CLONING AND CHARACTERIZATION OF A CHITOSANASE GENE FROM THE PLANT-PATHOGENIC FUNGUS FUSARIUM-SOLANI

The plant pathogenic fungus Fusarium solani f. sp. phaseoli SUF386 sec retes a chitosanase in the absence of exogenous chitosan. Based on par tial amino acid sequences of the purified chitosanase, two degenerate oligonucleotides were synthesized and used as reverse transcription-me diated PCR (RT-PCR) primers to amplify a 500-bp fragment of correspond ing cDNA. The PCR product was used as a probe to isolate the genomic c opy of the gene (csn). F. solani csn has an open reading frame encodin g a polypeptide of 304 amino acid residues with a calculated molecular mass of 31,876 Da and containing a putative 19-amino acid residue sig nal sequence. Comparison between the genomic and cDNA sequences reveal ed that three introns are present in the coding region. Southern blot analysis results indicated that csn is present as a single copy in the genome of F. solani SUF386, The cDNA fragment corresponding to the ma ture enzyme was introduced into E. coli using an expression vector dri ven by the T7 promoter. The resulting E. coli transformant overproduce d proteins with chitosanolytic activity.