• Food Science and Biotechnology
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• 제17권2호
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• pp.219-227
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• 2008

Bacillus thuringiensis was isolated as a pathogen of the sotto disease of silkmoth larvae about a hundred years ago. Since then, this bacterium has attracted attentions of not only insect pathologists but also many other scientists who are interested in its strong and specific insecticidal activity. This has led to the recent worldwide development of B. thuringiensis-based microbial insecticides and insect-resistant transgenic plants, as well as a landmark discovery of par asp orin, a cancer cell-specific cytotoxin produced by B. thuringiensis. In this review, we describe examination of interaction between inclusion proteins of B. thuringiensis and brush border membrane of insects using a surface plasmon resonance-based biosensor, identification and characterization of parasporin-4, the latest parasporin produced by the B. thuringiensis A1470 strain, and an effective method for preparing the parasporin-4 from inclusion bodies expressed in the recombinant Escherichia coli cells.

• 농약과학회지
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• 제15권2호
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• pp.166-176
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• 2011

제주도의 녹차 밭에 서식하는 딱정벌레목인 청동풍뎅이 (Anomala albopilosa)의 사체와 녹차 밭 토양에서 분리한 Bacillus thuringiensis CAB530 균주의 생물효과를 검토하였다. 이 균주는 몇 종류의 해충에 대한 살충활성에서 난방제 농업해충 가운데 하나인 파밤나방에 높은 효과를 나타냈다. 파밤나방 2령 유충에 대한 살충활성 검정에서 CAB530 균주는 $LC_{50}$값이 $1.49{\times}10^4$(cfu/$m{\ell}$)으로 고활성을 보였다. 이 균주가 생산하는 살충성 독소단백질의 SDS-PAGE에서는 파밤나방에 살충활성이 있는 기존의 B. thuringiensis subsp. kurstaki와 비슷한 130kDa의 밴드를 나타내었다. 또한 파밤나방 중장액으로 반응을 시킨 후에 약 65kDa의 활성 독성단백질을 확인할 수 있었다 PCR수행에서 CAB530 균주는 cry1Aa, cry1Ab, cry1C, cry1D, cry1F 그리고 cry1I등 6 개의 유전자가 존재하는 것으로 밝혀졌으며, B. thuringiensis subsp. kurstala기준 균주와 차이가 있었다. 딱정벌레목에서 분리 선발한 B. thuringiensis CAB530균주는 crystal의 형태와 SDS-PAGE의 결과는 B. thuringiensis subsp. kurstaki와 유사하게 나타났지만, 살충활성 검정과 PCR product 전기영동 결과는 B thuringiensis subsp. aizawai와 유사하게 나타났다.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.509-514
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• 2004

Six new serovarieties of B. thuringiensis carrying specific H-antigen have minor differences in biochemical characteristics and morphological characteristics of crystals, which are commonly resistant against four antibiotics. The B. thuringiensis serovar. coreanensis is nontoxic to silkworm larvae, but it is moderately toxic against the Culex pipiens larvae. The B. thuringiensis serovar. konkukian and leesis are nontoxic against mosquitos larvae, but are toxic against silkworm larvae. The B. thuringiensis serovar. seoulensis, sooncheon, and yosoo are highly toxic to B. mori larvae and moderately toxic to C. pipiens larvae. The six serovarieties harbor different plasmid DNA patterns. A 102-kDa protein is a major crystal protein in the four serovarieties and a 86-kDa protein is in one serovariety.

• 한국미생물·생명공학회지
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• 제14권3호
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• pp.259-264
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• 1986

The compositions of the four madia and their pHs for delta endotoxin production by B. thuringiensis serovar kurstaki 3a3b were examined. In the M-4 media out of the 4 media at pH8, the production of the endotoxin and spore formation were maximal. The mean generation times of the bacterium were 53.4 minutes in the M-1 media, 98 in the M-2, 132 in the M-3, and 127.5 in the M-4. The proper pHs of the media for the endotoxin production appeared to be pH 7 to 8. In the M-4 media, the lag time lasted about 5 hours.

• 한국잠사곤충학회지
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• 제36권2호
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• pp.157-161
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• 1994

B. thuringiensis subsp. kurstaki Cry-B에서 cryIA(b) 유전자 promoter 조적을 받는 cryIA(c) 유전자와 그 자신의 promoter 조절을 받는 cryIIA 유전자의 발현 여부와 내독소 단백질의 형태를 관찰하기 위하여, 이들 두 내독소 단백질 유전자를 B.thuringiensis - E. coli shuttle vector를 이용하여 발현벡터 pKC1A와 pKC2A를 각각 제작하였다. 발현벡터 pKC1A와 pKC2A를 B. thuringiensis subsp. kurstaki Cry-B 균주에 형질전환시키고, 이들 형질전환체로부터 각각 bipyramid형과 cuboid형의 정상적인 내독소 단백질이 발현되었음을 확인하였다.

