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

A basic chitinase was secreted into culture medium of pumpkin cell suspensi on cultures. The chitinase was purified from the culture medium. A cDNA enc oding the pumpkin chitinase was cloned by reverse transcription (RT)-PCR an d rapid amplification of cDNA ends (RACE) methods. The chitinase gene was s trongly expressed in pumpkin callus cells, but little or not at all in matu re leaf, young leaf, cotyledon, stem, hypocotyl and root of pumpkin. No chi tinase mRNA was detected in intact pumpkin fruit tissues. However, chitinas e was induced during callus formation from sliced pumpkin fruit tissues. In duction also occurred in the absence of 2,4-D, a chemical causing callus fo rmation, suggesting that it may be independent of the presence of 2,4-D, Pe rhaps, induction is caused by osmotic or wounding stress, Levels of chitina se mRNA markedly increased at 4 h after transfer of pumpkin callus cells in to fresh culture liquid medium. They were also high at later stages of cell suspension culture. In transgenic tobacco BY-2 cells, into which the pumpk in chitinase cDNA was introduced, the recombinant pumpkin chitinase was exp ressed and secreted into the culture medium, suggesting that the signal pep tide of pumpkin chitinase also functions for secretion from tobacco BY-2 ce lls.