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Detection and identification of vegetative insecticidal proteins vip3 genes of Bacillus thuringiensis strains using polymerase chain reaction-high resolution melt analysis.
Curr. Microbiol.
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In this study, vegetative insecticidal proteins vip3 genes from Bacillus thuringiensis strains were detected based on polymerase chain reaction-high resolution melt (PCR-HRM) analysis. A pair of primers was designed according to the conservative sequences in 150 bp region of the known vip3 subfamily. The 150 bp regions of difference vip3 genes have only a few nucleotide difference vip3 genes were detected in 8 of 11 standard B. thuringiensis strains, and vip3Aa genes, vip3Af genes and vip3Ba gene can be distinguished as different melting curves by this method. The results demonstrate the utility of the HRM assay for mutant screening using vip3 gene. The PCR-HRM method may be a valuable and reliable tool for specific detection and identification of vip3 genes.
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