ISOLATION AND SEQUENCE OF AN ENDOCHITINASE-ENCODING GENE FROM A CDNA LIBRARY OF TRICHODERMA-HARZIANUM

There are no reports of gene sequences coding for extracellular chitin olytic enzymes from filamentous fungi, even though these enzymes are c onsidered critical to the biological control of plant pathogenic fungi . The purpose of this paper was to report the isolation of a gene (ThE n-42) encoding endochitinase (Ech) from Tr ichoderma harzianum strain P1, describe its sequence, and to determine whether it was related to genes coding for enzymes with similar functions from prokaryotic or ot her eukaryotic sources. A clone containing a 1096-bp foreign cDNA frag ment was isolated from thalli grown under induced conditions. This cDN A molecule was sequenced and found to lack a portion of the 5' terminu s. Polymerase chain reaction (PCR) was used to isolate a fragment from the lambda gt11 library which contained the 5' terminus plus an overl ap region with the 1096-bp cDNA clone. The full-length cDNA sequence, consisting of 1554 bp, contained an open reading frame (ORF) expressin g a protein of 424 amino acids (aa). Southern analysis of genomic DNA indicated that there is only a single gene in strain P1 with sequence identity to the sequence described in this report. One region within t he protein, thought to be required for catalytic activity of the enzym e, was highly conserved between genes coding for Ech from Th, Serratia marcescens, Bacillus circulans, Streptomyces plicatus, Vibrio parahem olyticus and Kluyveromyces lactis.