Abstract:[Background] The filamentous fungus Trichoderma reesei is a eukaryotic microorganism and smaller than any other multicellular, which is widely used in industry, and T. reesei QM9414 were one of the most abundant cellulase producing mutant. [Objective] In order to study cellulase activities and expression regulation in T. reesei, we constructed siRNA interfered vectors and overexpressed vectors to inhibit and enhance HKMT expression in T. reesei QM9414, respectively. [Methods] The siRNA interference fragment was designed according to the hkmt gene sequence of T. reesei and the overexpressed hkmt gene fragment was obtained by reverse transcription. The two fragments were cloned into the constitutive expression vector and the two vectors were transformed the protoplasm of T. reesei QM9414. The hyphae growth of the recombinant strains was observed through a fluorescence microscope. In addition, cellulase activities (CMC enzyme activity and filter paper enzyme activity) of recombinant strains were detected and related gene expressions were also detected by quantitative PCR. [Results] The mycelial morphology of the interfered and the overexpressed recombinant strains were not significantly different from the original strain. Quantitative PCR results showed that the interfered vector and the overexpressed vector can silence and promote the expression of hkmt gene, respectively. The enzyme activities of FPA and CMC in the interfered recombinant strains were increased by an average of 2.5 times compared with the original strain. In addition, the expression levels of cellulase-related genes and activators in interfered recombinant strains increased. However, the recombinant strains for overexpressing HKMT showed the opposite patterns in the genes’ expression levels and enzymatic activities. [Conclusion] These findings indicate that HKMT in T. reesei is involved in the negative regulation of cellulase expression and production.
