| Trichoderma Reesei is a filamentous fungus that is mainly used in the production of cellulases in the industry,but its yielding of cellulase is regulated by the carbon catabolic repression,so it cannot synthesize cellulase even in the medium rich in carbon sources.Current studies have found that proteins involved in the repression of carbon metabolism in filamentous fungi include CREA,CREB,CREC and CRED in A.nidulans,which are highly homologous to the CRE1,CRE2,CRE3 and CRE4 proteins in T.reesei.The previous studies on CRE1 and CRE2 have been reported but there are no reports on CRE3,CRE4 and the synergistic functions of these four genes in T.reesei are still not clear.In this paper,we constructed a multi-targeted si RNA expression vector to perform multi-targeting silencing of these four genes to investigate the regulatory mechanism of silencing carbon catabolic repressors on the expression of cellulase by T.reesei.In the previous studies,we had known that the best si RNA of cre2,cre3 and cre4,so we just need to find out the best si RNA targeting on the gene cre1.A si RNA expression vector targeting on the gene cre1 was constructed and transformed into the protoplasm of T.reesei.This research was proved that silencing the cre1 can improve the expression of cellulases in T.reesei,and the best si RNA silencing the gene cre1 was selected.The results lay a foundation for the further study of simultaneous silencing of multi-targeting carbon catabolite repressor genes cre1,cre2,cre3 and cre4.Next,we screened of the four best si RNA sequences for the genes cre1,cre2,cre3 and cre4,a multi-targeted expression vector system A and B were designed and constructed to silence multiple target genes in T.reesei.As a result,we found that the multi-target gene interference of T.reesei can more fully eliminate the carbon repression inhibitory effect than one-single gene interference.At the inducing incubation for 120 h,enzymatic activities reached its peak,the average increase of CMCase and FPUase in the multi-targeted gene silencing strain was 1.14 and 1.33 times compared with that of the single gene silencing strain T.reesei-cre1-T10.The average increase of expression levels of cbh1,egl1 and xyr1 genes was 1.53,1.36,and 1.40 times compared with that of T.reesei-cre1-T10.In the B system,T.reesei-cre4132 showed an increase of 1.50 times and 1.25 times increase in CMCase and FPUase respectively compared to that of single gene silencing strain T.reesei-cre1-T10 at 120 h,and the expression level of genes cbh1,egl1 and xyr1,was respectively 2.07 times,1.59 times and 1.61 times higher than that of T.reesei-cre1-T10.There was no significant difference in the CMCase and the FPUase between T.reesei-cres and T.reesei-cre1-T10 in the same time,the CMCase of T.reesei-cres was even 27% lower than the T.reesei-cre1-T10 and the expression level of genes cbh1,egl1 and xyr1 were also lower than that of the T.reesei-cre1-T10.The relative expression levels of cre1,cre2,cre3 and cre4 genes in T.reesei-cre4132 and T.reesei-cre1423 strains in the B system were 70%?53%?69% and 65% lower than those in the T.reesei QM9414 at inducing incubation for 120 h.The relative expression levels of cre1,cre2,cre3,and cre4 genes in T.reesei-cres strains in the A system were reduced by 27%,34%,26%,and 30%,compared to T.reesei QM9414.In this thesis,it showed that carbon catabolic genes cre1,cre2,cre3 and cre4 in T.reesei were simultaneously silenced,and the cellulase activities were much higher than that of the starting strain.The production of cellulase and cellulase-related genes expression were also increased.It is suggested that the silencing multi-targets of the carbon-repressing factor in T.reesei is beneficial to release the glucose effects and increase the utilization of non-reducing sugars to increase the expression and production of cellulase.The results provide evidence and techniques for study of regulation of carbon catabolite repressor genes and cellulase expression in Trichoderma reesei. |
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