• 한국미생물·생명공학회지
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• 제26권4호
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• pp.369-372
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• 1998

Microbial insecticide formulations were prepared by liquid and semi-solid fermentations using Bacillus thuringiensis subsp. kurstaki, HL-106 (BTK-HL106), B. thuringiensis subsp. israelensis HL-63 (BTI-HL63) and B. sphaericus 1593 (BS-1593) strains. The liquid fermentation medium contained molasses 2%, dextrose 1.5%, peptone 2%, D-xylose 0.025%, CaCl$_2$ 0.1%, K$_2$HPO$_4$ 0.1%, KH$_2$PO$_4$ 0.1%, MgSO$_4$$.$7H$_2$O 0.03%, FeSO$_4$$.$7H$_2$O 0.002%, ZnSO$_4$$.$7H$_2$O 0.02%. The composition of the semi-solid fermentation medium was rice bran 45.2%, zeolite 31%, yeast powder 0.02%, corn powder 5%, dextrose 3%, lime 0.3%, NaCl 0.06%, CaCl$_2$ 0.02%, and H$_2$O 15.42%. Insecticide formulations produced in the liquid fermentation named BTK-HL106, BTI-HL63 and BS-1593 pesticides and those in the semi-solid fermentation were designated as BTK-HL106-1, BTI-HL63-1 and BS-1593-1 pesticides, respectively. The number of spore (endotoxin crystals) was 2.65${\times}$10$\^$9/ spores per $m\ell$ in the BTK-HL106 and 3.5${\times}$10$\^$10/ in the BTK-HL106-1 3.8${\times}$10$\^$9/ spores in the BTI-HL63 and 7.0${\times}$10$\^$10/ in the BTI-HL63-1, and 7.5${\times}$10$\^$9/ in the BS-1593 and 1.4${\times}$10$\^$10/ in the BS-1593-1. The spores in the BS-1593 formulation was produced two times more than the other formulations. The spores in the BTI-HL63-1 were contained twice than those in the BTK-HL106-1, and five times than those in the BS-1593-1. The results indicated that spore (endotoxin crystals) productions in the semi-solid fermentation increased about ten times than those in the liquid fermentations. $LC_{50}$s of the BTI-HL63 and BS-1593 were 4.5 $\mu\textrm{g}$, and those of the BTI-HL63-1 and BS-1593-1 were 1.5 $\mu\textrm{g}$. $LC_{50}$ of the BTK-HL106 was 1.5 mg and that of the BTK-HL106-1 was 0.9 mg. The $LC_{50}$s of the formulations in the semi-solid fermentations showed about two to three times higher than those in the liquid fermentations.

• Genomics & Informatics
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• 제21권3호
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• pp.35.1-35.12
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• 2023

The Bacillus cereus group, also known as B. cereus sensu lato (B. cereus s.l.), is composed of various Bacillus species, some of which can cause diarrheal or emetic food poisoning. Several emerging highly heat-resistant Bacillus species have been identified, these include B. thermoamylovorans, B. sporothermodurans, and B. cytotoxicus NVH 391-98. Herein, we performed whole genome analysis of two thermotolerant Bacillus sp. isolates, Bacillus sp. B48 and Bacillus sp. B140, from an omelet with acacia leaves and fried rice, respectively. Phylogenomic analysis suggested that Bacillus sp. B48 and Bacillus sp. B140 are closely related to B. cereus and B. thuringiensis, respectively. Whole genome alignment of Bacillus sp. B48, Bacillus sp. B140, mesophilic strain B. cereus ATCC14579, and thermophilic strain B. cytotoxicus NVH 391-98 using the Mauve program revealed the presence of numerous homologous regions including genes responsible for heat shock in the dnaK gene cluster. However, the presence of a DUF4253 domain-containing protein was observed only in the genome of B. cereus ATCC14579 while the intracellular protease PfpI family was present only in the chromosome of B. cytotoxicus NVH 391-98. In addition, prophage Clp protease-like proteins were found in the genomes of both Bacillus sp. B48 and Bacillus sp. B140 but not in the genome of B. cereus ATCC14579. The genomic profiles of Bacillus sp. isolates were identified by using whole genome analysis especially those relating to heat-responsive gene clusters. The findings presented in this study lay the foundations for subsequent studies to reveal further insights into the molecular mechanisms of Bacillus species in terms of heat resistance mechanisms.

