Isolation And Identification Of Bacillus Thuringiensis And Characterization The Bt Strain Against Henosepilachna Vigintioctomaculata

Bacillus thuringiensis is a spore-forming entomopathogenic bacterium that produces parasporal crystals composed of proteins calledδ-endotoxins during sporulation. There are two known classes ofδ-endotoxins in B. thuringiensis: the insect-specific insecticidal crystal (Cry) proteins and the Diptera-specific cytolytic (Cyt) proteins. Currently, the Cry proteins have been shown to be highly active against a wide variety of insect larvae. These toxins are widely sought after for controlling agricultural pests due to both their specificity and their applicability in transgenic plants. Although any particular toxin has a desirably restricted host range, there is a large number of different toxins, each showing toxicity to one of many diverse pests. For these reasons there is currently great interest in isolating novel strains of B. thuringiensis with either unique host specificity or elevated toxicity. New B. thuringiensis strains with cry7 gene were isolated and identified in this paper. Furthermore, characterization of the new Bt strain with cry7 gene against Henosepilachna vigintioctomaculata was studied. The results were as the following.1. The detection of cry gene of these isolates was done by a method based on polymerase chain reaction (PCR). 56 Bt isolates were identified to contain cry7 gene in which 10 isolates possessed cry7 gene and other cry gene, 46 isolates had only cry7 gene. SDS–PAGE analysis indicated that most of 56 isolates produced 130kDa protein bands, but Bt BQZ-12 produced 80kDa and 70kDa proteins, Bt BQZ-8 produced 130kDa and 80kDa proteins. The results of bioassay showed that just only Bt WZ-9 strain was toxic to larvae of H. vigintioctomaculata.2. Bt WZ-9 strain produces a bipyramidal crystal consisting of a 130 kDa protein. A novel gene, cry7Ab3, encoding a polypeptide of 1138 amino acids with a deduced molecular mass of 129.6 kDa was identified from Bt WZ-9 strain and submitted to GenBank with accession numbers ABX 24522. The results of bioassay demonstrated that Bt WZ-9 crystal proteins had high toxicity to larvae of H. vigintioctomaculata (LC500.209 mg/mL).3. The cry7Ab3 gene was successfully expressed as 130kDa protein in Escherichia coli BL21 and Bt acrystalliferous strain HD73 cry-. The expressed Cry7Ab3 proteins all had significant activity against second instar larvae of H. vigintioctomaculata, the estimated 50% lethal concentrations (LC50) were 0.460mg/mL and 0.205 mg/mL,respectively. Bt recombinant HD73-Cry7Ab3 produced bipyramidal crystals which were larger than those from Bt WZ-9 strain. The crystal volume were 0.86×1.23μm and 0.73×1.00μm.4. To determine the minimal active fragment of Cry7Ab3 protein, the four different cry7Ab3 fragments gene were cloned and expressed in E. coli BL21 respectively. Bioassay results showed that the toxicity (LC50=0.114 mg/mL) of expressed protein of E. coli Cry7Ab3-1(Met1-Phe657) was one time higher than that from Bt WZ-9. The expressed protein from E. coli Cry7Ab3-3(Asn30-Phe657) and E. coli Cry7Ab3-4(Lys87-Phe657) were no toxic to H. vigintioctomaculata. The minimal activated Cry7Ab3 toxin fragment was located at N-terminal of Cry7Ab3 between position Met1 and Phe657.5. The trypsin and gut juice proteases from H. vigintioctomaculata larvae all could proteolyse 130kDa Cry7Ab3 protoxin into 75kDa active toxin. The toxicity of 75kDa processing products against H. vigintioctomaculata was the higher than that of Cry7Ab3 protoxin. Histopathological observations indicated that Cry7Ab3 ingestion by H. vigintioctomaculata larvae caused acceleration in the blebbing of the midgut epithelium cells into the gut lumen and eventual lysis of the epithelium cells resulting in larval death. Ligand blotting experiment demonstrated that Cry7Ab3 toxin bounded a 220kDa BBMV protein from midgut of H. vigintioctomaculata larvae. This putative receptor protein was identified as cadherin by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS).Highlights of this paper are finding of a novel Cry7Ab3 toxin specific to H. vigintioctomaculata larvae, identifying the minimal active fragment of Cry7Ab3 and putative receptor protein of Cry7Ab3 in BBMV of H. vigintioctomaculata larvae. The data obtained may contribute to a better understanding of the mechanism of Cry7Ab3 toxin against H. vigintioctomaculata larvae and a better usage of Cry7Ab3 for controlling H. vigintioctomaculata larvae.