• International Journal of Industrial Entomology and Biomaterials
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• 제11권2호
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• pp.125-127
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• 2005

In order to improve the transformation efficiency of the Bacillus thuringiensis (Bt)-Escherichia coli (E. coli) shuttle vector, pHT3101, we intended to minimize replication origin of Bt in pHT3101. For this, two modified shuttle vectors, pHT1K and pHT261, in which 2.9 kb of replication origin of Bt were shortened to 1 kb and 261 bp, respectively as previously reported. Whereas the pHT1K could efficiently transform Bt into the antibiotic resistant, no transformants were obtained with pHT261. Furthermore, pHT1K showed higher transformation efficiency compared to that of parent vector, pHT3101. Therefore, pHT1K might be a very useful Bt-E. coli shuttle vector carrying minimal replication origin of Bt.

• 한국응용곤충학회지
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• 제16권2호
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• pp.87-92
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• 1977

1963년부터 중부지방에 큰 피해를 주고 있는 맥류의 바이러스병을 동정하기 위하여 전자현미경에 의한 바이러스입자 관찰과 매개충을 이용한 전염시험 및 기주범위조사를 한 결과 정상입자의 평균 크기는 길이가 300-370nm였고 폭이 57-60nm였다. 매개충은 애멸구 Laodelphax striatellus(Fallen)로서 충체내 잠복기간은 7-19일이였고 대부분 10일간이었다. Host range는 보리, 옥수수, 밀, Rye맥, 구리 등으로 옥수수외의 기주식물들은 포장에서도 발병이 되고 있다. 그러므로 중부지방에서 발생되고 있는 맥류 바이러스병은 대부분이 북지모자익바이러스병으로 매년 $10\%$내외의 감염율을 보여주고 있으며 춘파맥에서는$100\%$ 감염 될 때도 있다.

• International Journal of Industrial Entomology and Biomaterials
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• 제8권1호
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• pp.77-80
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• 2004

For the development of the alternative control system against the major forest pests, Beauveria spp. F-101, isolated from a dead larva of Thecodiplosis japonensis, was selected because this isolate showed high pathogenicities against T. japonensis and Acantholyda parki. Beauveria spp. F-101 had irregular clustered conidio-phores and conidia borne on a distinctive apical zigzag extension, and it showed typical characteristic of the genus, Beauveria in morphology. For molecular based-identification, the ribosomal ITS region of Beauveria spp. F-101 was amplified with ITS1 and ITS4 primers, and cloned into pGEM- T Easy vector. The amplified PCR product was 569 bp in size and completely sequenced. The similarities of the cloned ITS sequence were 99 ％ and 97％ to those of B. bassiana and B. brongniartii, respectively. In comparison to other species among the genus Beauveria, the ITS region of Beauveria spp. F-101 showed a similarity of 95％ to B. amorpha, 95％ to B. tenella, 89％ to B. vermiconia and 69％ to B. alba, respectively. In addition, in comparison to different genus, it had 95％ similarities to Cordyceps militaris and 91％ to Paecilomyces tenuipes. Accordingly, the current result suggests that Beauveria spp. F-101 was a variant of B. bassiana and it seems to be a new isolate considering sequence variation in ITS region.

• International Journal of Industrial Entomology and Biomaterials
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• 제2권1호
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• pp.19-26
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• 2001

Transformed Spodoptera frugiperda (Sf9) cells expressing baculovirus very late factor (VLF-1) were constructed by using Autograha nuclear polyhedrosis virus (AcNPV) immediate earthy gene (ie1). Neomycin-resistance gene as a selectable marker was introduced under the control of AcNPV ie1 promoter, and Bombyx mori nuclear polyhedrosis (BmNPV-K1) vlf-1 gene was introduced under the control of the Drosophila heat shock protein gene (hspr70) promoter to yield dual expression plasmid with two independent transcription units. It was transfected into Sf9 cells and cell clones expressing vlf-1 were selected by G4l8 treatment. Genomic DNA from transformed cells was isolated and integration of AcNPV iel harboring vlf-1 was confirmed by PCR using AcNPV iel-specific primers and Southern blot analysis. The transformed cells expressing VLF-1 in an infection-independent manner expressed foreign gene product of recombinant baculovirus in the earlier stage of infection compared with control Sf9 cells. These results suggest the possible to develop highly efficient transformed insect cells for baculovirus expression vector system.

