A molt-associated chitinase cDNA from the spruce budworm, Choristoneura fumiferana.

Chitinase (CfChitinase) cDNA from the spruce budworm, Choristoneura fumiferana, was cloned using reverse transcription PCR and cDNA library screening. The CfChitinase cDNA was determined to be 2856 nucleotides long with the longest open reading frame made up of 1671 nucleotides that encoded a protein that was 557 amino acid long with a predicted molecular mass of 62 kDa. The deduced amino acid sequence showed 76-79% identity with other lepidopteran chitinases. Northern blots revealed that transcripts of CfChitinase appeared prior to each molt and peaked on the day of ecdysis from the second instar to the pupal stage but disappeared immediately after the molt. No transcripts could be detected in the early first instar prior to the spinning of the hibernaculum or in the diapausing second instars or during the intermolt periods of the other instars. Western blot analysis revealed that the protein appeared 12 h prior to ecdysis and disappeared 12 h after ecdysis from the sixth instar to pupal stage. The 20-hydroxyecdysone analog, tebufenozide (RH5992), induced expression of CfChitinase in the early stage of the sixth instar and caused a precocious and incomplete molt into an extra larval stage. During the sixth instar to the pupal molt, transcripts could be detected only in the epidermis and fat bodies, but not in the midgut. Western blots showed that the protein was present in the epidermis and midgut, but not in the fat bodies. The recombinant protein expressed in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) showed high levels of chitinolytic activity with an optimal pH range 6-9. Glycosylation appeared to be necessary for the chitinolytic activity and secretion of the recombinant protein.