• 한국응용곤충학회지
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• 제47권2호
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• pp.175-184
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• 2008

국내의 난방제 해충에 선택적으로 생물활성을 나타내는 균주를 선발하기 위하여 작물재배 지역의 토양으로부터 채취한 115개의 토양샘플 중 46개의 Bacillus thuringiensis 균주를 분리하였다. 이러한 B. thuringiensis균주를 사용하여 난방제 농업해충에 생물활성을 검정한 결과 배추좀나방(Plutella xylostella)에 CAB119을 포함한 35균주, 파밤나방(Spodoptera litura)에는 CAB128, CAB141균주가, 담배거세미나방(Spodoptera exigua)은 CAB133과 CAB159균주가 효과를 나타냈으며 CAB162균주는 3종류의 모든 해충에 높은 활성을 보였다. 분리된 B. thuringiensis CAB133 균주는 H serotype에 의한 혈청학적 동정과 SDS-PAGE를 통한 독소 단백질 패턴에서 B. thuringiensis subsp. kurstaki (3abc)로 동정되었다. 담배거세미나방 2령 유충에 대한 활성검정의 결과 B. thuringiensis subsp. kurstaki (3abc) CAB133, CAB162의 두 균주에 대한 $LD_{50}$ 값은 각각 0.089, $3.144{\mu}g/ml$로 높은 활성을 나타났다. 담배거세미나방의 령기에 따른 생물실험에서 CAB133 균주 $1.5{\times}10^6(cfu/ml)$에서 1령의 경우 3일, 2령은 5일에 100%의 사충율을 보였다. 담배거세미나방의 경우 B. thuringiensis를 섭식 후 먹기를 중단하여 성장이 멈추면서 $5{\times}7$일 후에 사망하였다. 그러므로 B. thuringiensis의 결정성독소단백질$(1.267{\mu}g/ml)$를 섭식하고 성장하지 못하는 담배거세미나방 유충의 무게는 대조군보다 5일후 약 30배정도 차이로 나타나서 사망하였다.

• Food Science and Biotechnology
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• 제15권4호
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• pp.625-630
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• 2006

Polymerase chain reaction (PCR) method was developed for the specific detection of insect-resistant rice containing cry1Ac gene derived from Bacillus thuringiensis (Bt). Primers were designed from the 35S promoter, NOS terminator, cry1Ac gene, and sucrose phosphate synthase (SPS) for general screening of Bt rice. By sequencing the PCR products from the two putative kinds of Bt rice, we designed a specific primer from the junction region between the cry1Ac gene and the NOS terminator that had been inserted into Bt rice. The construct-specific primer was employed to amplify a 147 bp product in the two lines of Bt rice. No amplified products were observed from the other Bt crops with various Bt genes introduced. In qualitative PCR analysis, the limit of detection was 0.005 ng from genomic DNA of Bt rice. In addition, PCR analysis was performed on 64 kinds of rice presently available in the Korean market, and no Bt rice was detected. This method presented in this paper can be used as a highly sensitive and specific detection method of Bt rice.

• 한국응용곤충학회지
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• 제48권4호
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• pp.509-517
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• 2009

