Preparation Of The Antifungal Protein From Paenibacillus Polymyxa WY110 And The Coding Gene Cloning And Expression

An antifungal protein P2 from Paenibaciilus poiymyxa WY 110 which was a plant growth-promoting bacteria strain isolated from rice plant was purified and characterized. The P2 protein-coding gene was cloned and sequenced. A full expression of the P2-coding gene in E. coil was achieved. With SDS-PAGE, antagonistic activity tracing, DEAE-sephadex A-SO and Sephacryl S-200 chromatography, P2 protein was prepared and purified. It was showed with antagonistic activity on PDA plates that the growth of Piricuiaria oryzae was inhibited by 1.5 pg of P2 protein effectively. The antiserum of P2 protein was prepared with purified P2 protein as the antigen. N-terminal an residues analysis showed a twenty four an residues sequence: H2N-Ala-Asn-Val-Phe-Trp-Pro-Ser-Tyr-Tyr-Asn-Pro-Ser-Thr-Trp-Gln-Lys-Ala-Gly- Tyr-Ser-Asn-. Using this an sequence as a target, the similarity of P2 protein was searched with BLASTP program on internet. It was showed that a high homology between the P2 protein and the precursor of 13-1,3-1,4-glucanase from Bacillus. The activity of ~3- 1,3-1 ,4-glucanase for P2 protein was identified with the specific substrate lichenan. This is the first report about the antagonistic activity to fungi for I~- 1,3-1 ,4-glucanase. According to the N-terminal an residues sequence of P2 protein and the conservation sequence of C-terminal an residues sequence of ~-1 ,3- 1 ,4-glucanase, the primers for both terminals were synthesized. The full sequence of P2-coding gene was amplified by using the chromosome DNA of WY 110 as the template with PCR technique followed by cloning on pMD18-T vecter. The nuclear acid sequence determination showed the 72 nuclear acids sequence on 5?terminal with 636 nt was identical with the known 24 an sequence on N-terminal of P2 protein. Comparing with a ~3-1,3-1,4-glucanase from a paenibaciilus polymytxa (Gosalbes M J, 1991), the sequence homology for nuclear acids and deduced amino acid residues were 84% and 88.7% respectively. III For constructing the expression vector of a fusion protein and obtain a target protein with full identity on aa sequence of a natural 13- 1,3-1 ,4-glucanase, with the recombined plasmid DNA harbouring the target gene as template, the primers designed with restriction sites for both terminals and enterokinase recognition site, followed by PCR amplification, was induced to the target gene. The sequencing result showed that the linker and the ORF were correct. The target gene was digested by two restriction enzymes and ligated with GST fusion protein expression vector PGEX-3X, followed by transforming to E.coli JM1O9. Thus the fusion protein expression vector was constructed successfully. With IPTG for inducing the fusion protein expression followed by bacterial cell ultrasonication, the total cell lysate was obtained. The expected protein band was showed on SDS-PAGE pattern analysis. The GST fusion protein was purified by glutathione affinity chromatography. The target protein released by digesting with enterokinase was identified as the products of the target gene by western-blotting with the specific immuno-biological reaction of the P2 protein antibody. The biological assay showed this target protein with remarkable antagonistic activity to Piricularia oryzae. The cloning of P2 protein-coding gene would be a new potential objective gene for plant gene engineering.