• Journal of Microbiology and Biotechnology
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• 제21권9호
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• pp.937-946
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• 2011

Several isolates of Bacillus thuringiensis (Bt) were screened for the vegetative insecticidal protein (Vip) effective against sap-sucking insect pests. Screening results were based on $LC_{50}$ values against cotton aphid (Aphis gossypii), one of the dangerous pests of various crop plants including cotton. Among the isolates, the Bt#BREF24 showed promising results, and upon purification the aphidicidal protein was recognized as a binary toxin. One of the components of this binary toxin was identified by peptide sequencing to be a homolog of Vip2A that has been reported previously in other Bacillus spp. Vip2 belongs to the binary toxin group Vip1-Vip2, and is responsible for the enzymatic activity; and Vip1 is the translocation and receptor binding protein. The two genes encoding the corresponding proteins of the binary toxin, designated as vip2Ae and vip1Ae, were cloned from the Bt#BREF24, sequenced, and heterologously expressed in Escherichia coli. Aphid feeding assay with the recombinant proteins confirmed that these proteins are indeed the two components of the binary toxins, and the presence of both partners is essential for the activity. Aphid specificity of the binary toxin was further verified by ligand blotting experiment, which identified an ~50 kDa receptor in the brush border membrane vesicles of the cotton aphids only, but not in the lepidopteran insects. Our finding holds a promise of its use in future as a candidate gene for developing transgenic crop plants tolerant against sap-sucking insect pests.

• Journal of Microbiology and Biotechnology
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• 제26권10호
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• pp.1774-1780
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• 2016

Vegetative insecticidal proteins (Vips) are insecticidal proteins synthesized by Bacillus thuringiensis during the vegetative stage of growth. In this study, Vip3Aa protein, obtained by in vitro expression of the vip3Aa gene from B. thuringiensis WB5, displayed high insecticidal activity against Spodoptera litura aside from Spodoptera exigua and Helicoverpa armigera. Bioassay results showed that the toxicity of Vip3Aa protein against S. litura larvae statistically decreased along with the increase of the age of the larvae, with LC₅₀ = 2.609 ng/cm² for neonatal larvae, LC₅₀ = 28.778 ng/cm² for first instar larvae, LC₅₀ = 70.460 ng/cm² for second instar larvae, and LC₅₀ = 200.627 ng/cm² for third instar larvae. The accumulative mortality of 100% larvae appeared at 72 h for all instars of S. litura larvae, when feeding respectively with 83.22, 213.04, 341.40, and 613.20 ng/cm² of Vip3Aa toxin to the neonatal and first to third instar larvae. The histopathological effects of Vip3Aa toxin on the midgut epithelial cells of S. litura larvae was also investigated. The TEM observations showed wide damage of the epithelial cell in the midgut of S. litura larvae fed with Vip3Aa toxin.

• 환경생물
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• 제37권4호
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• pp.585-591
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• 2019

본 연구는 LMO 유전산물의 위해성평가를 위해 바실러스로부터 증폭된 Vip3Aa 유전자를 이용하여 대장균에서 단백질 순수분리 하였으며, MALDI-TOP 분석법을 통해 기존의 알려진 살충성 Vip3Aa 단백질과 동등한 단백질임을 증명하였다. 순수 분리한 Vip3Aa 단백질을 이용하여 꿀벌 과독성 급성섭식독성평가를 수행하였다. 그 결과 무처리군, Hepes buffer, Vip3Aa 단백질 처리군 모두 치사 및 일반 중독증상을 보이는 개체는 발견되지 않았다. 이 결과를 통해 Vip3Aa 단백질은 꿀벌에 위해성을 나타내지 않는다는 결론을 얻을 수 있었다. 본 연구 결과는 향후 국내 LMO 유전자 산물 위해성평가에 유용하게 활용될 것이라 사료된다.