Abstract
A novel cDNA encoding the subtilisin-like serine protease gene CDEP2 was isolated from Beauveria bassiana by reverse transcription polymerase chain reaction (RT-PCR). It contained an 1137 bp ORF that predicted a protein of 379 amino acids with M = 38863 Da and pI = 8.21. In an attempt to improve insecticidal activity, the CDEP2 gene and the cry1Ac gene from Bacillus thuringiensis were co-fused into the vector pHT315 as pHAc-CDEP2 plasmid by Red/ET homologous recombination. The co-fusion gene was attempted under the control of the native cry1Ac promoter. Plasmid pHAc-CDEP2 was electro-transformed into the B. thuringiensis subsp. kurstaki Cry(-)B. Analyzed by SDS-PAGE and Western blotting, the transformant Cry(-)B-pHAc-CDEP2 strain produced a 130 kDa Cry1Ac protein and 39 kDa CDEP2 protein. The 50% lethal concentration values (LC(50)) of Cry(-)B-pHAc-CDEP2 strain (8.5 microl/ml) to Helicoverpa armigera third instars larvae was clearly higher than the Cry(-)B-pHAc strain (16.7 microl/ml) at 72 h.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Bacillus thuringiensis / genetics*
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Bacillus thuringiensis Toxins
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Bacterial Proteins / biosynthesis*
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Bacterial Proteins / genetics
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Bacterial Proteins / toxicity
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Beauveria / enzymology
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Beauveria / genetics
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Cloning, Molecular
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DNA, Complementary / chemistry
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DNA, Complementary / genetics
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DNA, Complementary / isolation & purification
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DNA, Fungal / chemistry
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DNA, Fungal / genetics
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DNA, Fungal / isolation & purification
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Endotoxins / biosynthesis*
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Endotoxins / genetics
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Endotoxins / toxicity
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Genes, Fungal
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Genetic Engineering*
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Hemolysin Proteins / biosynthesis*
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Hemolysin Proteins / genetics
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Hemolysin Proteins / toxicity
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Larva / drug effects
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Lepidoptera / drug effects
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Lethal Dose 50
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Molecular Sequence Data
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Molecular Weight
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Open Reading Frames
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Recombinant Fusion Proteins / biosynthesis
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / toxicity
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Recombination, Genetic*
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Sequence Analysis, DNA
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Serine Endopeptidases / biosynthesis*
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Serine Endopeptidases / chemistry
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Serine Endopeptidases / genetics
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Serine Endopeptidases / toxicity
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Survival Analysis
Substances
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Bacillus thuringiensis Toxins
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Bacterial Proteins
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DNA, Complementary
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DNA, Fungal
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Endotoxins
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Hemolysin Proteins
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Recombinant Fusion Proteins
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insecticidal crystal protein, Bacillus Thuringiensis
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Serine Endopeptidases