Biochemical And Multiplex Pcr Analysis Of Toxic Crystal Proteins To Determine Genes In Bacillus Thuringiensis Mutants (original) (raw)
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PCR analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis
Applied and environmental microbiology, 1994
A method allowing rapid and accurate identification of different subgroups within the insecticidal crystal CryI protein-producing family of Bacillus thuringiensis strains was established by using PCR technology. Thirteen highly homologous primers specific to regions within genes encoding seven different subgroups of B. thuringiensis CryI proteins were described. Differentiation among these strains was determined on the basis of the electrophoretic patterns of PCR products. B. thuringiensis strains, isolated from soil samples, were analyzed by PCR technology. Small amounts of bacterial lysates were assayed in two reaction mixtures containing six to eight primers. This method can be applied to rapidly detect the subgroups of CryI proteins that correspond with toxicity to various lepidopteran insects.
World Journal of Microbiology & Biotechnology, 1998
A new cry gene (cry1Ea4) was cloned and sequenced from a Bacillus thuringiensis isolate native to Mexico (LBIT-147). The gene coded for a 133kDa protoxin which had greater than 99% homology with the holotype Cry1Ea1, as only four mismatches were found between the two amino acid sequences. When the Cry1Ea4 toxin was expressed in a crystal-negative strain of B. thuringiensis, bipyramidal crystals were produced. Purified crystals from this recombinant strain and from the holotype (Cry1Ea1) were bioassayed against first instar larvae of the tobacco hornworm. Statistically different mean LC50 values indicated that Cry1Ea4 was more toxic than its holotype. This increase in toxicity may be attributed to the three amino acids which differ from the holotype sequence in the toxic fragment.
PCR-based identification of Bacillus thuringiensis pesticidal crystal genes
Fems Microbiology Reviews, 2003
The polymerase chain reaction (PCR) is a molecular tool widely used to characterize the insecticidal bacterium Bacillus thuringiensis. This technique can be used to amplify specific DNA fragments and thus to determine the presence or absence of a target gene. The identification of B. thuringiensis toxin genes by PCR can partially predict the insecticidal activity of a given strain. PCR has proven to be a rapid and reliable method and it has largely substituted bioassays in preliminary classification of B. thuringiensis collections. In this work, we compare the largest B. thuringiensis PCR-based screenings, and we review the natural occurrence of cry genes among native strains. We also discuss the use of PCR for the identification of novel cry genes, as well as the potential of novel technologies for the characterization of B. thuringiensis strains.
Molecular characterization and PCR-based screening of cry genes from Bacillus thuringiensis strains
3 Biotech, 2017
Novel cry genes are potential candidates for resistance management strategies, due to their different structures and modes of action. Therefore, it is desirable to clone and express novel cry genes from several new isolates of Bacillus thuringiensis (Bt). In the present study, 28 Bt strains were characterized at morphological and molecular level. All these strains are Gram positive, endospore forming and had shown different crystal morphologies when viewed under the microscope. The ARDRA (16S rDNA PCR-RFLP technique) with AluI, HaeIII, HinfI and TaqI produced unique and distinguishable restriction patterns used for the molecular characterization of these isolates. Based on UPGMA clustering analysis, Bt strains showed significant molecular diversity and the dendrogram obtained differentiated 28 Bt strains into 1 major cluster at a similarity coefficient 0.56. PCR analysis demonstrated that the Bt strains showed diverse cry gene profiles with several genes per strain. The Bt strain G3C1 showed the presence of maximum cry-type genes by PCR. The toxicological characterization of these cry genes will have huge importance in transgenic technology and will be useful in transgenesis of crop plants for better resistance management. Keywords Bacillus thuringiensis Á Delta-endotoxin Á cry genes Á PCR Á 16S rDNA Á ARDRA Á Screening Abbreviations Bt Bacillus thuringiensis Cry ptrotein Crystal protein PCR Polymerase chain reaction ARDRA Amplified ribosomal DNA restriction analysis UPGMA Unweighted pair-group method for arithmetic average & Devendra Jain
Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System
On the basis of the known cry gene sequences of Bacillus thuringiensis, three sets of primers were designed from four conserved blocks found in the delta-endotoxin-coding region. The primer pairs designed amplify the regions between blocks 1 and 5, 2 and 5, and 1 and 4. In silico analyses indicated that 100% of the known three-domain cry gene sequences can be amplified by these sets of primers. To test their ability to amplify known and unknown cry gene sequences, 27 strains from the CINVESTAV (LBIT series) collection showing atypical crystal morphology were selected. Their DNA was used as the template with the new primer system, and after a systematic amplification and sequencing of the amplicons, each strain showed one or more cry-related sequences, totaling 54 different sequences harbored by the 27 strains. Seven sequences were selected on the basis of their low level of identity to the known cry sequences, and once cloning and sequencing of the complete open reading frames were done, three new cry-type genes (primary ranks) were identified and the toxins that they encode were designated Cry57Aa1, Cry58Aa1, and Cry59Aa1 by the B. thuringiensis Toxin Nomenclature Committee. The rest of the seven sequences were classified Cry8Ka2, Cry8-like, Cry20Ba1, and Cry1Ma1 by the committee. The crystal morphology of the selected strains and analysis of the new Cry protein sequences showed interesting peculiarities.
