Cloning And Prokaryotic Expressing Of Chitinase Encoding Genes From Biocontrol Agent Strain Bacillus Atrophaeus CAB-1

As the problems of plant pathogen resistant to chemical fungisides and fungicide residue getting worse and worse, biocontrol was paid more attention to plant diseases management. Chitin was the main component of higher fungi cell wall, and could be degraded by chitinese effectively. In this way the plant pathogens with chitin in their cell wall would be destroyed by the chitinolytic microorganisms. Therefore, screening of antifungal chitinolytic microorganism not only was helpful to develop the higher efficient biofungicides but also provided antifungal genes for transgenic plants and engineered microorganism strains.1. Bacterial strain CAB-1 was isolated according to its chitinolytic activity. Antifungal activities against Botrytis cinerea and other plant pathogenic fungi were verified. Strain CAB-1 was identified as Bacillus atrophaeus based on the biochemical characters and the sequences analysis of 16s rDNA and gyrB gene.2. Through sequence analysis and function predicting based on it whole genome sequences, two chitinase genes named chit1 and chit2 were located and cloned from strain CAB-1. The results showed that the chit1 gene was 1791 bp, which encoded 596 amino acids residues with a molecular weight of 65.8 KDa. The chit2 gene was 2091 bp, which encoded 696 amino acids residues with a molecular weight of 77.8 KDa. There was a 69 bp gap between the two genes. In comparison with the reported chitinase gene (CP002207.1) from Bacillus atrophaeus 1942 and chit gene (AF069131.1) from B. subtilis sequences, the nucleotide sequence homology of the cloned chit1 gene was 99%. The chit 2 sequence shared 96% homology with Bacillus pumilus chitinase operon sequence. Through amino acids sequence analysis, chit1 had a 36 amino acids residues signal peptide at the N-terminal. There was three domain including chitinase catalytic domain, Fibronectin typeⅢlike domain and chitin binding domain. Chit1 was recognized as GH-18 chitinase family B subfamily protein. There was also a signal peptide at the N-terminal which was 36 amino acids residues in chit2. Only chitinase catalytic domain could be found in chit2.3. These two cloned chitinase genes were ligated into the prokaryotic expression vector and fusion proteins were induced. The target proteins were tested by SDS-PAGE. Chitinase activities of the induced protein Chit1 and Chit2 were testified by plate transparent cycle method and DNS method. Chitinese activity of Chit1 was stronger than Chit2. In the meantime, only Chit1 protein showed antifungal activity against B. cinerea. Chit2 did not show this antifungal activity. The expression condition of Chit1 protein was optimized. The result showed that incubating at 30℃in shaker and induced by using 0.5 mmol/L IPTG was recognized as the optimized condition for Chit1 protein production.