Purification And Cloning Of Antibiotic Protein Produced By Bacillus Subtilis Strain Tpb55 And Studies On The Sequence

Bacillus subtilis Tpb55 ,an antagonistic bacteria isolated from the phyllosphere of tobacco,strongly inhibits the growth of Alternaria alternata .An antifungal protein Bst1 from Tpb55 was purified and characterized.The N-terminal eighteen aa residues sequence of protein Bst1 was analyzed.With DEAE Sepharose Fast Flow ,Swphadex G-75 chromatography ,antagonistic activity tracing ,and SDS-PAGE ,the Bst1 protein is purified .It was showed with antagonistiv activitity on PDA plates that the growth of Alternaria alternate was inhibited by Bst1 protein effectively .The N-terminal aa residues analysis showed a eighteen aa residues sequence Met- Glu-Ile- Phe-Lys-Tyr- Met-Glu- Thr- Tyr- Asp-Tyr-Glu- Gln - Leu-Val- Phe- Cys- .Using the aa sequences as a target ,the similarity of Bst1 protein was searched with BLASTP program on internet .It was showed that a high similarity between the Bst1 protein and the Leucine Dehydrogenase from Bacillus subtilis .This is the first report about the antagonistic activity to Alternaria alternate for the Leucine dehydrogenase.According to the N-terminal aa sequences of Bst1 protein and the conservation sequence of C-terminal aa sequences of Leucine dehydrogenase ,the primers for both terminals were synthesized .The full sequence of Bst1-coding gene was amplified by using the chromosome DNA of Tpb55 as the template with PCR technique followed by cloning on pEASY-T1 cloning vecter .The nuclear acid sequence determination showed that the full-length of the Bst1 gene was 1095bp ,deducing amino acid 365nt long .The fifty four nuclear acids sequence on 5'terminal with 1095bp was identical with the known eighteen aa sequences on N-terminal of the Bst1 protein .The clonging of Bst1 protein–coding gene would be a new potential objective gene for tobacco plant gene engineering .The biological assay showed that the Bst1 protein with remarkable antagonistic activity to the Alternaria alternate .The cloning of Bst1 protein-coding gene would be a new potential objective gene for tobacco gene engineering.