Applied and Environmental Microbiology, 1995
DNA dot blot hybridizations with a cryV-specific probe and a cryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific) insecticidal crystal protein gene contents, respectively. The cryV-specific probe hybridized to 12 of the B. thuringiensis strains examined. Most of the cryV-positive strains also hybridized to the cryI-specific probe, indicating that the cryV genes are closely related to cryI genes. Two cryV-type genes, cryV1 and cryV465, were cloned from B. thuringiensis subsp. kurstaki HD-1 and B. thuringiensis subsp. entomocidus BP465, respectively, and their nucleotide sequences were determined. The CryV1 protein was toxic to Plutella xylostella and Bombyx mori, whereas the CryV465 protein was toxic only to Plutella xylostella.
Diversity of Insecticidal Crystal proteins (ICPs) of indigenous Bacillus thuringiensis strains
Tribhuvan University Journal of Microbiology, 2018
Objectives: The purpose of this study was to characterize the indigenous Bacillus thuringiensis (Bt) isolated from the soil samples of Terai. Methods: A total of 50 soil samples were collected from cultivated and barren fi elds of Terai region. Isolation was carried out using the acetate selection protocol Nutrient broth (NB) was acetated by using 0.25M sodium acetate which is a selective enrichment method for isolation of Bt. Characterization of the isolate was done by phenotyping methods (microscopy and biochemical). Results: No distinct variation was observed between the isolates of cultivable and uncultivable lands. Bt were categorized into 7 different types based on colony morphology. The dominant colony was fried egg type identical with the reference strain, followed by fl at white type of colony. The result showed that even though the colony morphology was same but the ICPs (Insecticidal crystal proteins) shapes produced by them varied, rod shapes (53.57%), spherical (10.71%), ovoid (8.3%), amorphous (17.85%), capheaded (9.5%). ICPs morphology revealed the cry1, cry2, cry3, cry4, cry8, cry 9, cry10 and cry11 types of gene may be present in the native isolates. Conclusion: This study represents the fi rst report of several indigenous Bacillus thuringiensis strains with signifi cantly different ICPs producing strains from hot tropical climate.
Pakistan Journal of Zoology, 2013
Bacillus thuringiensis (Bt) is endospore former, Gram positive bacterium and makes parasporal crystals (Cry proteins), which kill particular target pests of different crops. Bt isolated from different localities of Pakistan were screened for cry1Ca and cry1Cb domain III genes by polymerase chain reaction. Among all the screened Bt strains, five were positive for cry1C domain III gene, 3 of which were positive for cry1Ca and 2 for cry1Cb. Confirmation was done by colony PCR, restriction analysis, nucleotide sequencing and alignment on BLAST. The complete gene (3.6kb) encoding cry1Ca endotoxin of one of the Bt isolate (MS-SBS Bt1) was amplified and analyzed for the nucleotide sequence. The nucleotide and deduced amino acid sequences of endotoxin gene was compared with that of Bt strains available in the literature. The genes amplified from the positive strains had 99% similarity and had 100% same deduced amino acid with that of Bt strains reported in the Gene Bank. Full gene sequence was submitted in Gene Bank.
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 0 -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 0 -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 0 -truncated cry1Ab gene. Insecticidal activity assay showed that the recombinant 3 0 -truncated cry1Ab gene product was toxic to larvae of both Helicoverpa armigera and Spodoptera litura.
Pakistan journal of zoology
The toxic region (2.0 kb) of cry4B gene amplified from six different local isolates of Bacillus thuringiensis (DAB Bt 1-6) was cloned in pTZ57R/T. E. coli DH5α were transformed with this recombinant plasmid. The toxic region was restricted with EcoR1 and HindIII, and ligated in the expression vector pT7-7. E. coli BL21C were transformed with the recombinant DNA. The expression profile of recombinant organism containing cry4B gene was studied. The expression conditions were optimised with respect to IPTG concentration, time of induction and incubation temperature. It was found that the high level of expression occurred at 0.5mM of IPTG, at 37 o C for 3 hours. The toxicity of genetically modified organisms and crude recombinant Cry4B proteins was determined against third instar larvae of mosquito Anopheles stephensi. The LC 50 of E. coli transformed with cry4B gene isolated form six Bt isolates against 3 rd instar larvae of Anopheles stephensi ranged between 175±3.34-288±3.02 µg/ml, w...