• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1057-1062
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• 2004

The entomopathogenic bacterium Bacillus thuringiensis is the most widely used biopesticide. Insecticidal proteins, coded by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. We isolated a Bacillus thuringiensis strain having insecticidal activity against larvae of the house fly (M. domestica) from the soils at a pig farm in Korea, and named it Bacillus thuringiensis SM. The culture filtrate from Bacillus thuringiensis SM showed strong lethality (83.3%) against M. domestica larvae. The parasporal crystal is enclosed within the spores' outermost envelope, as determined by transmission electron microscopy, and exhibited a bipyramidal form. The crystal proteins of strain SM consisted of five proteins with molecular weights of approximately ~130, ~80, ~68, ~42, and ~27 kDa on a 10% SDS-PAGE (major band, a size characteristic of Cry protein). Examination of antibiotic resistance revealed that the strain SM showed multiple resistant. The strain SM had at least three different plasmids with sizes of 6.6, 9.3, and 54 kb. Polymerase chain reactions (PCRs) revealed the presence of cry1, cry4A2, and cry11A1 genes in the strain SM. The cry1 gene profile of the strain SM appeared in the three respective products of 487 bp [cry1A(c)], 414 bp [cry1D], and 238 bp [cry1A(b)]. However, the strain SM has not shown the cry4A2 md cry11A1 genes. In in vivo toxicity assays, the strain SM showed high toxicity on fly larvae (M. domestic) [with $LC_{50}$ of 4.2 mg/ml, $LC_{90}$ of 8.2 mg/ml].

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.240-245
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• 1991

We have constructed a promoter-probe vector pKU20 using pKT230, a derivative of broad-host-range plsmid RSF1010, as a base. The pKU20 contains structural gene for aminoglycoside phos-photransferase (aph), without promoter, and a multiple cloning site upstream the aph. Using this vector, a 412base pairs (bp) PstI fragment showing strong promoter activity both in Escherichia coli LE392 and Pseudomonas putida KCTC1644 has been cloned from Pseudomonas fluorescens chromosomal DNA on the basis of streptomycin resistance. The nucleotide sequence of the 412 bp fragment has been determined and the putative - 35 and -10 region was observed. Insecticidal protein gene of Bacillus thuringiensis subsp. kurstaki HD-73 inserted on downstream of the promoterlike DNA fragment was efficiently expressed in E. coli and P. putida. The toxin protein was efficiently synthesized in an insoluble form in both strains.

• 한국유기농업학회지
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• 제31권4호
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• pp.427-439
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• 2023

The silverleaf whitefly, Bemisia tabaci, is a major pest distributing worldwide damaging over 900 host plant species, and is highly resistant to chemical pesti- cides. Due to the high pesticide resistance of whitefly, there is a need for alternatives to chemical control. Entomopathogenic fungi are candidates for biological pesticide that can overcome the resistance problem of chemical pesticide. Therefore, in this study, we tested pathogenicity of the entomopathogenic fungi to select high insec- ticidal activity against whitefly. As a result, IPBL-C (Cordyceps fumosorosea) and IPBL-F (Metarhizium pinghaense) isolates showed high insecticidal activity against whitefly. Additionally, as a result of culturing the selected isolates on spent coffee grounds medium, the conidia of IPBL-F produced on coffee grounds medium showed five times higher heat stability after heat treatment at 45℃ for one hour than conidia produced on PDA medium.

• 한국응용곤충학회지
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• 제31권3호
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• pp.247-255
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• 1992

A laboratory bioassay that incorporates Bacillus thuringiensis (Bt) purified crystal protein toxins into an artificial diet has identified three toxins, CryIA(b), CryIA(c), and CryIIA, to by effective against the yellow stemborer, Scirpophaga incertulas(Walker). Research is aimed at engineering rice that incorporates genes of one of or more of these toxins so as to mimic the insecticidal action of the insect to Bt. The paper discusses potential strategies for slowing the rate of adaptation that include the use of multiple Bt toxins, promoters that express the toxins only in specific plant tissues at specific times, and mixing transgenic and non-transgenic plants.

• Molecules and Cells
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• 제28권2호
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• pp.131-137
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• 2009

Plant defensins are small (5-10 kDa) basic peptides thought to be an important component of the defense pathway against fungal and/or bacterial pathogens. To understand the role of plant defensins in protecting plants against the brown planthopper, a type of insect herbivore, we isolated the Brassica rapa Defensin 1 (BrD1) gene and introduced it into rice (Oryza sativa L.) to produce stable transgenic plants. The BrD1 protein is homologous to other plant defensins and contains both an N-terminal endoplasmic reticulum signal sequence and a defensin domain, which are highly conserved in all plant defensins. Based on a phylogenetic analysis of the defensin domain of various plant defensins, we established that BrD1 belongs to a distinct subgroup of plant defensins. Relative to the wild type, transgenic rices expressing BrD1 exhibit strong resistance to brown planthopper nymphs and female adults. These results suggest that BrD1 exhibits insecticidal activity, and might be useful for developing cereal crop plants resistant to sap-sucking insects, such as the brown planthopper.