• 한국식품영양학회지
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• 제9권4호
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• pp.459-465
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• 1996

지금까지 많은 연구가 되어 있지 않던 Bacillus thuringiensis serovar. darmstadiensis의 내독소를 Renografin-76 단계적 기울기 원심분리로 분리하여 전자 현미경으로 관찰하여 이중피라미드 구조를 가진 독소 단백질을 확인하였으며 B. thuringiensis serovar. kurstaki HD1의 독소 생성유전자와 B. thuringiensis serovar. darmstadiensis의 유전자가 유사성이 있다는 보고를 근거로 하여 B. thuringiensis serovar. HD1의 독소 생성유전자를 가진 프로브(pUYBT 9044)로 이용하여 colony hybridization 및 Southern hybridization 한 결과 2.6Kb EcoRI 단편 및 3.6Kb Hind III 단편을 선발할 수 있었다. 이들 단편들은 B. thuringiensis serovar. kurstaki HD1 독소 유전자와 hybridization시 유사성이 있었다. 특히 3.5Kb HindIII 단편은 2.6Kb EcoR I 단편에 클로닝되어 있는 1.8Kb의 HD1 독소 유전자와 유사성이 있는 부분을 공유하고 있었으며 1.0Kb 정도의 EcoR I-HindIII 부분이 더 삽입한 것을 알 수 있었다.

• Journal of Microbiology
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• 제42권4호
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• pp.340-345
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• 2004

To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis $Cry^-B$ strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the crylAc gene (pProAc-GFP). The B. thuringiensis $Cry^-B$ strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immu­noblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single spe­cies, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis $Cry^-B$ strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuring­iensis.

• Journal of Microbiology and Biotechnology
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• 제4권3호
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• pp.171-175
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• 1994

To find new crystal protein genes, we screened 42 Bacillus thuringiensis strains of serovar standards by Southern hybridization with a cryI-specific probe which was amplified from B. thuringiensis subsp. kurstaki HDl by polymerase chain reaction (PCR). Two strains, B. thuringiensis subsp. entomocidus HD9 and subsp. subtoxicus HD109, generated weak signals under the low-stringency hybridization conditions. Further analysis with Southern hybridization revealed that the two strains contained cryIB genes which are slightly different from those of B. thuringiensis subsp. thuringiensis HD2. These results were confirmed by PCR with cryIB-specific primers followed by the restriction analysis of PCR products.

• International Journal of Industrial Entomology and Biomaterials
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• 제1권2호
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• pp.115-122
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• 2000

A Bacillus thuringiensis designated NT0423, belonging to B. thuringiensis subsp. aizawai (H 7), was isolated from samples of dust and soil of sericultural farms. B. thuringiensis NT0423 having dualspecificity against Lepidoptera and Diptera produced bipyramidal inclusions consisting of two major polypeptides of approximately 130- and 70-kDa. Proteolytic processing by trypsin and gut juice of Bombyx mori yielded predominant proteins with molecular masses of about 66-kDa. The whole crystal protein of B. thuringiensis NT0423 immunologically was related to that of B. thuringiensis subsp. aizawai. PCR analysis showed that B. thuringiensis NT0423 has at least five crystal protein genes including cryIA(a), cryIA(b), cryIC, cryID and cryIIA, and southern blot was determined the location of each gene on intact and enzyme-digested plasmid DNA fragments. Except for cryIA(a) gene on the high molecular weight plasmid of 165-kb, all of four genes were located on the plasmid of 66-kb. The production of $\beta$-exotoxin from B. thuringiensis NT0423 was identified by the HPLC analysis. In addition, the $\beta$-exotoxin showed its ability to prevent pupation of treated larvae of house flies (Musca domestica) from developing into normal adults.

• 한국응용곤충학회지
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• 제36권3호
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• pp.264-269
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• 1997

뽕나무하늘소(Apriona germari) 및 왕똥풍뎅이 (Aphodius apicalis) 사충으로부터 4종의 Bacillus thuringiensis를 분리하였다. B. thuringiensis의 편모 항원에 의한 동정 결과, 4종의 분리된 B. thuringiensis 중에서 1종은 darmstadiensis 아종으로 판명되었으나, 나머지 3종은 33종의 어느 B. thuringiensis 편모 항체와도 반응하지 않았다. 분리된 균주의 포자와 내독소 단백질 혼합물을 이용하여 뽕나무하늘소와 왕똥풍뎅이, 누에(Bombyx mori) 및 빨간집모가(Cules pipiens pallens) 유충에 대하여 생물검정한 결과, 이들 분리주들은 검정된 곤충에 대하여 독성을 갖지 않는 것으로 나타났다. 아울러 SDS-PAGE와 agarose gel electrophoresis를 이용하여 분리된 4종의 B. thuringiensis의 내독소 단백질과 plasmid DNA 패턴을 조사한 결과, darmstadiensis와 이미 보고된 20종의 무독성 B. thuringiensis와 차이를 보여 새로운 무독성 균주로 사료된다.