• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.04a
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• pp.72-72
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• 2003

A new Bacillus thuringiensis strain (Kl), having high toxicities to Plutella xylostella and Spodoptera exigua was isolated from Korean soil sample. It was determined to belong to subsp. kurstaki (H3a3b3c) and produced bipyramidal inclusion. PCR-RFLP analysis showed that this isolate contains three novel cryl-type crystal protein genes in addition to crylAa and crylE genes. (omitted)

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1498-1503
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• 2007

To identify novel crystal proteins, Bacillus thuringiensis 2385-1 was isolated from Korean soil samples and characterized. The H-serotype of 2385-1 was identical to that of subsp. kenyae (H4a4c), and its crystal toxin was bipyramidal-shaped. However, 2385-1 showed a much higher toxicity towards Plutella xylostella and Spodoptera exigua larvae than subsp. kenyae. In addition, the crystal protein profile and plasmid DNA pattern of 2385-1 differed from those of subsp. kenyae. To verify the crystal protein gene types of 2385-1, a PCR-RFLP analysis was performed, and the results revealed that 2385-1 contained two novel cry1-type crystal protein genes, cryl-5 and cry1-12, in addition to the crylJal gene. The deduced amino acid sequences of cryl-5 and cry1-12 showed a 97.9% and 75.7% sequence similarity with the CrylAb and CrylJa crystal proteins, respectively. Among the novel crystal proteins, Cry1-5 showed a high toxicity towards P. xylostella and S. exigua larvae. In conclusion, B. thuringiensis 2385-1 is a new isolate in terms of its gene types, and should be a promising source for an insecticide to control lepidopteran larvae.

• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.322-326
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• 1994

Bacillus thuringiensis strain BT-14 was isolated from alfalfa dust in Korea. The strain BT-14 produced one bipyramidal crystal and one spore in the cell. The biochemical characteristics of the strain BT-14 were similar to those of Bacillus thuringiensis subsp. kurstaki HD-l. Examination of its antibiotic resistance revealed that while the strain BT-14 was less resistant than BTK HD-l to ampicillin, gentamycin, neomycin and tobramycin, it was more resistant to amikacin than BTK HD-l. The $\delta$-endotoxin crystal of strain BT-14 consisted of a single protein with a high molecular weight of ca 135 KD on a 10% SDS-PACE. The strain BT-14 contained at least nine different plasmids with sizes of 2.9, 5.3, 5.8, 6.2, 9.4, 15.1, 18.1, 23.1 and 79 Kb. In insect bioassay, the isolated strain BT-14 showed lethality of 67% against Plutella xylostella larvae at dilution of 5$\times$$l0^{-4}$ (5$\times$l0 to 3$\times$$l0^2$ spores/ml), which is, almost equivalent to that of BTK HD-l.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1093-1098
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• 2001

The gene coding for a Lepidoptera-specific insecticidal crystalline (or control) protein (ICP), recognized as cryIAc, from Bacillus thuringiensis subsp. kurstaki HD-73, was cloned into the vector pBluscript ll SK-, and then transformed in Escherichia coli $DH5{\alpha}$. The clone was named EBtIAc and the chimeric phagemid, as pEBtIAc. Hyperexpression of CryIAc protoxin was observed in the extract of the culture of E. coli harboring pEBtIAc. Crystalline protoxin was purified by differential solubility. It was dissolved in alkaline pH, and exposed to trypsin to be activated. The molecular weights of the pro- and activated toxins on SDS-PAGE were estimated to be ca. 130 kDa and 60 kDa, respectively. The toxicity was tested by force-feeding larvae of gypsi moth (Lymantria diapar) with trypsinized protoxin. Using the batch of biologically active form of the toxin as an immunogen, anti-CryIAc antiserum was raised in a New Zealand white rabbit. Immunoglobulin G was fractionated from the seam by Protein-A sepharose affinity chromatography. Immunoreactivity of the antibody was examined by dot and Westerns blottings. It has been found that the anti- CryIAc antibody recognized the purified toxin at a level below a nanogram in terms of quantity. Using the antibody some of Bt-corns were able to be differentiated from tons of corn kernels which were imported from America as forage crops.

• BMB Reports
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• v.47 no.10
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• pp.546-551
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• 2014

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.547-559
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• 2007

Bacillus thuringiensis (Bt) was first described by Berliner [10] when he isolated a Bacillus species from the Mediterranean flour moth, Anagasta kuehniella, and named it after the province Thuringia in Germany where the infected moth was found. Although this was the first description under the name B. thuringiensis, it was not the first isolation. In 1901, a Japanese biologist, Ishiwata Shigetane, discovered a previously undescribed bacterium as the causative agent of a disease afflicting silkworms. Bt was originally considered a risk for silkworm rearing but it has become the heart of microbial insect control. The earliest commercial production began in France in 1938, under the name Sporeine [72]. A resurgence of interest in Bt has been attributed to Edward Steinhaus [105], who obtained a culture in 1942 and attracted attention to the potential of Bt through his subsequent studies. In 1956, T. Angus [3] demonstrated that the crystalline protein inclusions formed in the course of sporulation were responsible for the insecticidal action of Bt. By the early 1980's, Gonzalez et al. [48] revealed that the genes coding for crystal proteins were localized on transmissible plasmids, using a plasmid curing technique, and Schnepf and Whiteley [103] first cloned and characterized the genes coding for crystal proteins that had toxicity to larvae of the tobacco hornworm, from plasmid DNA of Bt subsp. kurstaki HD-1. This first cloning was followed quickly by the cloning of many other cry genes and eventually led to the development of Bt transgenic plants. In the 1980s, several scientists successively demonstrated that plants can be genetically engineered, and finally, Bt cotton reached the market in 1996 [104].