• Animal cells and systems
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• 제15권1호
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• pp.29-36
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• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.

• Journal of Microbiology and Biotechnology
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• 제31권4호
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• pp.529-539
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• 2021

The beet armyworm, Spodoptera exigua, is a serious insect pest infesting various vegetable crops. Two infectious insect viruses, baculovirus and iflavirus, are known to induce epizootics in S. exigua populations. Indeed, some laboratory colonies have appeared to be covertly infected by these viruses. Diagnostic PCR tests detected two different viruses: Spodoptera exigua multiple nucleopolyhedrosis virus (SeMNPV) and iflaviruses (SeIfV1 and SeIfV2). Viral extract from dead larvae of S. exigua could infect Sf9 cells and produce occlusion bodies (OBs). Feeding OBs to asymptomatic larvae of S. exigua caused significant viral disease. Interestingly, both SeIfV1 and SeIfV2 increased their titers at late larval stages. Sterilization of laid eggs with 1% sodium hypochloride significantly reduced SeMNPV titers and increased larval survival rate. Double-stranded RNA (dsRNA) specific to SeIfV1 or SeIfV2 significantly reduced viral titers and increased larval survival rate. To continuously feed dsRNA, a recombinant Escherichia coli HT115 expressing SeIfV1-dsRNA was constructed with an L4440 expression vector. Adding this recombinant E. coli to the artificial diet significantly reduced the SeIfV1 titer and increased larval survival. These results indicate that laboratory colony collapse of S. exigua is induced by multiple viral infections. In addition, either suppression of SeMNPV or SeIfV infection significantly increased larval survival, suggesting a cooperative pathogenicity between baculovirus and iflavirus against S. exigua.

• 농약과학회지
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• 제20권4호
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• pp.361-368
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• 2016

솔수염하늘소(Monochamus alternatus)는 우리나라와 일본의 소나무림에서 소나무재선충(Bursaphelenchus xylophilus)을 전파하는 매개충으로 알려져 있다. 본 연구는 곤충병원성선충을 이용하여 솔수염하늘소의 생물적 방제 가능성을 알아보기 위하여 수행하였다. 실험에 이용한 한국산 곤충병원성선충 4종(Heterorhabditis sp. Gyeongsan, Steinernema carpocapsae GSN1, S. glaseri Dongrae, S. longicaudum Nonsan 계통)은 소나무 고사목 내의 미끼곤충인 꿀벌부채명나방 유충을 치사시켰는데 S. carpocapsae GSN1의 병원성이 가장 높았다. 두 선충 모두 7.5 cm 깊이에 있는 꿀벌부채명나방을 치사시켰다. 곤충병원성선충은 솔수염하늘소 유충과 성충 모두에 병원성을 보였는데 성충에 대한 분무처리 시 S. carpocapsae GSN1 계통보다 Heterorhabditis sp. Gyeongsan 계통의 병원성이 높았다. 솔수염하늘소 유충이 서식하고 있는 소나무 벌채목에 대한 S. carpocapsae GSN1 계통의 수피면 분무처리와 벌채목 수침 처리 시 분무처리에서만 선충에 감염 된 유충이 확인되었다. 곤충병원성선충은 고사목 수체 내 솔수염하늘소 유충 방제의 실용성은 낮았지만 수체 내 해충에 대한 방제 가능성은 있어 추후 천공성 해충의 방제에 활용 가능성이 있을 것으로 생각된다.

• 한국응용곤충학회지
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• 제56권1호
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• pp.19-28
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• 2017

