The expression of a recombinant cry1Ac gene with subtilisin-like protease CDEP2 gene in acrystalliferous Bacillus thuringiensis by Red/ET homologous recombination.
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.
Xia
Liqiu
L
College of Life Science Key Laboratory of Microbial Molecular Biology of Hunan Province, Hunan Normal University, Changsha, PR China.
Zeng
Zhi
Z
Ding
Xuezhi
X
Huang
Fan
F
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000633:toxicity; D052982:Beauveria / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004731:Endotoxins / Q000096:biosynthesis* / Q000235:genetics / Q000633:toxicity; D005800:Genes, Fungal; D005818:Genetic Engineering; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000633:toxicity; D007814:Larva / Q000187:drug effects; D007915:Lepidoptera / Q000187:drug effects; D007928:Lethal Dose 50; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000633:toxicity; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D016019:Survival Analysis
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D018076:DNA, Complementary; D004271:DNA, Fungal; D004731:Endotoxins; D006460:Hemolysin Proteins; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D012697:Serine Endopeptidases