• 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.

• 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.

• 한국응용곤충학회지
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• 제35권1호
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• pp.85-90
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• 1996

국내에서 분리된 Bacillus thuringiensis NT0423은 나비목과 파리목 곤충에 독성을 보이는 130 kDa의 전형적인 다이아몬드형 내독소 단백질을 생성한다. 이 균주의 파리목에 대한 독성을 강화하기 위하여, B. thuringeinsis NT0423에 모기 유충에 강한 독성을 보이는 B. thuringiensis subsp. morrisoni PG-14의 cryVID유전자를 가지고 있는 pCG10 플라스미드를 electroporation 방법을 이용하여 형질전환하였다. 형질전환체인 B. thuringiensis PT1227내에서 cryIVD와 숙주가 생성하는 원래의 다이아몬드형 130kDa 내독소 단백질 유전자는 그 자신의 형태로 잘 발현되었다. 형질전환체의 모기 유충에 대한 독성은 원래 숙주의 내 독소 단백질과ㅏ 도입된 CryIVD의 상승효과에 의해 현저히 증가하였다.

• International Journal of Industrial Entomology and Biomaterials
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• 제2권2호
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• pp.185-189
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• 2001

Wild type Bacilus thuringiensis subsp. israelensis and B. sphaericus toxins have been used separately as active in ingredients for bacterial insecticides to control mosquito larvae due to their comparable toxicity to chemical insecticides. Cry11B, recently cloned from B. thuringiensis subsp. jegathesan, shows higher toxicity against three major species of mosquito larvae than Cry11A, one of the major component of B. thuringiensis subsp. israelensis inclusion body. To determine whether the combination of cry11B and B. sphaericus binary toxins is as toxic as B. thuringiensis subsp. israelensis parental strain, cry11B and B. sphaericus binary toxins genes were co-expressed as an operon using cytlA promoters/STAB-SD hybrid expression system in B. thuringiensis subsp. israelensis acrystalliferous strain 4Q7. However, unexpectedly, B. sphaericus binary toxins were barely produced, whereas relatively large amount of Cry11B was produced. When this strain was grown in four different media, NB+G and Peptonized Milk produced more toxin proteins and spores per unit of media than GYS and G-Tris. Toxicity of this strain against fourth instar Culex quinquefasciatus was ranged from of 8.3 to 45.7 ng/ml, with NB+G culture being the highest, and GYS culture was the lowest.

• International Journal of Industrial Entomology and Biomaterials
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• 제18권1호
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• pp.18-27
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• 2009

B. thuringiensis strain LY-99 belonging to subsp. alesti (H3a3c), was isolated from Chinese tobacco warehouse and showed significantly high toxicity to Plutella xylostella. For the identification of the cry1-type genes from B. thuringiensis LY-99, an extended multiplex PCRrestriction fragment length polymorphism (PCRRFLP) method was established by using two pairs of universal primers based on the conserved regions of the cry1-type genes to amplify around 2.4 kb cry1-type gene fragments. Then the DNA fragment was cloned into pGEM-T Easy vector and digested with EcoRI and EcoRV enzymes. Through this method, a known cry1-type gene was successfully identified from the reference strain, B. thuringiensis subsp. alesti. In addition, the RFLP patterns revealed that B. thuringiensis LY-99 included a novel cry1A-type gene in addition to cry1Aa, cry1Ac, cry1Be and cry1Ea genes. The novel cry1A-type gene was designated cry1Ah2 (Genbank accession No DQ269474). An inverse PCR method was used to amplify the flank regions of cry1Ah2 gene. Finally, 3143 bp HindIII fragment from B. thuringiensis LY-99 plasmid DNA including 5' region and partial ORF was amplified, and sequence analysis revealed that cry1Ah2 gene from LY-99 showed 89.31% of maximum sequence similarity with cry1Ac1 crystal protein gene. In addition, the deduced amino acid sequence of Cry1Ah2 protein shared 87.80% of maximum identity with that of Cry1Ac2. This protein therefore belongs to a new class of B. thuringiensis crystal proteins.