• 농업과학연구
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• 제46권1호
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• pp.113-124
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• 2019

Insect-resistant transgenic (Bt-9) rice was generated by inserting mCry1Ac1, a modified gene from the soil bacterium Bacillus thuringiensis, into the genome of a conventional variety of rice (Ilmi). With regard to potential problems such as safety, an evaluation of non-target organisms is necessary as an essential element of an environmental risk assessment of genetically modified (GM) crops. We studied the effects of the Bt-9 rice on the survival of cantor Daphnia magna, a commonly used model organism in ecotoxicological studies. D. magna fed on the Bt-transgenic rice (Bt-9) and its near non-GM counterparts (Ilmi) grown in the same environment (a 100% ground rice suspension). The Bt-9 rice was confirmed to have the inserted T-DNA and protein expression evident by the PCR and ELISA analyses. The feeding study showed a similar cumulative immobility and abnormal response of the Daphnia magna between the Bt-9 rice and Ilmi. Additionally, the 48 h-EC50 values of the Bt-9 and Ilmi rice were 4,400 mg/L (95% confidence limits: 3861.01 - 5015.01 mg/L) and 5,564 mg/L (95% confidence limits: 4780.03 - 6476.93 mg/L), respectively. The rice NOEC (No observed effect concentration) value for D. magna was suggested to be 1,620 mg/L. We conclude that the tested Bt-9 and Ilmi have a similar cumulative immobility for D. magna, a widely used model organism, and the growth of Bt-9 did not affect non-target insects.

• 농업과학연구
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• 제46권4호
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• pp.827-841
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• 2019

The effect of genetically engineered rice Bt-9 on the diversity and abundance of plant-dwelling insects and spiders was tested under field conditions. Genetically engineered rice Bt-9, expressing mCry1Ac1 from Bacillus thuringiensis, confers resistance to rice leaf roller (Cnaphalocrocis medinalis) and provides tolerance to the herbicide glufosinate (PPT). The study compared Bt-9 and two non-GM reference varieties, Ilmi-byeo and Dongjin-byeo, at LMO isolated fields in Gunwi (Kyungpook National University) and Jeonju (National Institute Agricultural Sciences) in Southern Korea in 2016 - 2017. A total of 40,817 individuals from 62 families and 11 orders were collected from the two living modified organism (LMO) isolated fields. From the three types of rice fields, a total of 13,982, 14,105, and 12,730 individuals from the Bt-9, Ilmi-byeo and Dongjin-byeo were collected, respectively. Throughout the study, the analysis of variance indicated no significant differences (p < 0.05). Multivariate analysis showed that the abundance and diversity of plant dwelling insects were similar. The data on insect species population densities were subjected to principal component analysis (PCA), which did not distinguish among the three varieties, Bt-9 and the non-GM, reference cultivars, during the cultivation years. However, the results of the PCA analysis were completely divided into four groups based on the yearly survey areas. Therefore, there was no evidence for a negative impact of Bt-9 on the above-ground insects and spiders.

• 농업과학연구
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• 제49권3호
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• pp.511-520
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• 2022

Insect-resistant transgenic rice (Bt-T) expresses a toxic protein (mcry1Ac1) derived from the soil bacterium Bacillus thuringiensis found in the rice cultivar Dongjin with an insecticidal property against rice leaf roller (Cnaphalocrocis medinalis). In this study, to investigate the impact of Bt-T on non-target organisms, the feed and oviposition preferences and biological parameters of brown planthopper (Nilaparvata lugens Stål) were comparatively analyzed in four rice cultivars: Dongjin (parent variety), Ilmi (reference cultivar), Chinnong (brown planthopper resistant cultivar) and Bt-T. In the Bt-T and Dongjin cultivars, the feed preferences were 32.4 ± 8.3 and 34.1 ± 6.8%, and the oviposition preferences were 32.5 ± 5.1 and 30.0 ± 5.3% respectively, and there was no statistical significance between these rices. Additionally, in the Bt-T and Dongjin cultivars, the total lifespans from egg to adult were 39.5 ± 6.9 and 40.0 ± 5.8 days, and the weights of adult females were 1.78 ± 0.14 and 1.72 ± 0.16 mg, respectively. Therefore, there was no statistical difference in the biological parameters between these two varieties. Overall, the results indicate that the insect-resistant transgenic rice (Bt-T) did not negatively affect the reproduction and life cycle of brown planthopper, a non-target organism.

