Studies On Regulation Of Cre4 Gene On Cellulase Expression In Trichoderma Reesei

Filamentous fungi Trichoderma reesei(T.reesei) is the main strain of cellulose enzyme production in industry,which is simple and cheap to cultivate and provided mature fermentation method. There is carbon catabolic repression(CCR) existing in T.reesei, involving a variety of cellulase genes expressing-induction and the positive and negative regulation of transcriptional regulation factor and carbon catabolic repressor. In T.reesei,regulatory proteins participating in CCR includes CRE1, CRE2, CRE3 and CRE4, homologous to the Aspergillus nidulans CREA, CREB, CREC and CRED respectively. CRE4/CRED is rarely reported, and CRE4 may be mediated by ubiquitin ligase which involved in cellulose enzyme regulation. This study used siRNA interference technique to suppres the expression of cre4 gene, and constructed CRE4 expression vector to study the regulation of cellulase expression effect,futher more explore CCR regulation network in T.reesei.Here is the results:We took cre4 as target gene to design siRNA interference fragment, and selected four siRNA(T2, T7,T10 and negative control Neg) respectively to construct interference expression vector p-Ppdc’-T2-Tcbh1, p-Ppdc’-T7-Tcbh1, p-Ppdc’-T10-Tcbh1 and p-Ppdc’-Neg-Tcbh1, and respectively integrated them into the genome of T.reesei QM9414. The genomic PCR identification and sequencing had shown that we obtained 4 interference recombinant strains: T.reesei-cre4-T2, T.reesei-cre4-T7, T.reesei-cre4-T10 and the negative control T.reesei-cre4-Neg.The fluorescence quantitative PCR results showed that, strains T.reesei-cre4-T2, T.reesei-cre4-T7, T.reesei-cre4-T10 after 48 h induction cre4 expression reduced 87%, 71%, 80%, compared to T.reesei QM9414. According to filter paper enzyme activity(FPA) and CMC enzyme activity(CMCA) results,we found interference recombinant strains enzyme activity both increased during induced grown, in 72 h reached a maximum of two kinds;FPA was averagely 2.0 IU/mL higher than T.reesei QM9414 meanwhile CMCA was averagely 4.0 IU/mL higher.The results showed that the interference fragment specifically suppressed the cre4 gene on the transcription level,and cre4 expression decreasing in recombinant strains may increase the cellulose enzyme genes expression.On the other hand, cre4 genes was amplified from T.reesei QM9414 genome through specific primers cre4-F/cre4-R,then was cloned to the modified vector p-Ppdc’’-Tcbh1.We constructed expression vector p-Ppdc’’-cre4 Tcbh1, which was integrated into the genome of T.reesei QM9414. The genomic PCR identification and sequencing showed that we obtained 2 overexpression recombinant strains: T.reesei-cre4-E5 and T.reesei-cre4-E14. According to the fluorescence quantitative PCR results, after 48 h induction strains T.reesei-cre4-E5 and T.reesei-cre4-E14 CRE4 expression quantity increased 4 times and 5 times respectively. SDS-PAGE and Western Blot results showed that CRE4 did overexpress in recombinant strains, and the molecular size of CRE4 is about 110 k Da. According to filter paper enzyme and CMC enzyme activity results,we found that the recombinant strains enzyme activity were lower than T.reesei QM9414, and FPA and CMCA decreased respectively 0.5 and 0.8 IU/m L. The above results showed that in T.reesei QM9414 CRE4 may influence the expression of cellulose as a repressor.In order to study how CRE4 regulates the cellulase expression in the whole regulation system, we used the fluorescent quantitative PCR to analyze metabolic repressor genes ace1, cre1, cre2, cre3 and cellulose enzyme gene cbh1, egl1 and xylanase activation factor XYR1 in both recombinant strains. The results showed that CRE4 has no significant regulation to repressors ACE1, CRE2, CRE3 and activating factor XYR1. Otherwise cre1 expression quantity reduced about 50%,so CRE4 positivly regulateed CRE1.The cellulose enzyme gene cbh1 and egl1 expression quantity in recombinant strains has increased about 2 times of T.reesei QM9414,therefore interfering cre4 gene could partially remove the inhibition from repressors to cellulose enzyme gene.In this paper, by using siRNA interference technology and constructing cre4 overexpression vector to study how cre4 gene regulates the expression of cellulose enzyme genes,we confirmed that CRE4 was a repressor to influence the expression of cellulose.The results help us to further illustrate cellulose enzyme regulatory mechanism of promotion and repression,to raise the yield of cellulose enzyme in T.reesei.