[Purification and cloning of an antifungal protein from the rice diseases controlling bacterial strain Paenibacillus polymyxa WY110].

Paenibacillus polymyxa WY110, a plant growth-promoting bacteria strain isolated from rice rhizosphere could suppress the growth of various plant pathogens effectively. With (NH4)2SO4 fractional precipitation, DEAE-Sephadex A-50 chromatography and Sephacryl S-200 chromatography followed by tracks of fraction antagonistic assay and SDS-PAGE, an antifungal protein P2 with in vitro anti-Pyricularia oxyzae activity was isolated and purified. It was showed with antagonistic activity on PDA plates that the growth of Pyricularia oryzae was inhibited by 1.5 microg of P2 protein effectively. N-terminal amino acid residues analysis showed 24 amino acid sequence: H2N-Ala-Asn-Val-Phe-Trp-Glu-Pro-Leu-Ser-Tyr-Tyr-Asn-Pro-Ser-Thr-Trp-Gln-Lys-Ala-Asp-Gly-Tyr-Ser-Asn-. Using this amino acid sequence as a target, the similarity of P2 protein was searched with BlastP program on Internet. It was showed a high homology between the P2 protein and the precursors of beta-1, 3-1, 4-glucanases from Bacillus. The beta-1, 3-1, 4-glucanase activity of P2 protein was identified with the specific substrate lichenan. According to the N-terminal partial sequence of P2 protein and the C-terminal conserved sequence of beta-1, 3-1, 4-glucanase, the primers for both terminals were synthesized. Using the genomic DNA of WY110 as the template, the full-length sequence of the gene encoding P2 was amplified by high fidelity PCR, then cloned into pMD18-T vecter. Sequence analysis showed the 72 nucleotide sequence on 5'-end matched with the known 24 amino acid sequence on N-terminal of P2 protein. The sequence (GenBank Accession Number: AF284449) was 636 bp in length encoding 212 amino acids. Comparing with a beta-1, 3-1, 4-glucanase gene (gluB) from Paenibacillus polymyxa, the sequence homology for nucleotides and deduced amino acids were 84% and 88.7% respectively. The cloning of the gene encoding P2 protein would be a new potential objective gene for plant gene engineering.