• Korean journal of applied entomology
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• v.43 no.1
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• pp.35-41
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• 2004

Bacillus thuringiensis produces crystal proteins toxic to medically and agriculturally important pests during sporulation. To improve the activity of insecticidal crystal protein in applying to mosquito larval control, an expression vector, pSyn4D harboring the mosquitocidal cry11Aa gene under control of psbA promoter of Amaranthus hybridus was constructed. This expression vector was transformed into Synechocystis PCC6803 and a transformant, Tr2C was selected with kanamycin. The mosquitocidal cry11Aa gene was stably integrated Into genomic DNA of Tr2C in PCR detection using cry11Aa-specific primers. The transformant expressed 72-kDa Cry11Aa protein and median lethal time (LT$\sub$50/) was approximately 2.1 days for Culex tritaeniorhynchus larvae and 0.7 day for Anopheles sinensis larvae, respectively. These results suggest this transformant can be used for mosquito larval control as a biological control agent.

• Korean journal of applied entomology
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• v.56 no.1
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• pp.19-28
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• 2017

Polydnaviruses (PDVs) are a group of double-stranded DNA viruses symbiotic to some endoparasitoid wasps. Cotesia plutellae bracovirus (CpBV) is a PDV symbiotic to an endoparasitoid wasp, C. plutellae, parasitizing young larvae of Plutella xylostella. An early expressed gene, CpBV-ELP1, plays an important role in the parasitism by suppressing host cellular immunity by its cytotoxic activity against hemocytes. This study aimed to test its oral toxicity against insect pest by expressing it in a recombinant tobacco plant. A recombinant CpBV-ELP1 protein was produced using a baculovirus expression system and secreted to cell culture medium. The cell cultured media were used to purify CpBV-ELP1 by a sequential array of purification steps: ammonium sulfate fractionation, size exclusion chromatography, and ion exchange chromatography. Purified rCpBV-ELP1 exhibited a significant cytotoxicity against Spodoptera exigua hemocytes. CpBV-ELP1 was highly toxic to the fifth instar larvae of S. exigua by injection to hemocoel. It also showed a significant oral toxicity to fifth instar larvae of S. exigua by a leaf-dipping assay. CpBV-ELP1 was cloned into pBI121 vector under CaMV 35S promoter with opaline synthase terminator. Resulting recombinant vector (pBI121-ELP1) was used to transform Agrobacterium tumefaciens LBA4404. The recombinant bacteria were then used to induce callus of a tobacco (Nicotiana tabacum Xanthi) leaves and subsequent generation (T1) plants were selected. T1 generation tobacco plants expressing CpBV-ELP1 gave significant insecticidal activities against S. exigua larvae. These results suggest that CpBV-ELP1 gene can be used to control insect pests by constructing transgenic crops.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.497-506
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• 1998

The expression in Escherichia coli of a cloned insecticidal protein (ICP) gene from Bacillus thuringiensis var. kurstaki HD1 in pHLN1-80 (+) and pHLN2-80(-) plasmids was investigated through deletions in promoters, transcription start point, and termination region. Six recombinant plasmids were constructed in an attempt to analyze the overexpression of the ICP in relations to its gene structure. The amounts of ICP produced from the recombinants were measured by SDS-PAGE and confirmed by Western blot analysis. One clone was not overexpressed which having only -80 bp (contained BtI promoter) part of the ICP gene promoter (without Plac promoter), the right-oriented ICP gene and the termination region. Removal of 350 bp from upstream region of the Plac of the clone pHLN2-80 (-) resulted in overexpression of the ICP. One clone was not overexpressed in which the clone consisted of -72 bp part of the ICP promoter without the transcription start point and the transcriptional termination region, and having the right-oriented ICP gene sequence. One clone consisting of the inverted ICP gene sequence, the -72 bp ICP gene promoter, and without the termination region caused overexpression. One clone which consisted of the inverted ICP gene, the -72 bp ICP gene promoter and the termination sequence was overexpressed. These results indicated that the Plac promoter, transcription termination region, the inverted ICP gene insertion, and the -80 bp or -72 bp part of the ICP gene promoters were concerned in the overexpression of the ICP gene in the recombinant plasmid, and also the overexpression mechanism might result from the disruption of the transcription-suppressing regions in the promoter regions.

• Korean journal of applied entomology
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• v.47 no.1
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• pp.87-93
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• 2008

Bacillus thuringiensis with selected high toxicities against tobacco cutworm, Spodoptera litura were isolated from domestic soils. When being observed under a phase-contrast microscope, the insecticidal crystal proteins were showed a bipyramidal crystal types. New CAB 109 isolate was identified to B. thuringiensis subsp. aizawai in the H serotype. As a results of insecticidal activities between CAB 109 isolate and 3 existing ready-made products against 3rd larva of S. litura, CAB 109 isolate showed 100% mortality with spore concentration $(1.3{\times}10^7cfu/ml)$. It was a very high insecticidal activity compared with a existing ready-made B. t. products. $LD_{50}$ values of CAB 109 isolate was $9.78{\times}10^5,\;6.87{\times}10^6\;and\;1.83{\times}10^7cfu/ml$ spore concentration against 2nd, 3rd and 4th larva of S. litura, respectively. Unlike Plutella xylostella, S. litura was slowly died after application up to 7 days. The weight of S. litura larva applied with CAB 109 isolate were 6-7 times less than controlled group. Even though it didn't die, it did not grow into next larva. The result observed with scanning electron microscope was that CAB 109 isolate of B. t. aizawai formed a typical bipyramidal crystal protein type. Otherwise, when CAB 109 isolate was examined with SDS-PAGE and with trypsin, there was no difference between CAB 109 strain and ready-made products of B. thuringiensis.