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

• Published in: Gene 2001-10, Vol.277 (1), p.199-208
• Main Authors: Donzelli, B.Giuliano Garisto, Lorito, M., Scala, F., Harman, G.E.
• Format: Article
• Language: English
• Subjects: 5' Flanking Region - genetics; Amino Acid Sequence; beta-Glucosidase - genetics; Biological control; Blotting, Northern; Blotting, Southern; Cloning, Molecular; DNA, Fungal - chemistry; DNA, Fungal - genetics; Exo 1,3-β-glucanase; Exons; Fungal growth and differentiation; Fungi - enzymology; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Genes - genetics; gluc78 gene; glucan 1,3-^b-glucosidase; Glucan 1,3-beta-Glucosidase; Introns; laminaran; laminarin; Molecular Sequence Data; Regulatory Sequences, Nucleic Acid - genetics; RNA, Fungal - genetics; RNA, Fungal - metabolism; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Transcription Initiation Site; Trichoderma - enzymology; Trichoderma - genetics; Trichoderma atroviride; Trichoderma harzianum; Trichoderma virens
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(01)00681-3

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.