Characterization of a basic chitinase which is secreted by cultured pumpkin cells


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A basic chitinase was secreted into culture medium of pumpkin cell suspension cultures. The chitinase was purified from the culture medium. A cDNA encoding the pumpkin chitinase was cloned by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE) methods. The chitinase gene was strongly expressed in pumpkin callus cells, but little or not at all in mature leaf, young leaf, cotyledon, stem, hypocotyl and root of pumpkin. No chitinase mRNA was detected in intact pumpkin fruit tissues. However, chitinase was induced during callus formation from sliced pumpkin fruit tissues. Induction also occurred in the absence of 2,4-D, a chemical causing callus formation, suggesting that it may be independent of the presence of 2,4-D. Perhaps, induction is caused by osmotic or wounding stress. Levels of chitinase mRNA markedly increased at 4 h after transfer of pumpkin callus cells into fresh culture liquid medium. They were also high at later stages of cell suspension culture. In transgenic tobacco BY-2 cells, into which the pumpkin chitinase cDNA was introduced, the recombinant pumpkin chitinase was expressed and secreted into the culture medium, suggesting that the signal peptide of pumpkin chitinase also functions for secretion from tobacco BY-2 cells.