- Expression of a Recombinant Cry1Ac Crystal Protein Fused with a Green Fluorescent Protein in Bacillus thuringiensis subsp. kurstaki Cry-B
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Jong Yul Roh , In Hee Lee , Ming Shun Li , Jin Hee Chang , Jae Young Choi , Kyung Saeng Boo , Yeon Ho Je
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J. Microbiol. 2004;42(4):340-345.
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DOI: https://doi.org/2101 [pii]
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 Abstract
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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 cry1Ac 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.
- Nosema sp. isolated from Cabbage White Butterfly (Pieris rapae) Collected in Korea
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Ji Young Choi , Jong Gill Kim , Young Cheol Choi , Tae Won Goo , Jin Hee Chang , Yeon Ho J e , Keun Young Kim
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J. Microbiol. 2002;40(3):199-204.
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A microsporidium, from cabbage white butterflies, Pieris rapae, collected in Korea, was purified and characterized according to its gene structure, spore morphology and pathogenicity. From the observation of the isolate by SEM and TEM, the endospores, exospores and nuclei, about 12 polar filament coils of the polar tube and posterior vacuoles were all identified. The nucleotide sequence was determined for a portion of genomic DNA which spans the V4 variable region of the small subunit rRNA gene. Comparison with the GenBank database for 15 other microsporidia species suggests that this isolate is most closely related to Nosema species. The pathogenicity against cabbage white butterflies was quantified by inoculating variable doses of spores to the second instar larvae. Peroral inoculation at a dosage of 10^8 spores/ml resulted in the death of all larvae prior to adult eclosion, but at lower spore dosages of 10^4 ?0^5 spores/ml, many adults successfully emerged. The median lethal dose (LD_50 ) was determined to be 4.6 X 10^6 spores/ml and the isolate also transmitted transovarially to the progeny eggs at a frequency of 92%.
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