국내에서 분리된 Bacillus thuringiensis subsp. aizawai CAB109균주가 난방제 해충으로 알려진 담배거세미나방과 파밤나방에 동시에 높은 독성을 보이는 것으로 나타났다. B.t. CAB109 균주의 활성을 평가하기 위해 혈청형이 aizawai이면서 미생물농약으로 시판중인 TB-WP제품 및 SC제품과의 살충활성을 비교한 결과, B.t. CAB109균주, TB-WP제품, SC제품은 담배거세미나방 2령충에 대한 반수치사농도($LC_{50}$)가 각각 $1.3{\times}10^5cfu/ml$, $2.3{\times}10^6cfu/ml$, $5.2{\times}10^5cfu/ml$으로 나타났고 파밤나방 2령충에 대한 반수치사농도는 $1.8{\times}10^4cfu/ml$, $1.3{\times}10^6cfu/ml$, $1.5{\times}10^6cfu/ml$으로 나타나 두 종 해충 모두에서 B.t. CAB109 균주가 독성이 더 높은 것을 볼 수 있었다. B.t. CAB109균주가 이미 알려져 있는 aizawai와 비교해 차이가 나는 새로운 유전자를 소유하는지 확인하기 위해 Plasmid DNA를 추출하여 전기영동 한 결과 B.t. subsp. aizawai HD-133과 다른 패턴을 보이는 것을 확인 할 수 있었고 Cry1-Cry5의 primer를 사용하여 PCR을 진행한 결과 B.t. subsp. aizawai CAB109균주는 Cry1Aa, 1Ab, 1C, 1D를 B.t. subsp. aizawai HD-133은 Cry1Aa, 1Ab를 가지고 있음을 확인 할 수 있었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제19권1호
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• pp.199-204
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• 2009

The newly cloned Bacillus thuringiensis cry1-5 gene showed high activity to both Plutella xylostella and Spodoptera exigua, while cry1Ac only showed high activity against P. xylostella but low to S. exigua. Through the alignment of amino acid sequences between Cry1Ac and Cry1-5, we found 12 different residues in domain I (6 residues) and domain II (6 residues). In this study, the modified cry1Ac gene, which is constructed according to a crop-preferring codon usage, was used as a template to construct mutant B. thuringiensis cry1Ac genes based on cry1-5 gene through multi site-directed mutagenesis. Total 63 various mutant cry genes were obtained at 12 positions randomly. Among them, ten mutant cry genes, whose domain I was totally converted and domain II was randomly, were selected to express in baculovirus expression system as a polyhedrin fusion form. The recombinant proteins were 95 kDa in size and were stably activated as 65 kDa by trypsin. The expressed mutant Cry proteins were applied to bioassays against P. xylostella and S. exigua. All mutants showed high insecticidal activity both to P. xylostella and S. exigua similar to cry1-5. These results suggest that these mutant cry genes might be expected of desirable cry genes for introduction to transgenic crops.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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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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• 제27권1호
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• pp.36-42
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• 2017

In this work, we isolated a surface layer protein (SLP) from a Bacillus thuringiensis (Bt) strain to evaluate it cytotoxic effects against MDA-MB-231 human breast cancer cells. AP11 was selected from a g roup of Bt strains using SLP olig onucleotides developed from Bacillus conserved regions. The AP11 strain was grown in Luria Bertani medium until the late exponential phase; an 86 kDa protein was extracted using 5 M LiCl and identified by liquid chromatography-tandem mass spectrometry. It corresponded to a multispecies SLP highly similar to previously described SLPs in Bt. The MDA-MB-231 breast cancer cells $LC_{50}$ was obtained using $0.25{\mu}g/ml$ of the isolated SLP. HaCat non-cancerous cells presented 90% survival using the same protein concentration. Our data suggest that SLP cytotoxicity against MDA-MB-231 could be induced by an interaction with the CDH11 cell membrane receptor.

• Applied Biological Chemistry
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• 제35권5호
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• pp.361-366
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• 1992

토양전염 병원성 사상균인 Fusarium oxyporum, Rhizoctonica solani에 대하여 길항력을 갖는 균주 Bacillus subtilis를 근권 토양에서 분리하여 동정한 후 이들 균주의 사상균에 대한 길항력, 발아율 및 작물의 생육에 미치는 영향력을 검토하였다. 또한 이 균주의 chromosome에 Bacillus thuringiensis(BT) 독소유전자를 삽입하여 균주의 형질변환을 유도하였다. BT 독소 유전자는 southern blotting에 의하여 확인되었으나 이의 최종 생성물인 독소 단백질은 SDS-PAGE에 확인되지 않았다. 이들 형질변환된 균주의 생리 및 생화학적 특성을 조사한 결과 모균주와 차이는 없었으며, BT 독소유전자가 삽입된 균주는 선충의 유충에 대한 생물학적 검정에서는 효과가 인정되지 않았으나 누에에 있어서는 1X 균체 희석액에서 10내지 20% 정도의 치사율이 관찰되었다. 모균주와 BT 독소유전자에 의하여 형질변환된 균주 모두 발아율 및 작물의 생육을 향상시켰다.

