• 식물조직배양학회지
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• 제24권5호
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• pp.305-311
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• 1997

Bacillus thuringiensis는 그람 양성 토양 세균으로 포자형성시 결정화된 내포체를 형성하는데, 이 내포체를 구성하는 결정단백질은 각 곤충에 대하여 특이적인 독성을 나타낸다. 살충성 결정단백질 중 Cry II A 결정단백질은 인시류와 쌍시류 곤충에 모두 특이적으로 작용한다. 결정단백질은 살충제로서 불안정하고 포장에서 지속성이 낮은 단점을 가지고 있으므로, 본 연구에서는 Cry II A 결정단백질 유전자가 형질전환된 담배 식물을 제작하고자 하였다. cry IIA 유전자가 삽입된 벡터를 대장균에 형질전환한 후, 알칼리 용액에 대한 용해도의 차이를 이용하여 Cry II A 결정단백질(70 kDa)을 분리하였고, 분리한 Cry II A 결정단백질을 trypsin 처리하여 활성화된 Cry II A (50 kDa)를 확인하였다. 식물 형질전환을 위하여 두 개의 CaMV 35S promoters에 의해 발현이 조절되는 식물 발현 벡터에 cry II A 유전자를 클로닝 하였다. 이 식물 발현 벡터를 Agrobacterium을 이용한 엽편형질 전환을 통해 담배(N. tabacum var. Petit Havana SRI)에 형질전환 시켰으며, 재분화 과정을 거쳐 여섯 개체의 형질전환 식물체를 얻었다. Southern blot을 통하여 분석한 결과 세 개체 내에 cry II A 유전자가 존재하였는데, 한 개체에는 하나의 cry II A 유전자가, 또 한 개체에는 두 개의 cry II A 유전자가, 다른 한 개체에는 잘려진 형태의 cry II A 유전자가 존재함을 확인할 수 있었다. 본 연구를 통하여 얻어진 cry II A 형질전환 식물체는 살충성 검정 등을 거친 후 내충성 식물 생산 및 후대 유전 양상 분석을 위한 기초 자료로 이용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제22권6호
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• pp.729-735
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• 2012

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.

• Journal of Microbiology and Biotechnology
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• 제8권5호
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• pp.517-522
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• 1998

The cry1 gene content of a collection of Bacillus thuringiensis strains, which include new isolates from Malaysia and Indonesia, was determined by a multiplex PCR using a set of eight oligonucleotide forward primers specific to cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1Ca, cry1Da, cry1Ea, and cry1Fa genes, and two reverse primers, one specific to cry1Ab and the other common to the remaining cry1 genes. Two-thirds of the 59 strains screened were cry1 positive and contained one to four different genes. The cry gene profiles correlated well with toxicities of the strains to lepidopteran insects. The method can be used for rapid screening of a large number of new isolates as the total DNA extracted by boiling cells from single colonies can be used directly in the PCR. However, it is not suitable for follow-up monitoring of specific commercial strains after application in the field as the PCR product profiles of these strains could not be differentiated from those of new isolates.

• 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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• 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 and Biotechnology
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• 제22권1호
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• pp.133-140
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• 2012

A novel cry2Ab gene was cloned and sequenced from the indigenous isolate of Bacillus thuringiensis subsp. kurstaki. This gene was designated as cry2Ab25 and its sequence revealed an open reading frame of 1,902 bp encoding a 633 aa protein with calculated molecular mass of 70 kDa and pI value of 8.98. The amino acid sequence of the Cry2Ab25 protein was compared with previously known Cry2Ab toxins, and the phylogenetic relationships among them were determined. The deduced amino acid sequence of the Cry2Ab25 protein showed 99% homology to the known Cry2Ab proteins, except for Cry2Ab10 and Cry2Ab12 with 97% homology, and a variation in one amino acid residue in comparison with all known Cry2Ab proteins. The cry2Ab25 gene was expressed in Escherichia coli BL21(DE3) cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Cry2Ab25 protein is about 70 kDa. The toxin expressed in BL21(DE3) exhibited high toxicity against Malacosoma neustria and Rhagoletis cerasi with 73% and 75% mortality after 5 days of treatment, respectively.

