Abstract
The tobacco BY-2 chitinases were induced in intra and extracellular fractions by the addition of the pathogenic fungus Alternaria alternata cultured medium. We isolated three of these intracellular chitinases (TBC-1,2,3) by heat treatment, acid treatment, colloidal chitin affinity, ion-exchange chromatography, and hydrophobic chromatography. These molecular masses were estimated to be 28.8, 33.3, and 28.5 kDa, respectively. N-terminal amino acid sequences were analyzed. Based on this sequence, proper primers were designed used for successive TBC-1 cDNA analysis. TBC-1 cDNA was cloned encording 1013 bp full-length cDNA including 831 bp putative ORF resion. Compared with those of other proteins, TBC-1 showed homology to class IV chitinases of other plants but not already reported tobacco chitinases. Therefore, TBC-1 was thought to be a novel chitinase of tobacco. By northern blot analysis, TBC-1 gene expression was induced by a fungal elicitor from A. alternata.