• 한국잠사곤충학회지
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• 제38권2호
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• pp.154-159
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• 1996

전국 토양, 앙장 농가, 저곡 창고, 시설 원예 단지, 과수원, 산, 밭 등에서 분리한 토양과 먼지 등의 시료로부터 거세미나방속 해충에 강한 독성을 보이는 7개 균주를 분류하였다. 이들을 각각 STB-1에서 STB-7까지 명명하였으며 모두 약 130kDA의 내독소 단백질을 형성하였다. 편모항원성 검정 결과 STB-1, STB-2는 B. thuringiensis subsp. kurstaki와 STB-3, STB-4, STB-5는 subsp. kenyae와 반응하였으며 STB-6, STB-7은 기존의 편모항체와 반응하지 않았다. PCR로 유전자형을 분석한 결과 STB-1은 B. thuringiensis subsp. kurastaki에서는 보고되지 않은 cryIE 유전자를 가지고 있으며 STB-5는 subsp. kenyae와는 다른 유전자형을 보였다. 기존의 편모항체와 반응을 보이지 않은 STB-6와 STB-7은 cryIA(a), cryIA(b), cryIC, cryII를 가지고 있었다.

• Journal of Microbiology and Biotechnology
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• 제19권8호
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• pp.749-759
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• 2009

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDS-PAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.

• International Journal of Industrial Entomology and Biomaterials
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• 제8권1호
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• pp.89-93
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• 2004

Expression of a fusion protein between B. thuringiensis crystal protein, Cry1Ac1 and a scorpion insect toxin (AaIT, Androctonus australis Hector insect toxin) in acrystalliferous B. thuringiensis strain (Cry-B strain) was examined. The cry 1Ac1 gene was cloned in B. thuringiensis-E coli shuttle vector, pHT3101, under the control of the native cry 1Ac1 gene promoter (pProAc) and a gene encoding AaIT was inserted in XhoI site in the middle of the cry 1Ac1 gene (pProAc-ScoR). B. thuringiensis Cry-B strain carrying pProAc-ScoR (PyoAc-ScoR/CB) produced an inclusion body of irregular shape and the expressed fusion protein is approximately 65 kDa in size. Sporulated cells and spore-crystal mixtures of ProAc-ScoR/CB had insecticidal activity against Plutella xylostella larvae, showing $LT_50$ of ProAc-ScoR/CB (22.59 hrs) lower than that of ProAc/CB (30.06 hrs) at $1{\times}{10^7} {CEU/cm^2}$. These results suggest that the fusion protein including a B. thuringiensis crystal protein and an AaIT may be functionally expressed in B. thupingiensis. Moreover, we verified the additive toxicity of AaIT, which is a new feasible candidate for insect control.

• 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.

• 한국잠사곤충학회지
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• 제35권2호
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• pp.120-128
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• 1993

B. t k HD-1 균주에 0.002% SDS와 0.5$\mu$g/ml EtBr을 처리한 결과, 9개의 cry- 변이균주를 분리하였으며 또한 B. t k HD-1균주와 B. cereus 569균주를 혼합배양하는 방법으로 mating 실험을 수행하여 B. t k HD-1으로부터 일부 plasmid가 전이된 11개의 cry+ B. cereus와 2개의 cry=B. cereus를 분리하고 plasmid수와 분자량을 조사하였다. B. t k HD-1의 경우 9개의 plasmid가 존재하였고 일부 plasmid가 curing된 B. t k HD-1변이균주의 경우 29Md plasmid나 44Md plasmid가 반드시 존재하였으나, cry- 변이균주에는 29Md 이상의 모든 plasmid가 소실되어 내독소 단백질 합성에 관여하는 유전자가 기억된 plamid를 결정하였다.