• Plant Biotechnology Reports
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• 제4권4호
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• pp.293-301
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• 2010

The feasibility of two strategies for transgene pyramiding using Agrobacterium-mediated transformation was investigated to develop a transgenic potato (Solanum tuberosum L. cv. Iwa) with resistance to potato tuber moth (PTM) (Phthorimaea operculella (Zeller)). In the first approach, cry1Ac9 and cry9Aa2 genes were introduced simultaneously using a kanamycin (nptII) selectable marker gene. The second approach involved the sequential introduction (re-transformation) of a cry1Ac9 gene, using a hygromycin resistance (hpt) selectable marker gene, into an existing line transgenic for a cry9Aa2 gene and a kanamycin resistance (nptII) selectable marker gene. Multiplex polymerase chain reaction (PCR) confirmed the presence of the specific selectable marker gene and both cry genes in all regenerated lines. The relative steady-state level of the cry gene transcripts in leaves was quantified in all regenerated lines by real-time PCR analysis. Re-transformation proved to be a flexible approach to effectively pyramid genes for PTM resistance in potato, since it allowed the second gene to be added to a line that was previously identified as having a high level of resistance. Larval growth of PTM was significantly inhibited on excised greenhouse-grown leaves in all transgenic lines, although no lines expressing both cry genes exhibited any greater resistance to PTM larvae over that previously observed for the individual genes. It is anticipated that these lines will permit more durable resistance by delaying the opportunities for PTM adaptation to the individual cry genes.

• 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 Plant Biotechnology
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• 제35권2호
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• pp.147-153
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• 2008

국화에 담배거세미 나방 (tobacco cutworm; Spodoptera litura)에 저항성을 나타내 국화를 육성하기 위하여 cry1Ac 유전자를 형질전환하였다. Cry1Ac 유전자는 pCAMBIA2301를 포함하는 Agrobacterium C58C1을 통하여 국화 'Linneker salmon'에 도입하였다. Agrobacterium C58C1을 접종한 후 엽절편을 10 mg/L kanamycin이 함유된 재분화 배지 (MS + 1.0 mg/L BA + 0.5 mg/L IAA)에서 1차 선발을 하였고, 재분화 배지에 20 mg/L kanamycin이 첨가된 배지에서 2차 선발을 하였으며, 20 mg/L kanamycin이 첨가된 MS 배지에서 3차 발근선발을 하였다. 3차 발근선발까지 69개의 신초 (1.6%)가 생존하여 발근하였다. NptII primer로 PCR을 한 결과 그 중 36개의 신초 (0.8%)가 putative transformant로 확인되었고, Southern 분석을 한 결과, 35개체 (0.8%)가 nptII 유전자와 cry1Ac 유전자를 가진 형질전환체로 확인되었다. Cry1Ac 유전자의 형질전환율은 0.8%로 양호하였다. 온실에서 담배거세미 나방에 대한 저항성을 검정한 결과, 3개체의 형질전환체가 저항성을 나타내는 것으로 확인되었다.

• 한국응용곤충학회지
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• 제35권1호
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• pp.66-73
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• 1996

Bacillus thuringiensis subsp. morrisoni PG-14의 cryIVD 유전자를 포함하고 있는 발현벡터 pCYASK5-1을 제작하여 Synechocystis PCCC6803에 형질전환시킨 후, 학질모기(Anopheles sinensis)유충에 대한 독성을 검정하였다. Kanamycin이 포함된 BG-11배지에서 선발된 형질전환체의 cryIVD 유전자 발현은 SDS-PAGE와 West-ern blot분석으로 확인하였다. 형질전환체는 A. sinensis 유충에 대해 높은 독성을 나타내었으며, 성장은 야생주인 Synechocystis Pccc6803과 유사하였다. 또 수심에 따른 형질전환체의 분포도 조사에서 전체적으로 살충농도의 세포수로 분포함을 확인하였다. 이러한 결과들은 B. thuringiensis subsp. morrisoni PG-14의 cryIVD 유전자로 형질전환된 Synechocystis PCCC6803이 모기유충 방제에 효율적으로 이용될 수 있음을 나타내었다.