Isolation and Cloning of Cre1 Gene from a Filamentous Fungus Trichoderma Harzianum

Cellulases and hemicellulases are two important classes of enzymes produced by filamentous fungi and secreted into the cultivation medium. The production of these enzymes is under carbon catabolite repression (CCR), a general mechanism that prevents their synthesis in the presence of a preferred carbon source such as glucose. CRE1 causes the repression of transcription of cellulase and xylanase encoding genes. This study describes the isolation and cloning of a partial sequence of glucose repressor creI gene from Trichoderma harzianum E-58. The fungus was grown in Vogel’s medium at 28C, and pH 5.5 with glucose as a carbon source. Genomic DNA was subjected to the polymerase chain reaction (PCR) for amplification of cre1 gene by using degenerate primers. The PCR product was purified through agarose gel electrophoresis and ligated into pTZ57R/T vector. The ligation mixture was then transformed into E. coli DH10B and spread on the LB agar plate containing ampicillin. Clone analysis by PCR was done to confirm the positive transformants. These results will help study the regulation of cellulase and xylanase gene expression in the fungus in future.