The Role And Mechanism Of Intron Retention And NMD Pathway In Fungal Cellulase Production

Cellulose is an environmentally friendly and renewable resource commonly used in everyday life.It is inexpensive and can be degraded and used for the production of bio-refined products.Trichoderma reesei,as the most widely used fungus for industrial cellulase production,has a very high protein secretion,but its industrial application is hindered by its low β-glucosidase activity.Therefore,studying the cellulase production mechanism of T.reesei is significant for the construction of genetically engineered T.reesei with high β-glucosidase production and other specific needs.Intron retention is an important way to regulate fungal physiological processes,but exactly how it plays a role in fungal cellulase production has not been studied in depth.This thesis explores the roles and mechanisms of intron retention(IR),its induced nonsense-mediated mRNA decay(NMD),and unfolded protein response(UPR)in the cellulase production process of T.reesei.The following three main aspects of work have been carried out:1.The effect of NMD pathway on cellulase production during fungal cellulase production and its possible mechanism of action: The effect of NMD inhibitors on cellulase activities produced by T.reesei are investigated using the original strain RUT-C30 and knockout strain of T.reesei,using cellulose,lactose and glucose as carbon sources,respectively.The results show that the effect of caffeine on the cellulase activities is the most obvious among the NMD inhibitors,and the caffeine-binding receptors may exist in target of rapamycin(TOR)pathway.2.Fungal intron retention and nonsense degradation of mRNA decay(IR-NMD)posttranscriptional regulatory layer in the process of cellulase production in T.reesei: The changes of intron retention of T.reesei RUT-C30 in the presence of different carbon sources and inhibitors are observed to further investigate how intron retention and nonsense-mediated mRNA decay regulate the transcriptional level of cellulase.It is found that RUT-C30 exhibits a more active NMD pathway and lower IR levels on the cellulase condition compared to the glucose condition.3.The role and mechanism of hac1,the main gene responsive to the unfolded protein response in fungi,and its 5’ end and intron retention in the process of cellulase production and mechanism exploration: Four different strains overexpressing the gene hac1 are constructed.They are examined for cellulase activities during fermentation,abundance changes of 5’flanking region and 20 nt intron,and the transcriptional levels of UPR-related genes.We found that the 20 nt intron is retained in the mRNA to prevent translation of the hac1 gene by forming a hairpin structure with its 5’ flanking region and further regulating cellulase production.In summary,this thesis focuses on the cellulase production process of T.reesei.On the one hand,it investigates the effects of intron retention and nonsense-mediated mRNA decay on cellulase production,and elucidates the mechanism by which its constitutive IR-NMD posttranscriptional additional regulatory layer plays a role in the cellulase production process of RUT-C30.On the other hand,it takes the hac1 gene as an object of study and explores the effects of its intron retention and 5’ transcript level on cellulase activities,which complements and explores the fungal post-transcriptional regulatory layer.It is hoped that the work in this thesis can have reference value in the role of fungal intron retention and the mechanism of fungal cellulase production,and to a certain extent help researchers to have a meaningful understanding on the mechanism of fungal cellulase production process and the construction of genetically engineered T.reesei surrounding the mechanism.