• International Journal of Industrial Entomology and Biomaterials
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• 제8권2호
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• pp.145-149
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• 2004

The expression of a fusion protein comprised of the B. thuringiensis crystal protein, Cry1Ac, and enhanced green fluorescent protein (EGFP) in Escherichia coli XLl-blue was examined. Three recombinant plasmids were transformed into E. coli XL1-blue and named as ProAc/Ec, MuEGFP/Ec and ProMu-EGFP/Ec, respectively. All transformants were observed by light and fluorescence microscopy at mid-log phase. The expression in E. coli transformants, ProMu-EGFP/Ec and MuEGFP/Ec, exhibited bright enough fluorescence to be observed. Furthermore, ProMu-EGFP/Ec produced fluorescent inclusions, which may have been recombinant crystals between EGFP and Cry1Ac while MuEGFP/Ec expressed soluble EGFP in cell. In SDS-PAGE, ProAc/Ec had 130 kDa crystal protein band and MuEGFP/Ec had thick 27 kDa EGFP band. However, ProMu-EGFP/Ec had about 150 kDa fusion protein band. Accordingly, these results indicated that a fusion protein between the B. thuringiensis crystal protein and a foreign protein under the lacZ promoter was successfully expressed as granular structure in E. coli. It is suggested that the E. coli expression system by N-terminal fusion of B. thuringiensis crystal protein may be useful as excellent means for fusion expression and characterization of B. thuringiensis fusion crystal protein.

• Journal of Applied Biological Chemistry
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• 제54권4호
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• pp.296-302
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• 2011

해충저항성 Bacillus thuringiensis (Bt) 벼와 낙동벼의 비표적 생물체인 물벼룩(Daphniamagna)에 대한 급성독성시험을 실시한 결과, 해충저항성 Bt벼의 48시간-$EC_{50}$은 4,429.13 mg/L(95% 신뢰한계는 3908.130~5020.363 mg/L), 무영향 농도(NOEC)는 1,800 mg/L이었고, 낙동벼는 48시간-$EC_{50}$은 2,889.56mg/L (95% 신뢰한계는 1,073.407~6,854.321 mg/L), 무영향농도는 1,000 mg/L이었다. 처리기간 중 해충저항성 Bt벼와 낙동벼간의 급성독성에 영향을 미칠 수 있는 요인은 발생하지 않았다.

• Journal of Applied Biological Chemistry
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• 제59권4호
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• pp.373-377
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• 2016

An extracellular metalloprotease, Btmp, was partially purified from the culture supernatant of Bacillus thuringiensis 29-126, isolated from animal feces collected in a zoological garden in Japan, by ultrafiltration, ammonium sulfate precipitation, and a set of chromatography on Sephadex G-75 and High-Q. The molecular mass of the protease was estimated to be 60 kDa by SDS-PAGE. The enzyme showed optimum activity at $50^{\circ}C$ and pH 6.0, and had a half-life of 14 min at $50^{\circ}C$. The enzyme activity was not influenced by $Na^+$, $K^+$, $As^+$, $Mg^{+2}$, $Ca^{2+}$, $Ba^{2+}$, and phenylmethylsulfonyl fluoride, but it was moderately inhibited by $Zn^{+2}$ at a concentration of 1.0 mM, while the activity was significantly inhibited to less than 50 % by $Cu^{2+}$, $Co^{2+}$, $Cd^{2+}$, and ethylenediaminetetraacetic acid. Interestingly, the enzyme was activated to 178 % by 1.0 mM of $Mn^{2+}$. From these results, it may be suggested that the protease is a novel extracellular manganeseactivated metalloprotease.