• Plant Breeding and Biotechnology
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• 제5권4호
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• pp.269-281
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• 2017

With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in Bacillus thuringiensis (Bt)-resistant rice, one in resveratrol-producing rice, and two in beta-carotene-enhanced soybeans, were selected for molecular characterization. To merge NGS analysis and Southern blot-hybridization results, we confirmed the transgene insertion sites, insertion construction, and insertion numbers of these four transformation events. In addition, the read-coverage depth assessed by NGS analysis for inserted genes might provide consistent results in terms of inserted T-DNA numbers in case of complex insertion structures and highly duplicated donor genomes; however, PCR-based methods can produce incorrect conclusions. Our combined method provides an effective and complete analytical approach for whole-genome visual inspection of transformation events that require biosafety assessment.

• 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.

• 한국국제농업개발학회지
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• 제23권5호
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• pp.570-577
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• 2011

해충저항성 Bt벼와 낙동벼의 미꾸리(Misgurnus anguillicaudatus)와 잉어(Cyprinus carpio)에 대한 급성독성시험을 실시한 결과 48시간 및 96시간-LC₅₀은 1,000mg/L 이상으로 나타났다. 48시간 및 96시간 무영향농도(NOEC)는 1,000mg/L이었다. 급성독성 시험기간 중 해충저항성 Bt벼와 낙동벼간의 pH, DO, 수온, 체중 및 전장에 대한 유의적인 결과는 나타나지 않았다.

• Journal of Plant Biotechnology
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• 제41권3호
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• pp.127-133
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• 2014

Bacillus thuringiensis 균주로부터 분리한 cry3A 유전자는 딱정벌레목 해충에 살충성을 가지고 있어서 효율적으로 딱정벌레목 해충을 방제 할 수 있다고 알려져 있다. 국립농업과학원에서는 cry3A 유전자 형질전환벼를 육성하였고, GMO 포장 생물검정 연구를 통해 벼물바구미에 대한 내충성을 확인하였다. 본 연구에서는 벼물바구미 내충성 벼 4 계통에 대한 도입유전자 특성을 분자생물학적 방법으로 검정하여 유전자의 도입 사본 수와 도입유전자의 염기서열 등의 분석결과를 제시하였다. BT12R1 계통은 염색체 10번의 exon 부위에 도입유전자가 단일 사본으로 삽입되었으며, BT12R2 계통은 두 개의 사본으로 삽입되었고, BT12R3 계통은 LB 인근의 염기서열만 확인되었다. BT12R4 계통의 경우 단일 사본의 도입유전자가 염색체 5번의 24,516,607 ~ 24,516,636 위치에 30개의 염색체 염기서열이 결실된 상태로 삽입되었다. 이 도입위치는 벼의 내재 유전자가 발현하지 않는 intergenic 부위로 판명되었으며, 도입유전자 이외의 벡터 염기서열이 삽입되지 않음을 확인하였다. 이러한 벼물바구미 내충성벼 BT12R4 계통에 대한 분자생물학적 분석 결과는 차후 GM 작물 실용화의 환경 위해성 및 안전성 평가에 대한 기초자료로 활용할 수 있을 것으로 생각된다.

• 농약과학회지
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• 제20권3호
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• pp.181-188
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• 2016

비티플러스는 Bacillus thurigiensis와 면역억제가 혼합된 생물농약의 총칭이다. 본 연구는 모기류에 살충력이 높은 새로운 비티플러스 생물농약에 대해서 야외포장 조건에서 약효와 환경생물에 대한 안전성평가에 관해 수행되었다. 모기 유충에 대한 효과적 방제를 위해 두 가지 살포 처리에 따라 비티플러스의 방제 효과를 검증하였다. 비티플러스의 공중살포 ($3.3m^2$ 당 100 mL)에 따른 수계에 서식하는 모기 유충의 효과적 방제를 위해서는 최소 50%의 제제화 된 약제 농도가 요구되었다. 이에 반해 논생태계와 유사한 조건의 간이모형 조건에서 비티플러스를 수중살포한 경우 모기유충에 대한 비티플러스의 방제효과는 0.1%의 제제화 된 약제 농도에서 80% 이상의 살충 효과를 나타냈고, 0.2% 처리에서는 완전 방제 효과를 보여 주었다. 또한 유충에 비해서는 다소 낮지만 10% 설탕물에 포함된 0.1%의 비티플러스는 성충에 대해서도 40% 이상의 살충력을 나타냈다. 환경생물에 대한 안전성평가에서는 잉어 (Cyprinus carpio), 큰물벼룩(Daphnia magna), 꿀벌(Apis mellifera)을 대상으로 비티플러스에 대한 독성을 평가한 결과 급성적인 영향은 주지 않는 것으로 나타났다. 이러한 결과는 논생태계에 서식하는 모기 유충방제에 비티플러스의 수중살포 처리가 유효한 방제 처리라는 것을 제시하고 있다.