• Plant Biotechnology Reports
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• 제3권4호
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• pp.317-324
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• 2009

The insecticidal toxin gene of Bacillus thuringiensis (Bt) is one of the most commonly used in the development of genetically modified (GM) crops. In this research, we analyzed Bt rice showing lepidopteran pest-resistance. The Bt gene is a synthetic Cry1Ac composed of optimal codons for plants, and the Bt protein is targeted to the chloroplast by a transit peptide. Three Cry1Ac rice events (C103-3, C127-1, and C7-1) were analyzed for molecular characterization. C103-3 contains two copies of T-DNA where the left border (LB) region is truncated. Both C7-1 and C127-1 have a single copy of T-DNA, but a part of the vector backbone DNA is inserted into the genome of C127-1; thus, only C7-1 had intact T-DNA. Progenies of C7-1 crossed with the original cultivar, Nakdong, and double-haploid lines from anther culture of lines crossed with the elite cultivar, Dongjin, were analyzed for T-DNA flanking genomic DNA and genotyping. Results showed that an intact T-DNA region without the vector backbone was inserted into the genome and was stably inherited through generations. The C7-1 homozygous event could be used as breeding material to develop GM rice with pest resistance.

• 한국응용곤충학회지
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• 제57권1호
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• pp.43-50
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• 2018

4종의 diamide계열 살충제를 이용한 배추좀나방과 파밤나방의 지역계통별 감수성을 조사하였다. 배추좀나방의 경우 성주와 거창지역 집단에서 4종의 약제 모두 추천농도에서 100%의 살충활성을 보인 반면, 평창지역 집단에서는 chlorantraniliprole에 대해 42.3%의 낮은 살충활성을 보였다. 감수성계통 배추좀나방과 저항성비를 비교한 결과 cyclaniliprole은 비교적 4지역에서 유사하거나 낮은 저항성비(0.1~6.3배)를 보인 반면, 평창지역 집단은 chlorantraniliprole (1,196.3배)와 cyantraniliprole (105.6배), flubendiamide (191.6배)은 매우 높은 저항성비를 보였다. 파밤나방의 경우 청주와 진도, 영광지역의 집단 모두 4종의 약제에 낮은 감수성을 나타났는데, 특히 청주와 진도지역의 집단은 flubendiamide에 대해 100,000배 이상의 저항성비를 보였다. 채집 연도(2014년과 2017년)에 따른 파밤나방에 대한 chlorantraniliprole의 감수성을 비교한 결과 2014년에 채집된 집단들은 모두 100%의 살충률을 보인 반면 2017년에 채집된 집단들은 살충활성이 매우 낮게 나타났다. 본 연구결과는 diamide계열 살충제가 빠르게 저항성이 발현되고 있으며 작용기작이 다른 약제와의 교호살포 등 종합적 방제전략을 수립하는데 기초자료가 될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제4권4호
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• pp.322-326
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• 1994

Bacillus thuringiensis strain BT-14 was isolated from alfalfa dust in Korea. The strain BT-14 produced one bipyramidal crystal and one spore in the cell. The biochemical characteristics of the strain BT-14 were similar to those of Bacillus thuringiensis subsp. kurstaki HD-l. Examination of its antibiotic resistance revealed that while the strain BT-14 was less resistant than BTK HD-l to ampicillin, gentamycin, neomycin and tobramycin, it was more resistant to amikacin than BTK HD-l. The $\delta$-endotoxin crystal of strain BT-14 consisted of a single protein with a high molecular weight of ca 135 KD on a 10% SDS-PACE. The strain BT-14 contained at least nine different plasmids with sizes of 2.9, 5.3, 5.8, 6.2, 9.4, 15.1, 18.1, 23.1 and 79 Kb. In insect bioassay, the isolated strain BT-14 showed lethality of 67% against Plutella xylostella larvae at dilution of 5$\times$$l0^{-4}$ (5$\times$l0 to 3$\times$$l0^2$ spores/ml), which is, almost equivalent to that of BTK HD-l.

• 한국유기농업학회지
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• 제24권4호
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• pp.821-831
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• 2016

본 연구는 난방제 해충인 파밤나방을 농가에서 효과적으로 방제하기 위해 Bacillus thuringiensis와 고삼추출물 혼합처리에 의한 살충효과를 실내 포트 및 포장 실험을 통해 조사하였다. 실내 포트 실험에서 BT를 농도별($1{\times}10^5$, $1{\times}10^6$, $1{\times}10^7cfu\;mL^{-1}$)로 처리하였을 때 파밤나방의 살충율은 27.5%, 51.3%, 92.5%로 농도가 높아질수록 살충율이 증가하였다. BT ($1{\times}10^5$, $1{\times}10^6$, $1{\times}10^7cfu\;mL^{-1}$)와 0.033% 고삼추출물 혼합처리구의 살충율은 98.8%, 88.8%, 91.3%로 BT 단독처리보다 BT+고삼추출물 혼합처리구가 높은 살충율을 나타냈으며, 혼합된 BT의 농도가 낮을수록 살충율 증가가 높았다. 각 처리별 파밤나방의 식이저해 효과는 BT의 농도가 높아질수록 우수한 것을 확인하였다. 또한 포장 실험에서도 실내포트 실험과 유사하게 BT 단독처리구(14.4%, 26.7%, 66.7%)보다 고삼추출물 혼합처리구가 높은 살충율(72.2%, 67.8%, 61.1%)을 나타냈으며 식이저해효과 또한 실내실험과 유사한 결과를 나타냈다. 따라서 농가에서 파밤나방을 효과적으로 방제하기 위해서는 BT 단독처리보다 0.033% 고삼과 BT($1{\times}10^5cfu/ml$)를 혼합하여 처리하는 것이 효과적일 것으로 사료된다.

• 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.