폴리드나바이러스(polydnavirus: PDV)는 일부 내부기생봉에 공생하는 이중나선형 DNA 바이러스 분류군이다. Cotesia plutellae bracovirus (CpBV)는 프루텔고치벌(C. plutellae)에 공생하는 일종의 PDV이다. 프루텔고치벌은 어린 배추좀나방(Plutella xylostella) 유충에 기생한다. 기생 초기에 발현하는 CpBV-ELP1 유전자는 혈구세포에 세포독성을 발휘하면서 기주의 세포성 면역을 억제하여 기생에 중요한 역할을 담당하고 있다. 본 연구는 이 유전자를 담배 식물에서 발현하여 해충에 대한 경구독성을 분석하는 데 목적을 두었다. 재조합 CpBV-ELP1 단백질이 배큘로바이러스 발현시스템을 통해 합성되어 세포배양액에 분비되었다. 수거된 세포배양액은 일련의 단백질 분리과정(ammonium sulfate 단백질 분획, size exclusion 크로마토그래피, 이온교환 크로마토그래피)을 통해 CpBV-ELP1 단백질을 분리하는 데 이용되었다. 분리된 rCpBV-ELP1 단백질은 파밤나방(Spodoptera exigua) 혈구에 대한 뚜렷한 세포독성을 보였다. CpBV-ELP1은 파밤나방 5령충에 대해서 혈강 주입하여 살충력을 나타냈고, 엽침지법을 이용하여 경구독성을 갖고 있는 것을 확인하였다. CpBV-ELP1 유전자를 CaMV 35S 유전자 프로모터와 opaline synthase 유전자 전사종결신호를 갖는 pBI121 벡터에 클로닝하여 Agrobacterium tumefaciens LBA4404 세균에 형질전환을 유도하였다. 형질전환된 세균은 담배(Nicotiana tabacum Xanthi)잎에 감염하여 캘러스를 유도하게 하였고 이후 차세대(T1)를 확보하였다. T1 세대 담배는 파밤나방에 대한 해충저항성을 갖고 있음을 확인하였다. 이러한 결과는 CpBV-ELP1 유전자가 형질전환작물을 통해 해충방제에 응용될 수 있다는 것을 제시하고 있다.

• Journal of Plant Biotechnology
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• 제35권2호
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• pp.101-108
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• 2008

아그로박테리움을 이용한 형질전환은 대다수의 쌍자엽과 몇몇 단자엽 식물의 게놈내로 외래유전자를 도입하는 성공적인 방법이다. 해충저항성 CryIAc 유전자가 도입된 배추형 질전환체를 아그로박테리움 형질전환법을 통해 얻은 후, 도입유전자의 수를 Southern 분석을 통하여 확인하였다. 배추형질전환체 46개 중에서 29개는 1 copy의 CryIAc 유전자가 도입된 것으로 확인되었다. 식물체의 게놈내로 T-DNA 결합에 관한 정보를 얻기 위해서 LB 인접서열을 genome walking PCR 방법을 통하여 분석하였다. 46개의 배추형질전환체중에서 37개는 운반체의 backbone 염기서열을 지니는 것으로 확인되었다. 이러한 결과는 도입 운반체의 LB 지점에서 제대로 종결이 이루어지지 않아서 운반체의 backbone 염기서열이 운반된 것으로 보여진다. 운반체의 backbone 염기서열이 도입되지 않은 9개체의 배추형질전환체를 LB 인접서열을 분석한 결과, 모든 LB 부위는 절단부위가 보존되지 않았고, 절단부위에서 36bp까지도 결실이 확인되었다.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1223-1229
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• 2009

To construct a new recombinant strain of Bacillus velezensis that has antifungal and insecticidal activity via the expression of the insecticidal Bacillus thuringiensis crystal protein, a B. thuringiensis expression vector (pHT1K-1Ac) was generated that contained the B. thuringiensis cry1Ac gene under the control of its endogenous promoter in a minimal E. coli-B. thuringiensis shuttle vector (pHT1K). This vector was introduced into a B. velezensis isolate that showed high antifungal activities against several plant diseases, including rice blast (Magnaporthe grisea), rice sheath blight (Rhizotonia solani), tomato gray mold (Botrytis cinerea), tomato late blight (Phytophthora infestans), and wheat leaf rust (Puccinia recondita), by electroporation. The recombinant B. velezensis strain was confirmed by PCR using cry1Ac-specific primers. Additionally, the recombinant strain produced a protein approximately 130 kDa in size and parasporal inclusion bodies similar to B. thuringiensis. The in vivo antifungal activity assay demonstrated that the activity of the recombinant B. velezensis strain was maintained at the same level as that of wild-type B. velezensis. Furthermore, it exhibited high insecticidal activity against a lepidopteran pest, Plutella xylostella, although its activity was lower than that of a recombinant B. thuringiensis strain, whereas wild-type B. velezensis strain did not show any insecticidal activity. These results suggest that this recombinant B. velezensis strain can be used to control harmful insect pests and fungal diseases simultaneously in one crop.