Abstract:[Objective] In order to study carbon metabolic repressors on cellulase activities and expression regulation in Trichoderma reesei, we constructed multi-targeting siRNA expression vectors to perform silencing carbon metabolic repressor cre1, cre2, cre3 and cre4 by simultaneous producing multi-targeting siRNAs. [Methods] According to previous studies and screening, the four best siRNAs targeting the cre1, cre2, cre3 and cre4 were selected and they were constructed a multi-targeting siRNA expression vector A. Additionally, according to 5 overlap common sequence in the cre1, cre2, cre3 and cre4, we designed and constructed the vector B. The two vectors were transformed the protoplasm of Trichoderma reesei QM9414 and selected on the hygromycin selection medium. Cellulase activities (CMC activity and filter paper activity) of transformants were detected and related gene expressions were also detected by RT-qPCR after incubation for 48 and 120 hours respectively. [Results] RT-qPCR results showed that the cre1, cre2, cre3 and cre4 expression levels in Trichoderma reesei were simultaneously silenced, and the cellulase activities were much higher than that of the staring strain. The CMC activity and filter paper activity of the multi-targeted inhibitor strains were 1.95-fold and 2.66-fold higher than those of the original strains. The cellulase-related genes expression levels were also increased significantly. The expression levels of cbh1, egl1 and xyr1 were 3.83-fold, 3.95-fold and 2.78-fold higher than those of the original strain. [Conclusion] Our results indicated that simultaneous silencing multi-targets of the carbon metabolic repressors in Trichoderma reesei is beneficial to release the glucose repressor effects and increase the expression and production of cellulase. These results provide evidence and techniques for study of regulation of carbon catabolite repressor genes on cellulase expression in Trichoderma reesei.
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