Cloning, sequence and structure of a gene encoding an antifungal glucan 1,3-β-glucosidase from Trichoderma atroviride ( T. harzianum)

A gene ( gluc78) encoding an antifungal glucan 1,3-β-glucosidase was cloned from strain P1 of the biocontrol fungus Trichoderma atroviride (formerly T. harzianum). A putative regulatory sequence upstream from the coding region was cloned using single-strand extension from a primer in the known portion of the gene, circularized with T4 ligase, and then reamplified with PCR to generate double-stranded DNA. The entire genomic DNA sequence consisted of 3440 bp, with 559 and 579 bp, respectively, in 5′ and 3′ untranslated regions. The transcription unit contains a single intron, positioned in the 5′ untranslated region. The gene encodes for a protein of 770 aa, including a 40 aa signal peptide. Symmetry between the first and second halves of the mature protein was found. The gene is present as a single copy in T. atroviride and a similar gene also is present in T. harzianum and T. virens. The encoded protein has similarity to a small group of sequences from filamentous fungi and no significant similarity to 1,3-β-glucanases or glucosidases from other organisms. Northern analysis indicates that the gene is repressed in the presence of 3% glucose and expressed in media containing 0.1% of the sugar. Laminarin (0.1%) enhances expression after 18 h and other polymers such as scleroglucan and pustulan may enhance expression after 40 h of growth.