Study On The Bacillus Thuringiensis Ssp.wuhanensis 140 Strain

Bacillus churingiensis (Bt) is a spore-forming gram-positive bacterium. During sporulation, the Insecticidal Crystal Proteins (Cry proteions) are produced. Those proteins show toxicity to the larvae of Lepidopetera, Diptera and Coleoptera. The Cry Proteins from Bt have developed as one of the most successful biological insectidides.Atomic force microscope (AFM) was used to scanning the crystal proteins of ssp. wuhanensis 140 strain. The crystal proteins have polygon, similar squareness and anomalistic figure. These crystal proteins have high virulence to Prodenia litura (Fabricius) larvae. But these crystal proteins have medium virulence to Heliothis armigera larvae. The crystal proteins from ssp. wuhanensis 140 have synergistic effects with HD-1 to Heliothis armigera larvae, and they can strongly restrain the delovpment of larva. But the synergistic effects of these crystal proteins with HD-73 was not significant.On SDS-PAGE gels, a preparation of purified crystal proteins of ssp. wuhanensis 140 exhibited 135, and 60 kDa protein bands. These proteins were analyzed by MALDI-ToF/ToF. The internal amino acid sequence of the 135-kDa and 60-kDa major protein have similarity to the sequences of the Cry1Ab16,CryE1,CryIG and Cry2Ac of Bacillus thuringiensis. The internal amino acid sequences of 60-kDa protein of ssp. wuhanensis showed homology to CryIIC proteins in the databases. Based on this result, we have cloned a new cry-type gene from ssp. wuhanensis 140 with PCR products as probes. The deduced amino acid sequence of the clone is 97% identical to the holotype Cry2Ac1. The nomenclature assignment of this clone is, therefore, named Cry2Ac6 by the Cry Nomenclature Committee. Proteomic analysis of these proteins leads to the conclusion that the sequence data provided the protein databases of this new crystal protein of the ssp. wuhanensis.We analysed the sequences of chitinase gene of Bacillus thuringiensis that gotten from GenBank,EMBL,DDBJ and PDB databases. Based on the analysis of the sequences, a paire of primers were designed to detected the existence of chitinase gene. After PCR, we found that ssp. wuhanensis 140 strain contains chitinase gene. Then, another paire of primers were used to clone the Open Reading Frame(ORF).After sequenced, we found that the ORF was 100% identical to the chitinase gene from ssp. kurstaki (AY189740).We presumed that ssp. wuhanensis were relative to ssp. kurstaki.Our research revealed that ssp. wuhanensis 140 strain contains a lot of valuable genes. This strain has the distinct characteristic to HD-1 and HD-73 such as figure of crystal protein, toxicity and insecticide crystal protein gene. This strain values further research, and can be extensively used.